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The Insulin-Related Ovarian Regulatory System in Health and Disease

Division of Endocrinology, Department of Medicine (L.P.) and Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology (Z.R.), New York Presbyterian Hospital and Weill Medical College of Cornell University, New York, New York 10021; and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Stanford University Medical Center (N.A.C., L.C.G.), Stanford, California 94305

	Abstract

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 

I. Introduction

II. Insulin and Insulin Receptor

A. Structures of insulin and insulin receptor

B. Presence of insulin and insulin receptor in the ovary

C. Insulin action and the ovary

D. Summary

III. IGFs and Their Receptors

A. IGF peptides and receptors

B. Expression of IGFs and IGF receptors in the ovary

C. Role of IGFs in ovulatory function and steroidogenesis

D. Summary

IV. IGF-Binding Proteins (IGFBPs) and Proteases

A. Structural relationships among IGFBPs

B. IGFBP expression in the ovary

C. IGFBP proteases in the ovary

D. IGFBP actions in the ovary

E. Role of IGFBPs in follicular development and atresia

F. Summary

V. Polycystic Ovary Syndrome (PCOS)

A. Clinical features

B. Theories of pathogenesis

C. Insulin resistance in PCOS

D. Alterations of IGFs and IGFBPs in PCOS

E. Summary

VI. The Insulin-Related Ovarian Regulatory System: Implications for Therapy

A. Treatment of PCOS

B. Therapeutic use of IGF-I and IGF-II

C. Use of GH in ovulation induction

VII. Summary and Conclusions

	I. Introduction

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 
INSULIN, a pancreatic peptide hormone produced in the ß-cells of the islets of Langerhans, plays a major role in the regulation of carbohydrate, fat, and protein metabolism (1). The classical target organs for insulin action are muscle, adipose tissue, and liver (2). Until approximately a decade ago, insulin was not thought to play a significant role in the regulation of ovarian function, despite suggestions of the "gonadotropic" function of insulin (3) in observations of abnormal ovarian function in young women with type 1 diabetes mellitus by Joslin et al. (4), which predated the discovery of insulin more than 75 years ago (5). A resurgence of interest in the ovarian effects of insulin was stimulated by observations of severe ovarian hyperandrogenism in women with syndromes of extreme insulin resistance (6, 7), which led to the hypothesis that high levels of circulating insulin may cause excessive androgen production in these patients (8, 9). The demonstration of insulin’s ability to stimulate steroidogenesis in ovarian cells in vitro (10) and the demonstration of insulin receptors in both stromal and follicular compartments of the human ovary (11, 12) established the ovary as another important target organ for insulin action.

This field was further expanded by studies of the ovarian production and ovarian effects of the insulin-like growth factors, IGF-I and IGF-II, by the discovery of ovarian type I and type II IGF receptors, and by the discovery of the ovarian production of binding proteins [IGF-binding proteins (IGFBPs)] for these two growth factors (13, 14, 15). Thus, in addition to insulin, a role for the structurally related IGFs in ovarian function has gained recognition. Over the last decade, a significant amount of information has accumulated about the role of insulin and IGFs in the ovary at the molecular, cellular, and clinical levels in a variety of normal and pathological conditions. Therefore, a need has arisen for a comprehensive review of what we term the insulin-related ovarian regulatory system. This system consists of the following components (Table 1): insulin; IGF-I and IGF-II; insulin receptor; type I and type II IGF receptors; IGFBPs 1–6; and IGFBP proteases.

This article reviews the role of each component of the insulin-related ovarian regulatory system in both normal ovarian physiology and in relevant pathological states, the interactions among the components of this system, and the therapeutic implications of this system for women with abnormal ovarian function.

	II. Insulin and Insulin Receptor

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 
A. Structures of insulin and insulin receptor
Detailed reviews of the structures of insulin and its receptor are available (1, 2, 39, 40, 41, 42), and thus only a brief overview will be presented here.

Insulin is a 5900 mol wt polypeptide secreted by the ß-cells of the pancreatic islets of Langerhans. The human insulin gene is located on chromosome 11 (39) and encodes pre-proinsulin, a 110-amino acid single-chain polypeptide that is the precursor of insulin (1). Pre-proinsulin is proteolytically converted to proinsulin, which consists of the A chain, B chain, and C peptide. Proinsulin is homologous with IGF-I and -II and can bind to the insulin receptor with approximately 10% of the affinity of insulin. Insulin is produced after the C-peptide is cleaved from proinsulin by endopeptidases active in the Golgi apparatus and in secretory granules. The endopeptidases preferentially cleave either at the C peptide/B chain junction, between Arg31 and Arg32 (endopeptidase type I), or at the C peptide/A chain junction, between Lys64 and Arg65 (endopeptidase type II). The resulting insulin molecule consists of an A chain (21 amino acids) and a B chain (30 amino acids), with three disulfide bridges: two between the A and the B chains (A7-B7 and A20-B12) and one within the A chain (A6-A11).

The insulin receptor is a heterotetramer consisting of two - (135 kDa molecular mass) and two ß- (95 kDa molecular mass) subunits (2). The gene for the insulin receptor is located on the short arm of chromosome 19 (43, 44, 45), contains 22 exons, is more than 150 kb in length, and encodes the proreceptor, a single-chain polypeptide with a molecular mass of 190 kDa that contains one and one ß-subunit. The mature ₂ß₂ heterotetrameric form of the receptor results from dimerization and several posttranslational processing steps, including proteolytic cleavage. An isoform of the receptor lacking 12 amino acids encoded by exon 11 results from alternative mRNA splicing. Insulin receptors lacking exon 11 may have biological properties somewhat different from those containing exon 11 (46), although no significant differences in insulin binding and insulin receptor kinase activity between these two variants were observed (47).

Insulin receptor -subunits are extracellular structures possessing cysteine-rich domains that serve as insulin-binding sites. Insulin receptor ß-subunits have extracellular, transmembrane, and intracellular domains, the latter containing an ATP-binding site and several tyrosine autophosphorylation sites. After insulin binds to the -subunits, the ß-subunits become phosphorylated on tyrosine residues and acquire kinase activity, initiating a cascade of intracellular protein phosphorylation (48, 49). The most important intracellular proteins phosphorylated under the influence of the insulin-receptor tyrosine kinase are the insulin receptor substrates (IRS), several of which have been described (50, 51, 52, 53, 54, 55, 56, 57, 58). IRS-1, the first of these to be discovered (2, 59), has a molecular mass of 131 kDa and possesses 14 potential tyrosine phosphorylation sites. IRS-1 appears to be important in insulin receptor function and its variant forms are sometimes associated with diabetes (60, 61). Mice deficient in IRS-2 develop a syndrome resembling type 2 diabetes (62). Some IRS-1 mutations are associated with insulin resistance and hyperinsulinemia (63), and codon 972 polymorphism of the IRS-1 gene is associated with impaired glucose tolerance, PCOS (64), and late onset of type 2 diabetes mellitus (65). IRS-1 binds phosphatidylinositol-3-kinase (PI-3 kinase), a src homology-2 (SH2) domain-containing enzyme, activation of which is necessary for the initiation of glucose transport (2, 59, 66, 67, 68, 69). In addition to PI-3 kinase activation, mitogen-activated protein kinase (MAPK) is also phosphorylated after insulin receptor binding (2, 49, 59, 70). MAPK activation is thought to be responsible for the growth-promoting effects of insulin (2). MAPK can be activated not only by the insulin receptor, but also by other tyrosine kinase receptors, such as the type I IGF receptor, and receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), as well as G protein-linked receptors (2, 71, 72). The molecular link between the MAPK cascade and the insulin receptor may be p21 Ras, a highly conserved protein involved in cell growth that may be a critical element in growth factor receptor and insulin receptor tyrosine kinase action (2, 49, 59).

Tyrosine kinase activation is believed to be the main signaling mechanism of the insulin receptor (48); it appears to be the earliest postbinding event and is necessary for many, although not all, of insulin’s effects, including transmembrane glucose transport (73, 74). Overexpression of tyrosine kinase-deficient insulin receptors in muscle causes insulin resistance in transgenic animals (75). Tyrosine kinase activity is required in vivo for phosphorylation of IRS-1 and for PI-3 kinase activation (76).

An alternative signaling pathway for the insulin receptor has also been described. It involves generation of inositolglycan second messengers at the cell membrane after insulin binding to receptor -subunits but independently of ß-subunit tyrosine kinase activation (77). This alternative pathway for receptor signaling may mediate some of insulin’s effects, including stimulation of ovarian steroidogenesis (78, 79, 80) (Fig. 1), but the role of this system in propagating the insulin signal for glucose transport and other insulin effects has not been fully established.

View larger version (19K): [in this window] [in a new window]   Figure 1. Insulin receptor, its signaling pathways for glucose transport, and hypothetical mechanisms of stimulation or inhibition of steroidogenesis. The main pathways for the propagation of the insulin signal include the following events: after insulin binds to the insulin receptor -subunits, the ß-subunit tyrosine kinase is activated; IRS-1 and -2 are phosphorylated; PI-3 kinase is activated; GLUT glucose transporters are translocated to the cell membrane, and glucose uptake is stimulated. An alternative signaling system may involve generation of inositolglycans at the cell membrane after insulin binding to its receptor. This inositolglycan signaling system may mediate insulin modulation of steroidogenic enzymes (see text for more details and references).

Insulin receptor-like proteins are present in lower organisms that do not produce insulin. For example, in certain species of worms, daf-2, a gene similar to that of the insulin receptor, regulates glucose metabolism and longevity (82). Mutation of the insulin receptor in Drosophila leads to small ovaries lacking oocytes, and thus sterility (83). Insulin receptor-like molecules are present in mosquito ovaries (84). The existence of these homologous proteins in insects suggests that the growth and regulatory functions of the insulin/IGF receptor family arose before the divergence of insects and vertebrates more than 600 million years ago (83). Conservation of the insulin receptor over this length of time in a variety of organisms indicates its importance for their survival. Indeed, mice with a genetic knockout of the insulin receptor die in the neonatal period (85).

B. Presence of insulin and insulin receptor in the ovary
Circulating insulin levels in the peripheral blood of normal women are approximately 10 µU/ml in the fasting state and up to 50 µU/ml within 1 h after an oral glucose load. In obese women, these levels are somewhat higher, averaging approximately 15 µU/ml in the fasting state and up to 60 µU/ml after a glucose load. In insulin-resistant hyperinsulinemic states such as PCOS or the early stages of type 2 diabetes mellitus, serum insulin levels range from 20–35 µU/ml in the fasting state to 120–180 µU/ml after a glucose load (9, 86). In patients with syndromes of extreme insulin resistance, circulating insulin levels may be as high as 200 µU/ml in the fasting state and up to 1400–2000 µU/ml after a glucose load (9).

Ovarian follicular fluid (FF) insulin concentrations range from less than 2 µU/ml to 65 µU/ml, with a mean value of approximately 16 µU/ml (87). These do not correlate with plasma insulin or FF estradiol (E₂) or androstenedione (A) concentrations, but do correlate directly with those of progesterone (P) (87). Insulin likely reaches FF from the circulation by transudation. To our knowledge, intrafollicular concentrations of insulin have not been reported in women with insulin resistance with or without ovulatory dysfunction.

Both in humans and in animal models, insulin receptors are widely distributed throughout all ovarian compartments, including granulosa, thecal, and stromal tissues (3, 11, 12, 88, 89, 90, 91) (Table 2). Ovarian insulin receptors have the same heterotetrameric ₂ ß₂ structure as insulin receptors in other organs. They possess tyrosine kinase activity (12) and may stimulate the generation of inositolglycans (79).

Insulin-induced hyperandrogenism is unlikely to result from an action of insulin through its own receptor, however, in disorders in which receptor expression or availability is significantly compromised, such as the type A syndrome of insulin resistance and acanthosis nigricans, caused by insulin receptor mutations, or the type B syndrome, associated with antiinsulin receptor antibodies (6, 7). In the latter two conditions, insulin receptors likely function as inefficiently in the ovary as in other organs, and another receptor, such as the type I IGF receptor, is more likely to mediate the effects of hyperinsulinemia in the ovary (9).

C. Insulin action and the ovary
Numerous actions of insulin on the ovary have been demonstrated both in vitro (Table 3) and in vivo (Tables 3 and 4), with no significant differences between humans and other species (3).

At this time, there is only limited knowledge about the specific effects of insulin on ovarian steroidogenic enzymes. A stimulatory effect of insulin on aromatase has been suggested by some studies of animal and human ovarian cells in vitro (102, 103, 104, 105), but one study (106) failed to confirm this finding. 17-Hydroxylase activity appears to be stimulated by insulin (29, 107, 108, 109), but a recent study of 28 women with PCOS and 18 normal controls found no correlation between insulin levels and 17-hydroxyprogesterone (17-OHP) levels after treatment with GnRH agonist (GnRHa) (110). Insulin increases P₄₅₀ side chain cleavage (scc) enzyme mRNA in porcine granulosa cells (111) and P₄₅₀scc activity in goldfish follicles (112). A similar effect could not be demonstrated, however, in a human ovarian thecal-like tumor line (101). In the latter study, insulin had no effect on the enzyme activity or mRNA concentration of 17-hydroxylase/17,20-lyase (P₄₅₀c17) or 3ß-hydroxysteroid dehydrogenase (HSD), but forskolin stimulation of 3ß-HSD mRNA was enhanced by insulin. In human luteinized granulosa cells, 3ß-HSD expression was found to be stimulated by insulin (106).

b. In vivo studies (Table 4).
It has not been consistently demonstrated that insulin stimulates ovarian steroidogenesis in vivo (113). Several studies have examined the in vivo effects of insulin on aromatase. In rats with experimental hyperinsulinemia, an increased estrone (E₁) to A ratio was demonstrated, consistent with a stimulatory effect of insulin on ovarian or peripheral aromatase (94). In women, an insulin infusion study has suggested a similar effect (114), and in hyperinsulinemic women with PCOS, an increased E₂/A ratio was seen after gonadotropin stimulation, compared with normoinsulinemic women with PCOS (115). Relatively insulin-deficient women with type 2 diabetes show reduced aromatase activity (116). The increase in circulating A level observed during insulin infusions in women (117, 118), on the other hand, suggests that insulin may inhibit aromatase. In short, it remains unclear whether or how insulin regulates aromatase in vivo.

The effect of insulin on ovarian androgen production in women has been extensively studied (Tables 3 and 4). In PCOS, a positive correlation has been reported between insulin and T or A levels (119, 120, 121, 122) in several studies, while more recent studies (123, 124, 125, 126, 127) failed to find such a relationship. In insulin infusion studies that maintained hyperinsulinemia for several hours, a stimulatory effect of insulin on ovarian androgen production has not been consistently found. Stuart and associates (117, 118, 128) demonstrated elevation of A and dehydroepiandrosterone (DHEA) in normal lean and obese women and in women with insulin resistance and acanthosis nigricans during a euglycemic, hyperinsulinemic clamp study. Micic et al. (129) demonstrated an increase of T in patients with PCOS during a 4.5-h insulin infusion. On the contrary, Diamond et al. (130) could demonstrate no change in total or free T or in A during either insulin or glucose infusion in normal women. Similarly, Nestler et al. (131) could not demonstrate a rise in T in normal women during insulin infusion. Dunaif and Graf (114) examined gonadotropin and sex hormone levels basally and during insulin infusion in normal and PCOS women. No effect on gonadotropins was demonstrated; E₂ levels rose in response to insulin in normal women. In PCOS women, A levels increased, but T, free T, and dihydrotestosterone (DHT) levels declined.

Another group of studies has examined the effects of food intake or oral or intravenous administration of glucose on circulating androgen concentrations. In normal women, Parra et al. (132) found an increase in free T and no change in A after breakfast, but a decline of free T after an oral glucose load. Elkind-Hirsch et al. (133) failed to demonstrate a rise of either T or A during a tolbutamide-enhanced intravenous glucose tolerance test (IVGTT). Smith et al. (134) found a positive correlation between insulin responses and A, T, and DHT levels during oral glucose tolerance testing (OGTT) in hyperandrogenic and normal women, but Tiitinen et al. (135) demonstrated no significant change in T or A in women with PCOS or weight-matched normal controls after an oral glucose load and Tropeano et al. (136) demonstrated a decline of T, A, and DHEA during an OGTT. On occasion, both a stimulatory response and the lack of it have been observed in the same study. For example, Anttila et al. (137) reported a tendency to increased serum T levels during OGTT mainly in a subgroup of PCOS patients with both hyperinsulinemia and elevated LH levels; most PCOS patients, however, showed a decline in T. Fox et al. (138) found that serum androgens declined in PCOS patients during OGTT, but A rose during a 2-h intravenous insulin infusion in obese controls. Since a decline of serum T in the course of a 3- or 4-h OGTT may be attributed to diurnal variations of T, the lack of an increase of T under these conditions argues against a significant acute stimulatory or inhibitory effect of insulin on ovarian androgen production in vivo.

While studies that raise circulating insulin concentration have produced variable effects on serum androgen levels, studies in which insulin levels were reduced have consistently demonstrated a decline in serum androgen levels in insulin-resistant hyperandrogenic women (139, 140) (see Section VI.A). Whether insulin levels are lowered with diazoxide (30, 141), octreotide (34, 142), metformin (29, 31, 108, 143, 144, 145, 146), troglitazone (35, 36), or through weight loss (147, 148, 149, 150, 151, 152, 153, 154, 155, 156), a decline in serum androgen levels is usually found and ovulatory function improves (Table 4). In contrast to the studies in which insulin levels were elevated acutely for several hours, the effect of the reduction of circulating insulin can be studied over many weeks. If insulin-induced stimulation of ovarian steroidogenesis requires a prolonged exposure to excess circulating insulin, the latter group of studies is more likely to be able to demonstrate, albeit indirectly, a stimulatory effect of insulin on circulating steroids. A confounding factor in some of these studies is a decline in circulating LH, which may be responsible, at least in part, for the reduced androgen secretion (157).

In summary, it appears that insulin may have stimulatory or inhibitory effects on ovarian steroidogenic enzymes, but the responses of specific enzymes may vary with cell type and possibly among species. Further studies are needed on the effects of insulin on steroidogenic enzymes in the ovaries both in vitro and in vivo.

2. Interactions with gonadotropins. Acting at the ovarian level, insulin appears to potentiate the steroidogenic response to gonadotropins, both in vitro and in vivo (96, 102, 157, 158, 159, 160, 161, 162, 163). In granulosa cells, this effect may be mediated by an increase in LH receptor number, since insulin in concert with FSH increases ovarian LH-binding capacity (13, 164). In addition, insulin may act on the pituitary to increase gonadotrope sensitivity to GnRH. Evidence for this effect comes both from in vitro studies (165, 166) and indirectly from studies in insulin-resistant patients treated with insulin sensitizers, in whom circulating LH declined concomitantly with insulin (29, 31, 35, 108). On the other hand, in rats with experimental hyperinsulinemia maintained over six 4-day estrous cycles, the response of gonadotropins to GnRH did not differ from that of controls (94). In normally cycling women, increasing body mass index (BMI) did not have an effect on gonadotropin secretion and in women with PCOS BMI and LH levels were inversely related (167, 168, 169), while gonadotropin responsiveness to GnRH did not change after insulin infusion (114). In summary, it remains unclear whether hyperinsulinemia significantly enhances gonadotrope responsiveness to GnRH in vivo, as it does in vitro.

3. Effects on ovarian growth and cyst formation. In a rat model, a synergistic interaction between LH/hCG and insulin on the ovary can be demonstrated directly during experimentally induced hyperinsulinemia, which enhances hCG-induced ovarian growth and cyst formation (28, 170) (Fig. 2). This synergistic action of insulin with LH/hCG is seen regardless of cotreatment with a GnRH antagonist, suggesting that the growth- and cyst-promoting effects of insulin are exerted directly on the ovary. Indeed, insulin can stimulate proliferation of both human and rat theca-interstitial cells in vitro (171, 172, 173). In humans, the ability of high insulin levels to stimulate ovarian growth in vivo has been suggested by a case report of a patient with the type B syndrome of insulin resistance, whose sonographically determined ovarian volume doubled during a prolonged insulin infusion (174). Furthermore, in women with PCOS, circulating insulin levels are correlated with ovarian volume (175, 176), and after gonadotropin stimulation, the increase in ovarian dimensions observed in hyperinsulinemic PCOS is greater than in normoinsulinemic PCOS (115).

View larger version (34K): [in this window] [in a new window]   Figure 2. The effects of 23 days of daily injections of normal saline (control), hCG, insulin, or insulin plus hCG and GnRHant on gross ovarian morphology in rats. Female Sprague-Dawley rats were randomized into the following treatment groups: vehicle; high-fat diet (to control for the effects of weight gain); insulin; hCG; GnRH antagonist (to control for possible central effects of insulin vs. direct effects on the ovary); GnRHant and HCG; insulin and GnRHant; insulin and hCG; insulin, hCG, and GnRHant. Ovarian morphology in the group treated with insulin and hCG (not shown) did not differ from that seen in the group treated with insulin, hCG, and GnRHant (shown above). [Reproduced with permission from L. Poretsky et al.: Metabolism 41:903–910, 1992 (170 ). ©W. B. Saunders Co.]

5. Effects on IGFBP-1 production. Another protein under the regulatory control of insulin is IGFBP-1. Insulin and BMI are the major determinants of circulating IGFBP-1 levels in both obesity (185, 186, 187) and PCOS (183, 188, 189, 190, 191, 192). Insulin inhibits IGFBP-1 production in the liver (193, 194, 195, 196, 197, 198), thereby reducing circulating IGFBP-1 levels. Insulin also inhibits IGFBP-1 production in ovarian granulosa cells (see Section IV.B), acting through its own receptor (199). A detailed discussion of the role of IGFBPs in ovarian function and their regulation in the ovary is presented in Section IV.D.

6. Ovulation in diabetes mellitus and in states of extreme insulin resistance. Insulin and IGFs have been shown to suppress apoptosis in ovarian follicles, thus reducing rates of their atresia (200, 201). A variety of clinical and experimental observations in patients with type 1 and type 2 diabetes mellitus and states of extreme insulin resistance suggest that insulin may be involved, either directly or indirectly, in the process of ovulation (3, 9, 202).

Insulin deficiency in type 1 diabetes has been associated with disordered ovulation (3, 202). In rats, streptozotocin-induced diabetes is associated with cessation of ovulatory cycles, which can be restored with insulin treatment (203). In mice with alloxan-induced diabetes, a similar reduction in ovulation rate has been reported (204). While the current availability of insulin therapy does not allow observation of a similar phenomenon in human type 1 diabetes, in the preinsulin era, girls who developed diabetes prepubertally failed to enter puberty (3, 4). It is difficult to determine whether it was insulin deficiency itself, the state of chronic diabetic ketoacidosis, the starvation diets used for treatment, or the dramatic weight loss that caused the failure of pubertal development in these girls. In patients with type 1 diabetes treated with insulin, the hypothalamic-pituitary-gonadal axis appears to be relatively hypoactive, mainly because of failure of the GnRH pulse generator (205, 206); low serum sex hormone levels, including low luteal-phase P levels, have been described (207, 208). Even with insulin treatment, up to one third of young women with type 1 diabetes may experience delayed menarche and oligomenorrhea of hypothalamic origin (205).

Hyperinsulinemia resulting from exogenous insulin administration is often present in treated patients with type 1 diabetes. If such patients gain excessive weight, their LH:FSH ratio increases, SHBG levels decrease, and more than 70% develop polycystic ovaries (209); the response of 17-OHP to GnRHa in oligomenorrheic diabetic adolescents is exaggerated, resembling the response reported in insulin-resistant patients with PCOS (29, 108, 210). Some patients with type 2 diabetes have mildly elevated androgen levels or increased androgen responses to GnRH stimulation (116, 202) as well as reduced SHBG levels (211), particularly in the early, hyperinsulinemic stage of the disease (116, 212). It should be noted that hyperinsulinemia in patients with diabetes is relatively mild, compared with that seen in patients with syndromes of extreme insulin resistance, and that significant hyperandrogenism is not characteristic of women with either type 1 or type 2 diabetes (9).

Hyperandrogenism and polycystic ovaries or ovarian hyperthecosis are commonly found in states of extreme insulin resistance (9, 140, 213). These conditions are sometimes caused by mutations of the insulin receptor gene (214, 215, 216) and include the type A syndrome (6), leprechaunism (9, 217, 218), Rabson-Mendenhall syndrome (9, 215), and syndromes characterized by defective insulin receptor signaling (74, 219, 220). Premenopausal patients with the type B syndrome (insulin resistance and acanthosis nigricans associated with the presence of antiinsulin receptor antibodies) also exhibit hyperandrogenism (7, 8).

Although there is evidence that hyperinsulinemia contributes to the development of hyperandrogenism, not all clinical conditions associated with hyperinsulinemia lead to ovarian androgen overproduction. For example, most women with type 1 diabetes, who are often hyperinsulinemic because of exogenous insulin administration but usually do not exhibit significant insulin resistance, do not become hyperandrogenic, but rather exhibit hypothalamic-pituitary-ovarian axis hypofunction. It is not clear why hyperinsulinemia developing in the setting of insulin resistance, rather than any form of hyperinsulinemia, is associated with ovarian hyperandrogenism, particularly since correction of hyperinsulinemia without correction of insulin resistance may improve ovarian function (38, 221, 222, 223).

Dissecting the effects of hyperinsulinemia from those of insulin resistance is difficult (224, 225). One can postulate, however, that because the postbinding insulin receptor pathways may diverge (2, 9, 226), in conditions characterized by hyperinsulinemia without primary insulin resistance all insulin receptor-signaling pathways are significantly down-regulated, whereas when hyperinsulinemia is caused by insulin resistance, only some of these pathways (e.g., glucose transport) may be deficient, while others may be hyperstimulated (9, 227, 228). Thus, if hyperinsulinemia promotes androgen production by activating insulin-signaling pathway(s) distinct from those involved in glucose transport, hyperandrogenism would be more likely to develop in the setting of insulin resistance and compensatory hyperinsulinemia.

7. Interactions of insulin with leptin; leptin-mediated effects on ovulation. New insights into the relationship between weight and ovulation and the role that insulin may play in modifying this relationship emerged with the discovery and characterization of leptin. Leptin is a 16-kDa protein produced by adipose cells (229, 230, 231, 232, 233). Circulating leptin levels are stimulated by estrogen and inhibited by androgens (234, 235, 236) and are directly proportional to adipose tissue mass (236, 237, 238, 239, 240, 241). Leptin regulates body weight by binding to specific receptors in the hypothalamus and thus decreasing food intake (242, 243, 244). Leptin is encoded by the ob gene, which is defective in genetically obese ob/ob mice (229, 231, 237, 245). These animals are also insulin resistant and infertile. Replacement of leptin in ob/ob mice produces weight loss, reverses metabolic abnormalities, and restores ovulation and fertility (246, 247). Db/db mice and Zucker fatty rats have a similar phenotype, which results from a genetic abnormality of the leptin receptor (237, 245, 248). A human kindred with an ob mutation has been described, in which two prepubertal cousins with a frameshift mutation in the ob gene suffer from massive obesity (249). It is not yet known whether they will develop reproductive abnormalities. Similarly, a mutation of the human leptin receptor gene associated with obesity has been reported (250).

A rise in circulating leptin levels is associated with and precedes puberty (251), and higher circulating leptin levels are associated with a younger age at menarche (252, 253), possibly because leptin serves as a signal for the initiation of an early pubertal gonadotropin-secretory pattern (254, 255, 256, 257). A rapid decline of circulating leptin levels is observed during caloric restriction (258) or starvation (244, 259, 260). A decline in leptin may be responsible for the activation of the hypothalamic-pituitary-adrenal axis and the inhibition of the gonadotropic axis observed with stress (261, 262), since these responses can be abolished in animals by leptin administration (233, 263).

Leptin receptors are present in the ovary (264, 265, 266). Their functional capacity and their role in both normal and abnormal ovarian function remain to be firmly established since two leptin receptor isoforms exist, one with a full-length and another with a truncated intracellular domain (267). While the action of leptin on gonadotropin secretion is stimulatory, the direct effects of leptin on ovarian steroidogenesis may be either inhibitory or stimulatory (264, 266, 268). For example, leptin inhibits insulin-induced P and E₂ production in bovine granulosa cells (264) and reduces synergism between FSH and IGF-I on E₂ production in rat granulosa cells (268). On the other hand, leptin appears to stimulate ovarian 17-hydroxylase (265).

Insulin stimulates secretion of leptin by adipocytes (269, 270, 271, 272). In addition, by promoting lipogenesis, insulin may increase adipose tissue mass, thereby further enhancing leptin production. However, there is no apparent acute effect of feeding on leptin levels (260, 273, 274) and no correlation between leptin and insulin sensitivity in vivo (273). Nevertheless, circulating leptin levels rise with acute massive overfeeding over a 12-h period (275).

Leptin inhibits insulin secretion from isolated pancreatic islets in some studies (276, 277), but stimulates insulin secretion in others, either by a direct stimulatory effect on pancreatic ß-cells (278) or because of its inhibitory effect on somatostatin (279). Leptin may affect pancreatic function through the autonomic nervous system (280) and was shown to improve insulin sensitivity in normal rats, reducing glucose and insulin levels (281). When administered intracerebroventricularly, leptin enhanced insulin-stimulated glucose metabolism (282). Leptin has been shown to possess antidiabetic properties in some studies (283, 284), but in other studies it did not affect glucose-stimulated insulin secretion and did not have a significant effect on glucose transport or insulin action in either adipocytes or muscle cells (285, 286). In some circumstances, as, for example, in the setting of obesity, leptin may contribute to the development of insulin resistance and diabetes (287, 288, 289, 290).

The above observations point to a complex relationship among insulin, leptin, body weight, ovarian steroidogenesis, and ovulation (Fig. 3). If a certain "threshold" level of leptin is needed to activate the hypothalamic-pituitary-ovarian axis, then a certain mass of adipose tissue must be present for ovulation to occur (291). In states characterized by hypoinsulinemia, such as starvation, weight loss, or untreated type 1 diabetes mellitus, amenorrhea may develop (292, 293), possibly because of a decline in circulating leptin (294) and a resultant deactivation of the hypothalamic-pituitary-ovarian axis (233, 293, 295). Thus, insulin deficiency may contribute to abnormalities of ovulatory function either directly, by affecting gonadotropins or the ovaries, or indirectly, by negatively influencing secretion of leptin. On the other hand, states characterized by insulin excess may be associated with higher circulating levels of leptin. Whether such putative leptin excess would play a role in the development of the hyperandrogenism or anovulation observed in hyperinsulinemic states remains to be determined.

View larger version (21K): [in this window] [in a new window]   Figure 3. The relationships among insulin, leptin, pituitary gonadotropins, and ovarian steroidogenesis. Insulin stimulates leptin secretion, enhances pituitary gonadotropin response to GnRH, and promotes ovarian steroidogenesis. Leptin stimulates the hypothalamic-pituitary-gonadal axis at the level of the hypothalamus and/or pituitary; it inhibits ovarian E2 and P production, but may stimulate androgen production by stimulating 17 -hydroxylase activity or expression. Leptin and insulin potentiate each other’s secretion, although leptin may inhibit insulin secretion under some circumstances. Ovarian sex steroids inhibit FSH production and either inhibit (E2, T, P) or stimulate (E1) LH responsiveness to GnRH.

View larger version (16K): [in this window] [in a new window]   Figure 4. [125I]IGF-I binding to ovarian homogenates from normal rats (A) and rats with experimentally induced hyperinsulinemia (B). Female Sprague-Dawley rats were treated with either vehicle (A) or insulin for 23 days. [125I]insulin (not shown) and [125I]IGF-I binding to ovarian homogenates was examined. In rats treated with insulin, a doubling of [125I]IGF-I binding was observed, suggesting amplification of the number of type I IGF receptors or hybrid insulin/type I IGF receptors. [Reproduced with permission from L. Poretsky et al.: Endocrinology 122:581–585, 1988 (94 ). © The Endocrine Society.]

D. Summary
The role of insulin in the ovary may be summarized as follows: 1) Insulin receptors are widely distributed throughout all ovarian compartments. Ovarian insulin receptors have a subunit structure identical to insulin receptors in other organs, possess tyrosine kinase activity, and are capable of stimulating the generation of inositolglycan second messengers. 2) At this time there is no convincing direct in vivo evidence that hyperinsulinemia acutely stimulates ovarian steroid production, but there is direct in vitro evidence and indirect in vivo evidence for a stimulatory effect of insulin on ovarian steroidogenesis. The in vitro evidence suggests that the stimulatory effect of insulin on steroidogenesis is mainly mediated by the insulin receptor and may involve the inositolglycan pathway. The in vivo evidence is largely derived from experiments in which a reduction in circulating insulin levels produces a decline of circulating androgens and from clinical observations in women with both insulin deficiency and insulin excess. 3) The effects of insulin on ovulation are complex. A threshold level of insulin is likely to be required for the normal function of the hypothalamic-pituitary-ovarian axis, either because of the direct stimulatory effects of insulin on this axis or because of the stimulatory effects of insulin on leptin secretion (both direct, with insulin stimulating adipocyte production of leptin, and indirect, because of insulin-stimulated lipogenesis). Leptin, in turn, participates in the initiation of puberty and activation of the hypothalamic-pituitary-gonadal axis. On the other hand, excessive circulating insulin, particularly in the setting of insulin resistance, may enhance ovarian androgen production and thus may contribute to the development of anovulation. 4) Insulin may amplify its own effects, the effects of IGFs, and those of gonadotropins by up-regulating type I IGF receptors and gonadotropin receptors, as well as by inhibiting production of IGFBP-1, both in the liver and ovary. In the setting of insulin resistance and hyperinsulinemia, therefore, a cycle of events that leads to a self-perpetuating amplification of the ovarian effects of insulin and IGFs can develop (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Figure 5. Hypothetical insulin/IGF self-enhancement mechanisms in the ovary. Hyperinsulinemia, acting through insulin receptors, type I IGF receptors, or possibly through hybrid insulin/type I IGF receptors increases the number of type I IGF receptors and/or hybrid insulin/IGF receptors and increases cellular pool of p21 Ras, which may be responsible for the mitogenic effects of insulin or of IGFs. Hyperinsulinemia also inhibits IGFBP-1 production, leading to a further increase in bioavailable IGFs. Thus, hyperinsulinemia may lead to a self-perpetuating cycle of events resulting in the exaggeration of the ovarian effects of both insulin and IGFs, leading to ovarian enlargement and excessive androgen production (please see the text for details and references). Solid arrow, action via a receptor; broken arrow, regulation of a receptor.

	III. IGFs and Their Receptors

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 
A. IGF peptides and receptors
1. IGF-I. IGF-I is a 70 amino-acid, single-chain polypeptide that shares significant sequence homology with IGF-II, proinsulin, and relaxin. The human IGF-I gene is located on chromosome 12. The major source of circulating IGF-I is the liver, but IGF-I is widely expressed in most tissues, especially during postnatal development (303). IGF-I was first known as somatomedin C and identified as a mediator of GH action (304). GH rapidly activates IGF-I gene transcription and also regulates changes in chromatin structure within the IGF-I gene, delineating a target within the chromatin for GH action (305). In addition to GH, other activators of IGF gene transcription include estradiol, experimental diabetes, and angiotensin II (306). Null mutants for IGF-I are severely growth restricted in utero but are fertile (307, 308).

2. IGF-II. IGF-II is a 7.5-kDa, 67-amino acid, single-chain polypeptide that is approximately 70% homologous with IGF-I and 50% homologous with proinsulin (14, 309, 310, 311, 312). The human IGF-II gene is located on chromosome 11, contiguous with the insulin gene. Pre-pro-IGF-II, the precursor of IGF-II, is a 22-kDa protein. Inactivation of the IGF-II gene in animals (308, 313) produces growth-deficient but fertile and otherwise normal individuals. IGF-II is highly expressed in fetal tissues and tumors, as well as in normal adult tissues. IGF-II can bind to type I and type II IGF receptors (see below), as well as to the insulin receptor (302, 314).

3. Type I IGF receptor. The type I IGF receptor precursor protein consists of 1367 amino acids, comprising both the - and ß-subunits of the receptor. The human type I IGF receptor gene is located on chromosome 15. The mature type I IGF receptor protein is a heterotetramer consisting of two - and two ß-subunits and is highly homologous with the insulin receptor (315, 316). The cysteine-rich regions of the -subunits of the insulin receptor and type I IGF receptor are 64–67% homologous, whereas the tyrosine kinase domains of the ß-subunits are 84% homologous. In addition to IGF-I, the type I IGF receptor can also bind IGF-II and insulin, although with somewhat lower affinity. In addition to binding IGF-I, IGF-II, and insulin, the type I IGF receptor has also been reported to interact with IGFBPs (317), but the significance of this finding remains to be determined. Type I IGF receptor postbinding events, similar to those of the insulin receptor, include tyrosine phosphorylation of receptor ß-subunits and IRS proteins, interactions with PI-3 kinase, and activation of MAPK (69, 315, 318, 319). Type I IGF receptor knockout mice weigh 45% of normal at birth and die immediately afterward (320). Patients with a deletion of the distal arm of chromosome 15 lack one copy of the IGF-I receptor gene and exhibit both intrauterine and postnatal growth restriction (321, 322).

4. Hybrid insulin/type I IGF receptors. Hybrid receptors that combine an /ß insulin hemireceptor and an /ß type I IGF hemireceptor have been reported in a variety of tissues, although not in the ovary (41, 323). These receptors can form in tissues coexpressing both insulin and type I IGF receptors, theoretically including the ovary. Hybrid receptors have properties similar to type I IGF receptors, binding IGF-I with high affinity and insulin with lower affinity. Interestingly, in situations that are characterized by insulin receptor down-regulation, the number of hybrid insulin/type I IGF receptors tends to increase (228).

5. Type II IGF receptor. The type II IGF receptor is identical to the mannose-6-phosphate (Man-6-P) receptor (309, 324, 325, 326). The gene for the type II IGF receptor is located on the long arm of chromosome 6. This receptor targets Man-6-P-containing enzymes from the Golgi apparatus to the lysosomes and also mediates the rapid internalization of IGF-II (309). The receptor is a single-chain polypeptide of approximately 300 kDa with a large extracellular domain containing IGF-II binding sites (325, 327). The cytoplasmic domain is very short and includes tyrosine, threonine, and serine phosphorylation sites. Type II IGF receptor knockout mice exhibit elevated IGF-II levels and die in utero (328, 329). Interestingly, if the IGF-II gene is knocked out at the same time, about 50% of the fetuses survive to birth (328). Type I/type II IGF receptor double-knockout mice differ from normal controls only in their patterns of growth (328). These observations, taken together, suggest that excessive activation of the type I IGF receptor by IGF-II may be lethal in utero.

The type II IGF receptor can be released from the cell membrane into the circulation. This mechanism may be principally responsible for its loss from the cell surface (330, 331, 332, 333). The circulating form of the IGF-II receptor retains its affinity for IGF-II (325, 334) and may participate in the local modulation of organ size in vivo. For example, overexpression of the soluble IGF-II/Man-6-P receptor in transgenic mice can significantly decrease the weight of their alimentary canal (335).

Although the type II IGF/Man-6-P receptor is important for IGF-II internalization and degradation, it is unclear whether this receptor actively mediates IGF-II signaling. Examples of such signaling have been reported, including stimulation of G-protein activation and of thymidine incorporation into rat hepatocyte DNA (325, 336, 337, 338). In most instances, however, the metabolic and growth-promoting actions of IGF-II appear to be mediated by the type I IGF receptor (339) or the insulin receptor (314). The type II IGF receptor, however, may mediate signals involved in angiogenesis (340) and other processes. Ligands for the type II IGF receptor, in addition to IGF-II and Man-6-P, include ß-galactosidase and other lysosomal enzymes, proliferin, renin, latent transforming growth factor (TGF)-ß (329), and leukemia-inhibitory factor (341). In the context of these observations, the functions of the type II IGF receptor within the ovary remain to be determined.

B. Expression of IGFs and IGF receptors in the ovary
1. Human and nonhuman primate. Distinctive features of IGF expression in the primate ovary include the predominance of IGF-II and its pattern of localization (Table 2). Other molecules that modulate IGF action, including the IGF receptors, IGFBPs, and IGFBP proteases, are also differentially expressed in the primate ovary (see below). While the majority of studies that examined the ovarian expression of IGFs and that of their receptors were done on human tissue, ovaries from cycling rhesus monkeys reveal similar expression patterns of IGF-I, IGF-II, and type I IGF receptor, and there is strong evidence that IGF-II, aromatase, and IGFBP-4 can be regarded as markers of the dominant follicle in the rhesus ovary (342).

In the human ovary, IGF peptide expression is follicle stage-specific and compartmentalized (Table 2). IGF-I mRNA is barely detectable in the adult ovary and not in the granulosa layer at any stage of follicular development (88, 89, 343). IGF-II mRNA is expressed in the theca and perifollicular vessels of all follicles and in the granulosa cells of some follicles. In small antral follicles, IGF-II mRNA and protein are detectable in both granulosa and theca (88, 89, 343). In atretic antral follicles, on the other hand, IGF-II is minimally expressed by the theca. IGF-II is abundantly expressed and secreted by granulosa cells of preovulatory follicles as well as by granulosa-luteal cells harvested during oocyte retrieval after controlled ovarian hyperstimulation (COH) (88, 90, 344, 345, 346, 347). These findings, plus the observations that granulosa cells do not express IGF-II prepubertally, but do so in a subpopulation of adult follicles, and that gonadotropins regulate IGF-II mRNA expression and secretion in human granulosa-luteal cells in vitro (344, 345), suggest that ovarian IGF-II gene expression is regulated by gonadotropins.

Follicular fluid (FF) constituents such as IGF peptides are derived from the circulation as well as from intraovarian production. In normally cycling women, FF IGF-I levels are similar in estrogen-dominant and androgen-dominant follicles and do not correlate with follicular size (348). In contrast, FF IGF-II levels are higher in estrogen- compared with androgen-dominant follicles and correlate positively with follicle size, cycle day, and E₂ and negatively with androgen-estrogen (A:E) ratio (348). In normally cycling women, simultaneous measurements of IGF-I, IGF-II, and insulin concentrations in ovarian and peripheral venous blood reveal an ovarian gradient only for IGF-II (349), and serum IGF-I and IGF-II levels in normally cycling women do not vary during the menstrual cycle (348). These data collectively suggest that FF IGF-I originates from serum by transudation and that FF IGF-II derives primarily from local production by the granulosa and possibly by the theca, in addition to some contribution from the circulation. After COH, FF IGF-II levels are about 8 times higher than those of IGF-I, and both IGF-I and IGF-II levels are lower than in serum (350, 351, 352, 353). In contrast to spontaneous cycles, these levels in COH do not correlate with follicle size, oocyte maturity, or FF E₂. FF IGF-I and IGF-II levels were noted to rise with increasing cycle day 3 serum FSH, an index of ovarian reserve (354).

Normal circulating levels of IGF-I are not a prerequisite for normal ovarian follicular development in women, as evidenced by cases of ovulation and fertility in individuals with Laron-type dwarfism, which results from GH receptor deficiency (GHRD) (355, 356, 357, 358). Furthermore, a normal follicular response to injected gonadotropins, leading to ovulation and conception, has been reported in women with GHRD, whose serum GH was markedly elevated and both serum and FF IGF-I barely detectable (355, 356). In such subjects, serum IGF-II levels were about 25% of normal (FF IGF-II was not measured). These clinical observations support the conclusion that IGF-I does not play an important role in the ovulatory process in women.

Both type I and type II IGF receptors are found in the human ovary (88, 298, 343, 359). By in situ hybridization, type I IGF receptor mRNA is predominantly expressed by granulosa cells and oocytes, with more intense expression in dominant compared with small antral follicles (88, 343). By this technique, theca and stroma are negative for type I IGF receptors, but stromal receptors with the specificity of the type I IGF receptor have been reported in ligand binding studies (298). Type II IGF receptors are localized to both granulosa and thecal layers, with more intense expression in the granulosa and in dominant, compared with smaller, antral follicles (88). By RT-PCR, both types of receptors were found to be expressed by granulosa, theca, and stroma and to persist upon culture of both granulosa and thecal cells (347).

2. Rodent. In the rat, ovarian IGF-I gene expression and protein production are granulosa specific (360, 361, 362); significantly, IGF-I is selectively expressed in the granulosa of only healthy antral follicles, not in atretic or luteinized follicles or in theca-interstitial cells (342, 360, 363, 364). IGF-II mRNA expression is limited to the thecal compartment and blood vessels (342, 362, 363), but the postnatal decline in ovarian IGF-II content (365) argues against a significant role for this peptide in rat ovarian physiology. While type I IGF receptor mRNA is abundantly expressed in granulosa cells (365), the corresponding protein is detected not only in the granulosa but also in the thecal compartment, regardless of the maturational stage or health status of the follicle (363), suggesting that regulation of the receptor is unlikely to play a major role in follicular maturation (366).

The patterns of IGF-I, IGF-II, and type I IGF receptor expression are essentially the same in rat and mouse ovary (342, 364, 367). IGF-I expression increases at the secondary preantral stage and is abundant in healthy follicles through the preovulatory stage. Type I IGF receptor is expressed constitutively, regardless of follicular developmental stage or health (367). These findings lay the groundwork for studies of ovarian function in transgenic mouse models with deletions of these components (368).

3. Livestock species. Porcine granulosa cells in culture secrete abundant immunoreactive IGF-I, which is increased by FSH, cAMP, GH, EGF, and TGF-. IGF-I is abundant in porcine FF, especially in large follicles. Its levels increase in response to PMSG and/or GH treatment (369, 370, 371). This finding suggests that gonadotropin and GH action on the granulosa cells of the developing porcine follicle is mediated in part by local induction of IGF-I. IGF-II in the porcine ovary is expressed mainly in the theca and is not under gonadotropin or GH regulation (15, 370, 372). FF IGF-II levels decline in response to GH (370, 372, 373). In the sheep ovary, at least four localization studies of IGF-I expression have been published, with divergent findings (374, 375, 376, 377). IGF-II is localized to the theca, and its levels in FF are 4-fold greater than those of IGF-I (377, 378). In the cow, IGF-I is produced by the ovary (379, 380), and its levels in FF increased with increasing E₂ concentrations and increasing follicle diameter in some (379, 381, 382, 383, 384), but not all (385, 386, 387), studies. IGF-II is exclusively expressed in the theca, with greater expression in dominant follicles, compared with subordinate or nonrecruited ones (388).

C. Role of IGFs in ovulatory function and steroidogenesis (Table 5)
1. Human. Studies of the effects of IGFs on human granulosa and thecal cells in vitro have primarily employed IGF-I, although as discussed above, the predominant endogenous locally produced ligand in vivo is IGF-II. IGF actions on the ovary include augmentation of DNA synthesis and steroidogenesis. IGF-I stimulates DNA synthesis and basal E₂ secretion in granulosa and granulosa-luteal cells and inhibits IGFBP-1 production (199, 389, 390, 391, 392, 393, 394, 395, 396). It also synergizes with gonadotropins in augmenting E₂ and P production (393, 397, 398, 399, 400). Several studies have been conducted recently of the effects of IGF-II on human ovarian cellular constituents. IGF-II stimulates basal P and E₂ secretion by human granulosa-luteal cells (353, 401). It also stimulates aromatization of androgen precursors (402) and inhibits IGFBP-1 (396) and IGFBP-2 (403) production by these cells. The effect of IGF-II on estradiol production is most pronounced if the cells are preincubated with insulin (402), possibly due to insulin-induced up-regulation of type I IGF receptors, formation of hybrid insulin/IGF-I receptors, or inhibition of IGFBP-1 production. IGF-II also stimulates granulosa-luteal cell DNA synthesis and proliferation in vitro (401, 404). In granulosa cells from both unstimulated and gonadotropin-stimulated preovulatory follicles, IGF-I, both alone and in synergy with gonadotropins, stimulates P450 aromatase mRNA expression and activity (405).

2. Rodent. IGF-I actions in rat granulosa and theca have been extensively reviewed (14, 23, 409, 410). IGF-I acts as a co-gonadotropin with FSH to stimulate granulosa cells to produce E₂ and P, and with LH to stimulate thecal androgen production. IGF-I stimulates LH receptor expression in granulosa and theca (13, 411, 412) and may be required for FSH receptor expression in granulosa (368); it also stimulates granulosa cell production of inhibin -subunit and augments the stimulation of this response by FSH (413, 414, 415). Stimulation of inhibin- expression in rat granulosa by FSH requires activation of protein tyrosine kinases by endogenously produced IGF-I, suggesting that IGF-I signaling is obligatory for this response (415). IGF-I also stimulates DNA synthesis in granulosa and theca-interstitial cells (171, 416).

In addition to its role in differentiation and proliferation of granulosa and theca, IGF-I also plays an important role in granulosa survival, since it can inhibit apoptosis (201). Granulosa cell apoptosis, associated with regular cleavage of nuclear DNA by endonuclease, is associated with follicular atresia (417). In vitro, this process is suppressed by IGF-I and gonadotropins and enhanced by the presence of IGFBPs (200). In the human ovary apoptosis is characteristic of androgen- but not estrogen-dominant follicles (418), but regulation of apoptosis by IGFs has not yet been demonstrated in human ovarian follicles or cellular components, as it has in the rat (201). To our knowledge, there are no studies examining specific effects of IGF-II in rodent ovaries.

3. Livestock species. In the sow, similar effects of IGFs on granulosa and thecal cell function have been reported as in humans and rodents (419, 420, 421). IGF-I stimulates granulosa cell proliferation and synergizes with FSH in granulosa cell differentiation (419). IGF-II enhances the delivery of cholesterol to the P₄₅₀ scc enzyme complex and enhances the functional activity of this first committed step in P biosynthesis (421). In sheep, IGF-I stimulates granulosa cells from small follicles to proliferate and those from larger follicles to produce P (422), an effect likely mediated through the type I IGF receptor (423). In the cow, IGF-I stimulates granulosa and thecal cell proliferation and steroidogenesis (379, 380, 424).

D. Summary
Although both IGF-I and IGF-II have been shown in vitro to have multiple ovarian effects in various species, IGF-II appears to be the predominant ovarian IGF in the human. The IGF-II gene is expressed in the human ovary, and the effects of IGF-II appear to be similar to those of IGF-I. The metabolic and growth-related effects of IGF peptides appear to be mediated under most circumstances by type I IGF receptors, which are present in all human ovarian compartments. Their numbers appear to be increased under the influence of insulin, as discussed in Section II.C. Type I IGF receptors may mediate the effects of insulin in the ovary in extreme insulin-resistant states with severe hyperinsulinemia. Clarification of the presence and the role of hybrid insulin/type I IGF receptors in the human ovary awaits further studies.

	IV. IGF-Binding Proteins (IGFBPs) and Proteases

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 
A. Structural relationships among IGFBPs
The bioavailability and, therefore, the actions of the IGFs are regulated, in part, by a superfamily of homologous proteins, called IGFBPs, that bind IGFs with high affinity. There are six IGFBPs, designated IGFBP-1 through IGFBP-6 (425, 426, 427), whose discovery, gene and protein structures, and mechanisms of actions have recently been reviewed (329, 428, 429).

All six IGFBPs have core molecular masses of 23–32 kDa. They are all at least 50% homologous, and for each IGFBP there is roughly 80% homology among species. The amino and carboxy termini are most highly homologous among the different IGFBPs, while the midsequence shows little similarity. The IGFBPs each contain at least 16 conserved cysteines, which are important in determining their conformation. There is also a group of proteins that share limited sequence homology with the IGFBPs and bind IGFs with low affinity. Due to their undefined roles as IGFBPs and limited structural homology to IGFBPs 1–6, they have been called IGFBP-related proteins (IGFBP-rPs) (427, 428). The high-affinity IGFBPs have dissociation constant (K_d) values for the IGFs in the range of 10^-9 to 10^-11 mol/liter, compared with 10^-6 to 10^-7 mol/liter for the IGFBP-rPs (428).

The genes for human IGFBP-1 and IGFBP-3 are located on chromosome 7, the IGFBP-2 and IGFBP-5 genes are on chromosome 2, the IGFBP-4 gene is located on chromosome 17, and the IGFBP-6 gene is on chromosome 12 (329, 430). IGFBP genes are in close proximity to homeobox (Hox) gene clusters (Hox A–Hox D), with which they appear to have coevolved. Hox genes encode DNA-binding proteins that are transcriptionally regulated by retinoic acid, as are some of the IGFBPs (430). IGFBP-1 and IGFBP-2 both contain the tripeptide motif Arg-Gly-Asp (RGD), which can bind to integrins, and their production and function are related to carbohydrate metabolism and metabolic homeostasis. In contrast, IGFBP-3, and likely the highly homologous IGFBP-5, are primarily involved in growth.

The IGFBPs have several functions, which include 1) to transport the IGFs in the circulation; 2) to regulate efflux of IGFs from the vascular space; 3) to prolong the half-life and metabolic clearance rates of the IGFs; 4) to prevent IGF-induced hypoglycemia; 5) to directly modulate interactions of IGFs with their receptors locally within target tissues; and 6) to directly modulate cellular function, independent of their ability to bind IGFs. All six IGFBPs have been shown to inhibit IGF action, likely by limiting bioavailable free IGFs from interacting with their receptors. IGFBP-1 and IGFBP-3 can also be stimulatory to IGF action, presumably by forming a pool of "slow-release" IGFs. IGFBP-1 and IGFBP-3 additionally have IGF-independent actions, including alteration of cellular motility and inhibition of DNA synthesis, respectively. IGFBP-4 and -5 may also have IGF-independent actions both in the human ovary (431) and in cell lines derived from other tissues (430, 432). Since the affinities of IGFBPs 1–6 for the IGFs are equal to or greater than the affinities of the type I and type II IGF receptors for the peptides, mechanisms have evolved to decrease IGFBP affinities and increase IGF bioavailability to the receptors. These mechanisms include phosphorylation, glycosylation, and proteolysis (329).

This review will focus on IGFBP expression and regulation primarily in the human and rat ovary and underscore the mechanisms of ovarian IGFBP production and regulation common to other species. Also discussed are IGFBP proteolysis by specific proteases, the regulation of these enzymes, and their putative functions in normal and pathological ovarian conditions.

B. IGFBP expression in the ovary
IGFBPs are expressed by granulosa and thecal cells and are present in the FF of every species studied. Significant differences exist in the patterns of ovarian expression and regulation of individual IGFBP species between the human and animal models.

1. Human (Table 2). The human ovary expresses mRNAs for IGFBP-1, -2, -3, -4, and -5. In situ hybridization shows distinctive patterns of mRNA expression for each of these IGFBPs in antral follicles, with parallel localization of immunostainable protein (89). IGFBP-1 is localized only to the granulosa cells of dominant follicles, not to theca or small antral follicles. IGFBP-2 is expressed by granulosa cells only in small, nondominant antral follicles, but by thecal cells in both dominant and nondominant follicles. IGFBP-3 expression is found in the theca of all follicles and the granulosa of only dominant follicles. IGFBP-4 is found in both granulosa and theca in all follicles, with a slight increase in granulosa expression in dominant compared with small follicles. IGFBP-5 has also been localized to both granulosa and theca; its expression is unaffected by follicular development. No IGFBP-6 mRNA or protein was localized by in situ hybridization (89), but expression was detected by RT-PCR (347). A recent study found IGFBP-4 to be expressed in luteal cells and in the granulosa and theca layers of only atretic antral, not healthy or preantral follicles (433). The expression of IGFBP-2, -4, and -5 by both granulosa and thecal cells has been confirmed by Northern analysis (347). Expression of IGFBP-1 has also been found in the corpus luteum (434).

The regulation of IGFBP production by the human ovary has been examined in cell culture studies. Two sources of tissue have been employed: antral follicles from surgically excised ovaries, and granulosa-luteal cells obtained at oocyte harvest for in vitro fertilization (IVF) after controlled ovarian hyperstimulation (COH). Granulosa cells derived from antral follicles in spontaneous cycles release IGFBP-2 and both core and glycosylated isoforms of IGFBP-4 and express the corresponding mRNAs (347, 435, 436). Cultures of thecal tissue derived from these follicles produce IGFBP-2, -3, and -4; theca from mature healthy follicles also produces proteolytic fragments of IGFBP-3 and -4 (436, 437, 438). Thecal IGFBP-3 accumulation, as determined by ligand blotting, was stimulated markedly by LH/hCG or GH in one study (438), but these effects were not noted by others (347, 437). Thecal expression of mRNA for IGFBP-5, but not IGFBP-1, -2, -3, or -4, is stimulated by LH (347).

Because luteinizing granulosa cells from IVF oocyte harvests are readily available, this model has been extensively employed to study human IGFBP production. These cells express mRNAs for IGFBP-1, -2, -3, -4, and -5 in culture and accumulate all of these proteins except IGFBP-5, as detected by ligand blotting of conditioned medium (403, 439, 440, 441, 442, 443). By metabolic labeling, they synthesize IGFBP-1 and -2 de novo, but evidence for IGFBP-3 synthesis is conflicting (403, 444, 445). Although IGFBP-5 mRNA is abundantly expressed (442), no immunoprecipitable IGFBP-5 protein has been detected in conditioned medium (443, 446). These findings suggest that human granulosa cells elaborate an IGFBP-5 protease as has been reported in the rat (447, 448).

Production of each IGFBP species by human luteinizing granulosa cells is uniquely regulated. IGFBP-1 production is inhibited by FSH, insulin, IGF-I, IGF-II, and the somatostatin analog octreotide, and increased by LH, EGF, PGs, and phorbol ester (199, 396, 439, 449, 450, 451, 452, 453, 454). The inhibition by insulin is mediated through its cognate receptor, not the type I IGF receptor (199). Both IGF-I and IGF-II inhibit IGFBP-1 production more potently than insulin (199, 449, 455) and apparently act via the type I IGF receptor. In fact, the concentrations of IGFs present in human FF completely inhibit in vitro granulosa cell IGFBP-1 production. This finding may explain the production of IGFBP-1 in cultured, but not in freshly obtained, human granulosa cells (347), as well as the observation that IGFBP-1 mRNA is not expressed in granulosa cells of small antral follicles (89). IGFBP-2 production is negatively regulated by LH/hCG through increased cAMP; this effect can be reversed by activin-A or interferon- (IFN-) (403, 443). IGF-II, but not IGF-I, decreases medium IGFBP-2, possibly through an action at the type II IGF receptor (403). In two studies, cAMP agonists promoted the accumulation of IGFBP-3 (403, 456), while a third found that FSH did not alter accumulation of immunoreactive IGFBP-3 but decreased its level on ligand blots, consistent with the action of an IGFBP-3 protease (451). In another study, IGFBP-3 detected by ligand blotting accumulated in conditioned medium during treatment with IGF peptides but not insulin, possibly reflecting release of IGFBP-3 from the cell surface upon binding ligand or protection from proteolysis (403). IGFBP-4 accumulation is inhibited by LH despite modest stimulation of its mRNA, apparently through elaboration of an IGFBP-4 protease (see Section IV.C below) (435, 436, 443, 457). IGFBP-5 mRNA expression is stimulated by activin-A (442).

IGFBPs found in human FF may either originate from local production or may reach the FF from an extraovarian source, such as the liver. FF IGFBPs have been measured both in antral follicles from cycling women and in hyperstimulated follicles aspirated for IVF, using both immunoassay and ligand blot techniques. FF from cycling women contains immunoassayable IGFBP-1, -2, and -3. IGFBP-1 levels range from 5–32 ng/ml, with levels positively correlated with follicular size and greater in dominant than cohort follicles (348, 446, 458). In one report, FF contained 15 ng/ml IGFBP-2, but the type of follicle studied was not stated (446). Mean immunoassayable IGFBP-3 in estrogen-dominant follicles (2995 ng/ml) was greater than in androgen-dominant follicles (2352 ng/ml); these levels were indistinguishable from those in hyperstimulated follicles (348). Immunoassays for IGFBP-4, -5, and -6 in these follicles have not been reported.

By ligand blotting, two distinct IGFBP profiles have been consistently observed in FF from cycling women (446, 459, 460). FF from estrogen-dominant, presumably healthy follicles contains low levels, while FF from androgen-dominant, presumably atretic follicles contains significantly greater levels of IGFBP-2 and both isoforms of IGFBP-4. The lower level of IGFBP-4 detectable by ligand blotting in FF from estrogenic compared with androgenic follicles results from the action of a serine metalloprotease found in estrogenic but not androgenic FF (see below) (435, 436, 457). An IGFBP-2 protease was also recently reported in estrogenic FF (436), but negative regulation of IGFBP-2 gene expression by gonadotropins (443) probably plays a more significant role in reducing IGFBP-2 levels in the healthy follicle. By contrast, IGFBP-3 levels are similar in FF from both types of follicles. In one study, IGFBP-3 levels in dominant follicles declined slightly but significantly with advancing follicle size and cycle day (446). IGFBP-1 has not been detected on ligand blots of FF from spontaneously cycling women (459).

FF obtained after hyperstimulation with menopausal gonadotropins followed by hCG contains IGFBP-1, -2, and -3, identified by immunoprecipitation (352, 434, 461). By immunoassay, mean IGFBP-1 levels are 90–160 ng/ml (434, 456, 462, 463), while mean IGFBP-3 levels are consistently near 2400 ng/ml (462, 464, 465), and IGFBP-6 levels are 170 ng/ml (466). By ligand blotting, IGFBP-1, -2, and -3 are detectable in FF from hyperstimulated cycles (352, 467).

2. Rodent. IGFBPs 2–6 have been detected in the rat ovary in both localization and cell culture studies (468, 469, 470). Studies of the cycling ovary revealed that IGFBP-4 and -5 are the predominant species expressed in granulosa cells of antral follicles. Both are preferentially localized to atretic follicles, with IGFBP-4 mRNA signal intensity increasing with the degree of atresia, and both IGFBP-4 and IGFBP-5 mRNA expression becoming more widespread in atretic follicles after the proestrous gonadotropin surge (468, 469, 470). In PMSG/hCG-treated rats, each gonadotropin treatment increased IGFBP-4 mRNA expression in small antral follicles, but no expression was seen in large follicles (471). Cultured granulosa cells from immature, diethylstilbestrol (DES)-treated rats secrete intact IGFBP-4 and IGFBP-5 into the medium (447, 448, 472). These cells respond to saturating doses of FSH by decreasing accumulation of both IGFBP-4 and IGFBP-5. These effects result from both decreases in mRNA expression and increases in elaboration of protease activities that degrade these IGFBPs into smaller, inactive fragments (448, 460, 473). Paradoxically, low doses of FSH (1–3 ng/ml) stimulate IGFBP-4 and -5 release (460). GnRH agonists, which induce follicular atresia (473) and granulosa cell apoptosis (474), stimulate basal IGFBP-4 accumulation without affecting IGFBP-4 protease activity and block the effect of FSH on both IGFBP-4 production and protease activity (473). IGF-I stimulates IGFBP-5 accumulation and decreases IGFBP-5 protease elaboration, while GnRH agonists can oppose the effects of FSH on both IGFBP-5 mRNA and protein expression and IGFBP-5 protease elaboration (447, 475, 476). Cytokines and growth factors known to block FSH-induced estradiol production, including TGF-ß, tumor necrosis factor (TNF)-, basic fibroblast growth factor, and interleukin-1, stimulate IGFBP-4 (477), suggesting that their effects on FSH action are due to the IGF-I-sequestering properties of IGFBP-4. Activin-A can decrease both IGFBP-4 and IGFBP-5 mRNA expression and IGFBP-5 protein accumulation (478).

In contrast to the expression of IGFBP-4 and -5 by granulosa cells, IGFBP-2 mRNA expression and production in culture are unique to theca-interstitial cells in the rat ovary. IGFBP-3 expression is limited to theca-interstitial cells and vascular and perivascular elements of corpora lutea, suggesting that it plays a role in the vascular control of luteal regression (468, 479, 480, 481). IGFBP-6 expression is limited to the thecal layer (422), while no IGFBP-1 expression has been detected (448, 468).

IGFBP production has also been examined in the mouse ovary. Notable differences from the rat include expression of IGFBP-2 by granulosa cells (364, 367), negative correlation of granulosa IGFBP-5 expression in antral follicles with atresia (367), and the failure of FSH to inhibit accumulation of IGFBP-4 and -5 in granulosa cell-conditioned medium (364, 367). In the mouse ovary, expression of IGFBP-4 was increased in granulosa cells of histologically atretic follicles and was correlated with positive staining for the DNA fragmentation characteristic of apoptosis (367).

3. Livestock species. The pig ovary expresses IGFBP-2, -3, -4, and -5, with granulosa cell IGFBP-2 localized by in situ hybridization to small follicles and IGFBP-4 to large follicles (482). IGFBP-2 mRNA and protein levels decline with advancing follicular development (483). Cultured porcine granulosa cells elaborate both IGFBP-2 and -3, with production of IGFBP-3 and IGFBP-2 stimulated by IGF-I and decreased by FSH (484, 485). Granulosa cells from medium-sized follicles also accumulate IGFBP-4 and -5. IGF-I stimulates, while FSH inhibits, IGFBP-5 mRNA and protein production. FSH stimulates elaboration of 22-kDa IGFBP-4 (484, 486). In porcine FF, follicular growth is accompanied by a slight increase in IGFBP-3 and a decrease in IGFBP-2 and IGFBP-4, as assessed by ligand blotting (487, 488, 489). While IGFBP-4 and IGFBP-5 are undetectable in FF from preovulatory follicles, atresia is associated with a marked increase of intrafollicular levels of IGFBP-2 and IGFBP-4 (487, 489, 490).

In the sheep, IGFBP-4 and -5 expression in healthy follicles is mainly limited to the theca (491, 492, 493). In atretic follicles, both IGFBP-2 and -5 are more strongly expressed in the granulosa layer than in healthy follicles, while both IGFBP-2 and -4 are more strongly expressed by the theca (493). FF content of IGFBP-2 and -4 declines, while IGFBP-3 slightly increases, with follicle growth. Atresia is associated with increased content of IGFBP-2, -4, and -5 (424, 493).

In the cow, as in the sheep, IGFBP-2, -3, -4, and -5 have been identified in FF by immunoprecipitation. By ligand blotting and mRNA expression analysis, IGFBP-2 and -4 are more abundant in estrogen-poor, atretic follicles than in estrogen-rich, healthy ones (384, 387, 494, 495, 496, 497). Within the dominant follicle, an increase in IGF-I and IGF-II with a concomitant decrease in IGFBP-2 may promote follicular dominance (388).

In summary, since granulosa cells from the pig, sheep, and cow express IGFBP-2, these three livestock species are better models for the human ovary than is the rat. The large animal models also permit the study of FF IGFBP content in relation to follicular functional status. In every species in which such studies have been reported, atretic follicles contain higher levels of IGFBPs -2, -4, and/or -5. Additionally, in cell culture models, gonadotropins universally decrease accumulation by granulosa cells of these small IGFBPs. These findings suggest that in a highly conserved mechanism, IGFBPs -2, -4, and -5 serve as IGF antagonists in follicles destined to undergo atresia, and that gonadotropins may exert their antiatretic action in part through down-regulation of IGFBP production. By contrast, IGFBP-3 may reach FF from thecal production or from the circulation; its level in FF is not affected by gonadotropins or atresia, but rather increases modestly with follicular maturation. By contrast to the smaller IGFBPs, IGFBP-3 appears not to function as an IGF antagonist within the follicle, possibly because it is saturated with ligand.

C. IGFBP proteases in the ovary (Table 2)
IGFBP protease activity was first demonstrated for IGFBP-3 in human pregnancy serum (498, 499). Subsequent reports of IGFBP-3 protease activity in pregnancy serum of other species (500, 501) were followed by nearly a decade of discovery of IGFBP proteases, which exist for most of the IGFBP species in a variety of biological fluids and are produced and secreted by a variety of cell types (329, 430, 502). The IGFBP proteases comprise a superfamily that includes several classes of proteases, including metal-dependent proteases, matrix metalloproteinases, disintegrin metalloproteinases, kallikreins, and cathepsins. These molecules likely represent enzymes with multiple active sites, multimeric proteins with subunit-specific active sites, or a cascade of enzymes with different activities. Several IGFBP proteases have been characterized with regard to their active sites and cofactor requirements, and the human pregnancy serum IGFBP-3 protease has been purified and characterized as a disintegrin metalloproteinase (503). Most IGFBP proteases are specific for particular binding-protein substrates. IGFBP-3 is the most susceptible to proteolysis by a variety of proteases, whereas IGFBP-1 appears to be the most resistant (504). Sequence analyses of IGFBP cleavage sites suggests that most proteolysis occurs in nonconserved regions (505).

The proteolysis of IGFBPs is likely to be an essential mechanism in the complex regulation of IGF action. IGFBP proteases partially proteolyze IGFBPs, resulting in lowered affinities of the IGFBP fragments for IGF peptides, thus increasing IGF binding to their receptors. In support of this concept, inhibitory effects of IGFBPs on IGF-stimulated DNA synthesis and mitogenesis are reversed in the presence of IGFBP protease activity in cultured chick embryo fibroblasts and prostatic epithelial cells, respectively (506, 507). In serum, proteolysis of IGFBP-3 releases IGFs for transport to the extravascular space, where they are likely bound to other IGFBPs, which are subsequently cleaved to promote release of the IGFs for action within the tissue. IGFBP-3 fragments may act at the cell membrane to augment the stimulatory effects of IGFs (508). Spatial and temporal regulation of IGFBP proteases is essential for controlled IGF actions, as well as the actions of IGFBP fragments.

It is remarkable that IGFBP-4 protease activity has been found in the ovaries of all species examined, including the pig, cow, and sheep. In these livestock species, the patterns of expression of low mol wt IGFBPs and their proteases in atretic and growing follicles are similar to those observed in follicles of other species. Likely this finding reflects a conserved mechanism that has evolved to regulate IGF bioavailability in the ovarian follicle (509, 510, 511). In the next sections, we will review the IGFBP protease activities that have implications for ovarian function in human and rat ovaries.

1. Human.
a. IGFBP-4 protease.
IGFBP-4 exists as a nonglycosylated 25-kDa form and a 32- to 34-kDa glycosylated protein. While some IGFBPs have inhibitory as well as stimulatory effects on IGF actions, IGFBP-4 appears to have exclusively inhibitory actions (429). IGFBP-4 mRNA and protein are abundantly expressed in small antral (androgen-dominant) follicles of normal and polycystic human ovaries (89, 343). As noted above, the apparent absence by ligand blotting of IGFBP-4 in FF from estrogen-dominant, compared with androgen-dominant, follicles (446, 459, 460, 512) was demonstrated to be due to an IGFBP-4 protease that decreases the affinity of IGFBP-4 for IGFs (457, 513). This protease is a metal-dependent enzyme with a pH optimum between 7 and 9 (436, 457), which is produced by nonluteinizing granulosa cells before the LH surge as well as by luteinizing granulosa (436, 443, 457, 513). The degree of proteolysis of IGFBP-4 is inversely proportional to the A:E ratio within the follicle (513). IGFBP-4 protease activity is stimulated by gonadotropins, IGF-I and -II, activin-A, and IFN- (435, 443, 513); FSH and IGF-II synergistically stimulate this activity in nonluteinizing granulosa cells (435).

When unsaturated with IGF peptide, IGFBP-3 inhibits proteolysis of IGFBP-4, whereas when saturated, it permits IGFBP-4 proteolysis (514). The implication of this finding is that in estrogen-dominant follicles, where IGF levels are high and IGFBP-3 is presumably saturated, IGFBP-4 proteolysis can increase IGF bioavailability from the pool of IGFs bound to this binding protein. In contrast, in androgen-dominant follicles, where IGFBP-3 is presumably unsaturated due to low levels of IGF production, any IGFBP-4 protease activity present is inhibited by the unsaturated IGFBP-3.

b. IGFBP-3 and IGFBP-2 proteases.
IGFBP-3 protease in estrogen-dominant FF (FFe) obtained at oocyte harvest from patients undergoing IVF was first demonstrated by Gargosky et al. (465). Iwashita et al. (515) also demonstrated a protease in FFe that cleaved radiolabeled IGFBP-3 into smaller fragments, whose activity in medium conditioned by luteinizing granulosa cells was stimulated by increasing doses of FSH. A 29-kDa fragment of IGFBP-3 was found in FF from dominant, compared with small antral, follicles, consistent with the presence of an IGFBP-3 protease (436, 465). With regard to IGFBP-2, immunoblotting revealed almost exclusively a 23-kDa IGFBP-2 fragment in FF from dominant follicles, compared with nearly exclusively intact IGFBP-2 and minimal fragments in FF from small cohort follicles (436). These observations are consistent with an IGFBP-2 protease in FFe, although specific IGFBP-2 proteolysis has not yet been demonstrated in these follicles. FSH action on luteinizing granulosa cells increases IGFBP-3 immunoreactivity in conditioned medium and apparently also increases IGFBP-3 proteolysis. These effects were found to be dose-dependent (515). These observations underscore the complexity of the mechanisms underlying control of IGF bioavailability within the human follicle.

c. Thecal and stromal proteases.
Limited information is available regarding IGFBP protease in the thecal or stromal compartments of the ovary of humans or other species. In human thecal cell-conditioned medium, LH decreases IGFBP-2, -3, and -4 levels, but no increase in low molecular weight forms consistent with proteolysis was seen. Conditioned medium contains an IGFBP-3 protease, which was partially inhibited by metal chelators. No difference was observed in theca from patients with normal or polycystic ovaries (438, 516).

In summary, since IGFs are potent stimulators of steroidogenesis and follicular growth in the human ovary, their regulation by IGFBPs and IGFBP proteases is temporally and spatially related within ovarian tissues. This is likely to provide timed promotion and inhibition of growth factor action during periods of follicular development and of limited follicular growth or steroidogenesis, respectively.

2. Rodent. Cultured rat granulosa cells secrete intact IGFBP-4 and IGFBP-5 into the medium (see above). When rat granulosa cells are cultured with FSH, there is a dose-dependent decrease in intact IGFBP-4 and an increase in a 17.5-kDa IGFBP-4 fragment, suggesting the stimulation of an IGFBP-4 protease by FSH (448, 473, 517). This proteolytic activity has a neutral pH optimum and is inhibited by EDTA, but not by other protease inhibitors, suggesting its dependence on a divalent cation (517). Some studies, however, failed to find IGFBP-4 protease activity in granulosa cell-conditioned medium, regardless of FSH stimulation (447, 518). FSH, but not IGF-I, also stimulates proteolysis of IGFBP-5. The granulosa-derived IGFBP-5 protease appears to be a zinc-dependent metalloprotease of molecular mass greater than 100 kDa, which is specific for IGFBP-5. The resulting degradation fragments were estimated at 18 and 14 kDa in one study (447) and 19.5 and 17.5 kDa in another (518). Under cell-free conditions, IGF-I attenuates IGFBP-5 proteolysis, suggesting that binding to IGF-I may be protective (447, 518). GnRH, which increases IGFBP-4 and IGFBP-5, does not induce protease activity for either of these IGFBPs under basal conditions, but it completely blocks the ability of FSH to inhibit IGFBP-4 and IGFBP-5 accumulation and stimulate protease activity (473, 476, 518). Since IGFBP-4 and IGFBP-5 are effective inhibitors of FSH action in rat granulosa cells, regulated production of their proteases is likely to be important in FSH-dependent control of follicle growth and development.

In summary, IGFBP proteases are produced by granulosa and theca cells at distinct times of follicle development in ovaries from a variety of species. This conservation of expression and their regulation by gonadotropins, IGFs, and other peptides and cytokines underscore the importance of IGFBP proteases in regulating IGF bioactivity at unique stages of follicle development. The striking absence of IGFBP-4 protease in androgen-dominant follicles and the presence of this enzymatic activity in estrogen-dominant follicles argue strongly for an important role for the IGF peptides as co-gonadotropins and for IGFBPs as antigonadotropins during follicular growth, steroidogenesis, and atresia.

D. IGFBP actions in the ovary
Studies of IGFBP actions in the ovary have largely employed IGFBPs purified from the FF of large animals or prepared by recombinant DNA technology, with cultured granulosa cells from DES-primed, immature rats as the target. When IGFBP-1, -2, -3, or -4 is added to cultured rat granulosa cells, each can inhibit FSH-stimulated steroidogenesis (471, 519, 520), while IGFBP-6 is ineffective (422). Porcine IGFBP-3 and IGFBP-2 inhibit FSH-stimulated E₂ and P release; their lack of efficacy in the presence of IGF-I antiserum or IGF peptide suggests that they act by neutralizing endogenous IGF-I (471, 519, 521). In this model, IGFBPs also decrease mitosis and cAMP generation. Human IGFBP-1, -2, -3, and -4 all similarly decrease FSH-stimulated P output (471, 522); human IGFBP-6 does not, possibly because of its lower affinity for IGF-I, the principal IGF produced by rat granulosa cells, compared with IGF-II (422). The physiological relevance of IGFBP actions on the granulosa is strongly suggested by in vitro studies showing the greater potency of IGF peptide analogs that do not bind to IGFBPs, compared with the native peptides, only under conditions of high-medium IGFBP levels (522). These observations have led to the conclusion that intrinsic IGF-I is an obligatory mediator of FSH-induced E₂ and P production by rat granulosa. Additional in vivo evidence for the biological relevance of IGFBP action on the ovary comes from studies showing that injection of IGFBP-3 into the rat ovarian bursa or introduction of IGFBP-3 into the in vitro perfusate of rabbit ovaries each can decrease the rate of follicular rupture at ovulation (523, 524), and from the recent observation that transgenic mice overexpressing IGFBP-1 have reduced numbers of ovulations per estrous cycle (525).

IGFBP actions on human granulosa cells are similar to those on cells from the rat. In cultured granulosa-luteal cells, IGFBP-1 and -3 decrease IGF-I-stimulated E₂ production; IGFBP-1 also decreases IGF-I-stimulated mitosis (390, 399, 513, 526, 527). IGFBP-3 fails to inhibit the steroidogenic effect of des(1, 2, 3)IGF-I, an analog that does not bind to IGFBPs. In granulosa cells obtained from women during unstimulated cycles, IGFBP-1 and IGFBP-3 inhibit IGF-I-stimulated E₂ and P production (399).

Recombinant human (rh) IGFBP-4 inhibits IGF-stimulated E₂ production by human granulosa cells (431, 435, 512, 513). Iwashita et al. (513) employed luteinizing granulosa cells, whereas Chandrasekher et al. (435) and Mason et al. (512) used nonluteinizing granulosa cells, showing that rhIGFBP-4 can inhibit both IGF-II- and FSH-stimulated E₂ production. This inhibition exceeded 80%, while in similar experiments IGFBP-2 or IGFBP-3 inhibited granulosa cell steroidogenesis by only about 20% (512). In contrast to the inhibitory effects of intact rhIGFBP-4 on E₂ production, addition of proteolyzed IGFBP-4 was without effect (513). These findings support an important role for IGFBP-4 and IGFBP-4 protease in the regulation of follicular steroidogenesis in the human ovary. IGFBP-4 inhibits FSH-stimulated E₂ production in the absence of added IGF peptide or in the presence of type I IGF receptor antibody, suggesting either IGF-independent action or antagonism of a locally produced IGF (431, 435, 512). Nevertheless, IGFBPs consistently display actions on cultured ovarian tissues opposite to those of IGF peptides and gonadotropins, suggesting that an excess of IGFBPs can be antigonadotropic (409, 528) and result in either follicular arrest (as in PCOS) or atresia.

In addition to regulating follicular differentiation and maturation, IGFs and IGFBPs also likely play a role in regulating apoptosis of granulosa cells, which is associated with follicular atresia (201). In a rat antral follicle culture system, both gonadotropins and IGF-I can prevent the apoptosis of granulosa cells that occurs spontaneously in serum-free medium, and IGFBP-3 reverses the protection from apoptosis afforded by hCG, FSH, GH, and IGF-I (200, 529). The restriction of IGFBP-4 expression in the mouse follicle to histochemically apoptotic granulosa cells (367) also supports a role for IGFBPs in promoting follicular atresia in vivo.

E. Role of IGFBPs in follicular development and atresia
In the growing estrogen-dominant follicle, a number of mechanisms have evolved to increase IGF peptide bioavailability and thereby amplify granulosa responsiveness to the growth-promoting, steroidogenesis-promoting, and antiapoptotic actions of FSH (Fig. 6). These include up-regulation of IGF receptors by gonadotropins and, in the rat, by estrogens (90, 530, 531); increase in IGF expression by gonadotropins (345, 532); inhibition by IGFs and gonadotropins of inhibitory IGFBP synthesis (403); and stimulation by gonadotropins and IGF-II of IGFBP protease activity (435, 513). The net result is maximum bioavailability of IGF peptides. In contrast, in the androgen-dominant follicle that is arrested in development or destined for atresia, these mechanisms are reversed (Fig. 6): FSH receptor numbers are low; IGF expression is almost undetectable; there is abundant expression of inhibitory IGFBPs (IGFBP-2 and IGFBP-4); and there is minimal detectable IGFBP protease activity. The net result is that aromatase is not induced, and thus precursor androgen persists in these follicles, in association with developmental arrest or atresia.

View larger version (22K): [in this window] [in a new window]   Figure 6. Model of IGF, IGFBP, and IGFBP protease actions in human ovary. In the estrogen-dominant, healthy growing follicle (shown at top left), granulosa cell IGF-II production increases, synergizing with FSH. IGF-II action is amplified by decreased synthesis and increased proteolysis of IGFBPs. In the androgen-dominant follicle (shown at top right), both increased IGFBP synthesis and decreased IGFBP proteolysis contribute to decreased FSH and IGF-II action on the granulosa, resulting in atresia or developmental arrest.

F. Summary
The high levels of expression of IGFs and low levels of expression of inhibitory IGFBPs in healthy follicles, and the reverse in atretic follicles, suggest that the level of bioavailable IGFs may play a role in regulating follicular growth, steroidogenesis, and apoptosis. IGFBPs and IGFBP proteases could thus assume importance in determining follicular destiny, since they can modulate the bioactivity of members of the IGF family.

	V. Polycystic Ovary Syndrome (PCOS)

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 
A. Clinical features
PCOS is a disorder of unknown, probably heterogeneous, etiology, characterized by chronic anovulation, biochemical and/or clinical evidence of hyperandrogenism, and enlarged, polycystic ovaries (535, 536). When first described by Stein and Leventhal (537) in 1935, the syndrome was defined by ovarian enlargement and multiple small cysts, in association with amenorrhea and hirsutism. PCOS affects between 5–10% of women of reproductive age (538, 539), and the onset of clinical manifestations often occurs at the time of puberty (191). In recent years, varying definitions of this syndrome have been used in studies of this disorder, with some investigators requiring polycystic ovaries on ultrasound for inclusion, and others requiring an elevation of serum LH or LH:FSH ratio (540). A consensus definition of PCOS was reached in 1990 under NIH auspices, which requires only hyperandrogenism of ovarian origin and oligomenorrhea or amenorrhea, with exclusion of other specific disorders such as steroid 21-hydroxylase deficiency (541). Other endocrine abnormalities that are inconsistently present in women with PCOS include obesity, peripheral insulin resistance and hyperinsulinemia, and elevations of serum PRL or DHEA-sulfate. Phenotypic differences among PCOS study populations may reflect underlying genetic differences in etiology or pathophysiology or in peripheral manifestations such as hirsutism (542, 543). Differences in diagnostic selection criteria can make comparison of studies on PCOS difficult.

PCOS is perhaps the most common disorder in which the association between insulin resistance and ovarian function appears to be important. Since several comprehensive reviews on this subject are available (26, 27, 140, 535), we focus herein on the controversial issues related to the pathogenesis of PCOS and the changes in the insulin-related ovarian regulatory system observed in PCOS. In the following section, we will review recent studies that have evaluated the use of inhibitors of insulin secretion and insulin-sensitizing agents in the therapy of PCOS.

B. Theories of pathogenesis
Determining the etiology or etiologies of PCOS has proven elusive. It was recognized as early as 1980 by Yen (544) that in PCOS a number of endocrine abnormalities perpetuate themselves in what has been described as a "vicious cycle." These include abnormal gonadotropin secretion, with excess circulating LH and low, tonic FSH levels; hypersecretion by ovarian thecal and stromal compartments of androgens, which were viewed as both disrupting follicular maturation and providing substrate for peripheral aromatization to estrogens in adipose and other sites; and negative feedback of this tonic estrogen production on the pituitary to decrease FSH secretion and thus trophic support of the granulosa cell (544). The vicious cycle concept was further supported by studies suggesting that normal ovulatory function can occur after disruption of this cycle, e.g., by ovarian wedge resection or cautery or during recovery from GnRHa-induced suppression (545, 546, 547, 548). The vicious cycle concept does not, however, provide an explanation of how the abnormalities become established. A number of endocrine disorders can produce similar anovulatory, hyperandrogenic states, such as functional or drug-induced hyperprolactinemia (549, 550) and adult-onset congenital adrenal hyperplasia resulting from 21-hydroxylase deficiency (551, 552). The primary abnormality in PCOS has been proposed to be of central, ovarian, adrenal, or peripheral metabolic origin. These theories will be briefly reviewed below.

1. Central hypothesis. Abnormalities in LH-secretory pattern and its regulation have been observed in PCOS. Women with PCOS often have both increased LH pulse amplitude and frequency, compared with ovulatory controls (168, 553, 554, 555). This results in increased or disordered LH secretion and may lead to an elevated serum LH:FSH ratio. These central alterations may be mediated by the altered steroid milieu of PCOS rather than being primary, since during recovery from GnRHa suppression no difference was seen between PCOS and normal women in the recovery of LH pulse frequency (556). On the other hand, while P normally slows GnRH pulse frequency, women with PCOS appear relatively resistant to this effect (557, 558), and chronobiological abnormalities of LH secretion can be observed in adolescent girls with features of PCOS (559), suggesting a primary abnormality of GnRH pulsatility in this disorder.

Abnormally rapid GnRH pulse generation is assumed to underlie abnormal LH secretion in PCOS. Abnormalities in other neuroendocrine modulators, such as the endogenous opioids, dopamine and leptin, have also been proposed as determinants of gonadotropin secretion in PCOS. Endogenous opioid excess may sensitize the gonadotrope to GnRH, particularly in association with hyperinsulinemia (37, 560). Decreased dopaminergic inhibition of LH release (561) and an increased incidence of an allelic form of the D3 dopamine receptor have been noted in women with PCOS (562). Recently, the possible role of leptin in PCOS has been examined. An initial report found serum leptin levels in a small subpopulation of women with PCOS greater than predicted from their BMI (563), but subsequent reports have failed to confirm this finding (564, 565, 566, 567, 568). In one study, hyperinsulinemia was associated with increased circulating leptin in PCOS subjects (141), although no association of serum leptin and insulin in women with PCOS was found in two other studies (567, 569). It seems unlikely that leptin is responsible for increased LH secretion in PCOS, since either an inverse (565, 570) or no relationship (563, 566) has been reported between serum leptin and LH levels. At this time, it is unclear whether leptin plays a role in the etiology of PCOS.

2. Ovarian hypothesis. An intrinsic ovarian functional defect has also been postulated as the source of the self-sustaining abnormalities in PCOS. Thecal hypertrophy and overproduction of androgens are recognized features of the PCOS ovary. When placed in culture, PCOS thecal cells continue to hypersecrete androgens, and when deprived of trophic support through GnRHa suppression, the PCOS ovary continues to hypersecrete 17-OHP in response to hCG in vivo (571, 572, 573). Dynamic short-term GnRHa testing in PCOS produces an exaggerated ovarian 17-OHP-secretory response (107, 573, 574). This response may reflect the increased thecal mass present in the ovary, but has been also interpreted as reflecting dysregulation of the activity of the steroidogenic enzyme P₄₅₀c17, which is responsible for both 17-hydroxylation of C21 steroids and for the 17,20-lyase activity necessary for androgen (C19) synthesis (575). The recent report that the lyase activity of P₄₅₀c17 can be promoted by serine phosphorylation of the enzyme (576) suggests a possible mechanism for abnormal steroidogenesis in PCOS. It is intriguing that excessive serine phosphorylation of the insulin receptor has been proposed as a cause of peripheral insulin resistance in some women with PCOS (577) (see below; Section V.C).

Granulosa cell steroidogenic and mitogenic abnormalities have also been found in PCOS. Aromatase activity is low in PCOS granulosa cells in vivo, reflecting decreased FSH activity, but is normal or exaggerated when they are cultured (105, 578). This observation led to the concept that the PCOS follicle contains excessive amounts of inhibitor(s) of FSH action. While IGFBP-2 and -4 are FSH antagonists (471, 521) that are abundant in FF from PCOS antral follicles, their expression in the PCOS ovary is indistinguishable from that in the cycling ovary (89, 533, 579) (see above), weakening the argument for an etiological role of these proteins. Other studies suggested that an inhibin -subunit-processing product, pro-C, can serve as an FSH antagonist and is found in FF (580, 581), but its presence and role in PCOS follicles are unknown. Granulosa cell mitosis also appears defective, in that granulosa cell numbers in PCOS follicles are lower than in healthy size-matched follicles from cycling women (582), but whether abnormal granulosa cell mitosis is important in the pathogenesis of PCOS has not been directly tested.

3. Adrenal hypothesis. Many women with PCOS develop irregular menses shortly after menarche. It has been hypothesized that excessive production of adrenal androgens, which increases at puberty, can supply substrate for extragonadal aromatization and result in tonic estrogen inhibition of FSH secretion (544). Premature adrenarche is associated with a higher incidence of both functional ovarian hyperandrogenism, with exaggerated 17-OHP response to GnRHa challenge (583, 584), and insulin resistance (585, 586). Hyperinsulinemia can stimulate adrenal as well as ovarian steroidogenesis (587). Since insulin resistance accompanies puberty and may contribute to adrenarche, an important unanswered question is why pubertal insulin resistance fails to resolve in adolescent girls who develop PCOS, and whether the effect of hyperinsulinemia on the adrenal, on the ovary, or on both of these organs is significant in the pathogenesis of PCOS.

C. Insulin resistance in PCOS
1. Putative causes and role in pathogenesis. A majority of women with PCOS demonstrate peripheral insulin resistance involving skeletal muscle and adipose tissue, which results in compensatory hyperinsulinemia (140). Insulin resistance does not appear to involve ovarian steroidogenesis, because granulosa and thecal cells from PCOS ovaries demonstrate a normal dose response to insulin in culture (96, 97). As a result, excessive insulin stimulation may promote thecal androgen hypersecretion.

Insulin resistance can be determined by measuring insulin levels during frequently sampled IVGTT (588) or by euglycemic, hyperinsulinemic clamp studies (589). Obese women with PCOS are more insulin resistant than weight-matched controls (589, 590, 591), suggesting that obesity and PCOS exert independent effects on insulin resistance. Many studies have found insulin resistance in lean as well as obese subjects with PCOS (120, 168, 589, 592), although at least one study failed to confirm this finding (593), and normal insulin sensitivity can be restored in some obese women with PCOS with weight loss (155).

The molecular basis of insulin resistance in PCOS is a subject of active research and has recently been reviewed (140, 594). Pedigree studies have suggested a genetic basis of PCOS in some kindreds, with premature balding as the male phenotype (595). In these families, linkage to the variable number of tandem repeats locus upstream of the insulin gene has recently been demonstrated (596). Mutations in the insulin receptor gene or defects in its intrinsic tyrosine kinase activity are rarely found, and insulin receptor binding is normal (216, 597, 598, 599, 600, 601, 602, 603, 604, 605). The defect of insulin action in PCOS appears to be at the postbinding level and to involve glucose transport (603); it may be observed only in some cell types (e.g., in adipocytes but not skin fibroblasts) (606) and may be accompanied by a defect in insulin-induced inhibition of lipolysis (605, 607). Several molecular mechanisms for the glucose transport defect have been suggested by recent studies. In one of these, abdominal adipocytes of PCOS subjects had a lower content of the GLUT4 glucose transporter than controls (608). Another noted that the insulin receptor in about half of women with PCOS is excessively phosphorylated on serine, a state that reduces signal transduction (577). In another report, PCOS adipocyte insulin sensitivity could be restored by an adenosine receptor agonist, suggesting that depletion of cellular adenosine may lead to insulin resistance (609). The correction of insulin resistance by a thiazolidinedione, troglitazone (35, 36), suggests that women with PCOS may be deficient in signal transduction through peroxisome proliferator-activated receptor- (PPAR-), the natural ligand for which appears to be a PG of the J series or an essential fatty acid (610, 611) (see below).

The potential links between hyperinsulinemia and the increased androgen production observed in PCOS (27, 612) have been discussed previously (Section II.C); they include direct stimulation of ovarian androgen secretion by insulin, possibly through stimulatory effects on the 17-hydroxylase/17,20-lyase and P₄₅₀scc enzymes; direct stimulation of LH secretion by insulin or sensitization of LH-secreting pituitary cells to GnRH stimulation; up-regulation of ovarian type I IGF receptors with the amplification of IGF-I, IGF-II, and insulin actions in the ovary; decreased levels of SHBG, with concomitant elevation of free androgens; decreased IGFBP-1 production, both in the liver and in the ovary, with concomitant elevation of free IGFs in the circulation and in the ovary; and the synergistic growth- and cyst-promoting action of insulin and LH.

In addition to these effects, an action of insulin on granulosa cells has been implicated in the follicular developmental disorder of PCOS. Granulosa cell numbers are decreased relative to follicle size in PCOS (582), and it has recently been suggested that acquisition of granulosa cell LH responsiveness too early in follicular development may have an antiproliferative effect on granulosa cells in PCOS (613, 614). Hyperinsulinemia could accelerate development of granulosa cell LH responsiveness by amplifying the induction of LH receptors (13, 96, 164, 613).

It has been proposed both that hyperandrogenemia may contribute to insulin resistance in PCOS and that hyperinsulinemia can promote hyperandrogenism (3, 9, 120). The results of pharmacological modification studies have suggested that the latter mechanism is more operative than the former. Androgen levels in PCOS have been reduced and their action blocked by the use of GnRHa and androgen receptor blockers. Suppression of ovarian or adrenal steroidogenesis has not improved insulin resistance (615, 616, 617), although in some studies, antiandrogens such as flutamide and spironolactone (618, 619, 620) have led to partial improvement. Ovarian cautery, which lowers androgen secretion, does not alter insulin resistance (621). Direct administration of androgens to oophorectomized women has no effect on insulin levels, though it increases circulating levels of IGF-I and suppresses SHBG (622). On the other hand, pharmacological reduction in the level of hyperinsulinemia, either by insulin sensitizers such as metformin or troglitazone or by insulin secretion inhibitors such as octreotide or diazoxide, has consistently improved circulating androgen levels (29, 30, 31, 34, 35, 36, 108, 142, 143, 221, 623, 624). Additionally, the occurrence of hyperandrogenism in states of extreme insulin resistance other than PCOS (9, 140) and in association with hyperinsulinemia induced by valproate therapy for epilepsy (625) supports a primary role for insulin excess in producing ovarian dysfunction.

In addition to decreased insulin sensitivity, insulin secretion in patients with PCOS also appears to be abnormal (137, 626). In particular, early insulin release after ingestion of glucose appears to be exaggerated (155, 627, 628). A decrease in the amplitude of meal-related insulin pulses and defective insulin clearance in peripheral tissues have also been reported (629, 630). Patients with PCOS exhibit abnormal entrainment of insulin secretory pulses in response to an oscillatory glucose infusion (626). These abnormalities, however, may be secondary to insulin resistance, since they can be reversed with the use of insulin-sensitizing agents (36). Both obese and nonobese women with PCOS appear to have inadequate insulin secretion for their degree of insulin resistance (631), placing them at an increased risk for the development of type 2 diabetes (538).

2. Role of obesity in PCOS. Some aspects of insulin action in obesity resemble those seen in PCOS (632, 633, 634, 635). Many patients with obesity are insulin resistant and hyperinsulinemic (636, 637, 638) and, when central obesity is present, often have reduced circulating levels of SHBG and mildly elevated androgen levels (639, 640, 641, 642, 643, 644). Because insulin resistance has not been consistently encountered in populations of lean women with PCOS, the existence of a cause of insulin resistance in PCOS distinct from that associated with obesity remains open to question. That obesity contributes significantly to both insulin resistance and hyperandrogenism in overweight women with and without PCOS is evident from the improvement in androgen levels usually seen with weight loss, sometimes to levels observed in weight-matched ovulatory women (121, 147, 148, 149, 150, 151, 152, 153, 154, 155, 632, 633, 639, 645, 646, 647). Anovulatory hyperandrogenemic adolescents and adults are more insulin resistant than weight-matched ovulatory controls (191, 586, 635, 648, 649, 650, 651). Since there is some evidence that androgens may contribute to insulin resistance, however (619, 620, 652, 653, 654), this finding fails to resolve the question of whether insulin resistance in PCOS is independent of obesity.

The cause of obesity-related insulin resistance is itself not well understood. As discussed above, obese individuals are usually insulin resistant, and in some individuals obesity may be a necessary factor for the development of diabetes. For example, Sigal et al. (655) recently demonstrated that glycine-arginine polymorphism in codon 972 of the IRS-I gene clusters with diabetes and obesity, suggesting that this polymorphism may predispose to the development of type 2 diabetes only if obesity is also present. A Pro115Gln activating mutation in the PPAR-2 receptor has been associated with obesity (656); activation of this receptor may reduce insulin resistance, and individuals with this mutation appear to have lower circulating insulin levels than obese individuals without this mutation. Recent studies have implicated the cytokine TNF- as a contributor to insulin resistance in obesity (241, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666). In Native American Pimas, in whom insulin resistance and obesity are highly prevalent, and in whom oligomenorrhea is common (212, 634), a mutation closely linked to TNF- has been associated with insulin resistance (667). TNF- is produced by adipose tissue and stimulates IRS phosphorylation on serine, which in turn appears to inhibit insulin receptor tyrosine kinase and PI-3 kinase activation (659, 662, 668, 669, 670, 671). Interestingly, TNF- may also interfere with the action of IGF-I, although this effect of TNF- may involve not only the inhibition of type I IGF-receptor tyrosine kinase, but also stimulation of IGFBP production (672). TNF- can also inhibit expression and signaling through PPAR- (673, 674), which serves as a major target for thiazolidinediones; it is controversial whether thiazolidinediones block TNF- inhibition of PPAR- expression (675). TNF- can also inhibit the synergism between insulin and FSH in stimulating steroidogenesis (676). Although all of these findings are of great interest, the ability of TNF- to induce insulin resistance in vitro or in vivo has not been firmly established (677, 678, 679). Further, circulating as well as FF TNF- concentrations in PCOS appear to be similar to those in normal women (680, 681). Leptin may also contribute to the insulin resistance of obesity via mechanisms similar to TNF-, but ob/ob mice, which lack functional leptin, develop insulin resistance (287). Furthermore, in the Zucker fatty rat, which lacks a functional leptin receptor, IRS-1 and -2 are down-regulated in the liver, leading to a dramatic reduction in PI-3 kinase activity in spite of the leptin resistance (289).

In summary, the cause of insulin resistance in women with PCOS appears to be, at least in part, related to obesity, and insulin resistance is not present in all women with PCOS (682). Whether there is a component of insulin resistance in PCOS independent of the insulin resistance of obesity will be clarified once the specific molecular mechanisms of insulin resistance in both of these conditions are better understood (670, 683, 684). It has been proposed that the pathogenesis of PCOS is different in obese and nonobese women, with insulin resistance and hyperinsulinemia playing a central role in obese patients, and abnormalities of the GH-IGF-I axis being important in PCOS in lean women (168, 685, 686).

D. Alterations of IGFs and IGFBPs in PCOS
1. Ovarian IGF production. By in situ hybridization and immunohistochemistry, the patterns of IGF-I and IGF-II mRNA and protein expression in the antral follicles of the PCOS ovary were identical to those of the small antral, nondominant follicles of cycling women (89). In human thecal cell cultures from PCOS ovaries, no differences were noted in IGF-I or IGF-II production compared with cultures derived from control women (438). In FF, levels of IGF-I in PCOS are similar to or slightly greater than in FF from cycling women (687, 688). To our knowledge, basal levels of IGF-II in FF have not been reported in PCOS. However, after gonadotropin stimulation for IVF, intrafollicular IGF-II levels are lower in PCOS than in control women, and IGF-II expression by granulosa cells is lower as well (689).

2. Ovarian IGFBP production. IGFBP production has been examined in the PCOS ovary. In an in situ hybridization study, each IGFBP displayed a pattern of mRNA expression identical to that seen in the small antral follicles of cycling women (89). In a recent study, IGFBP-4 localization in PCOS antral follicles correlated with insulin sensitivity: insulin-resistant women had greater IGFBP-4 staining in theca than in granulosa, while the reverse was seen in non-insulin-resistant subjects (433). Two groups have examined IGFBP production by cultured cells derived from PCOS ovaries. San Roman and Magoffin (579) reported the presence of IGFBP-3 in media conditioned by both granulosa and theca cell cultures from three women with PCOS, with levels declining after gonadotropin stimulation. TGF-ß increased IGFBP-3 production by granulosa cells and antagonized the effect of FSH. In a similar study, another group found no detectable IGFBPs by ligand blotting and no IGFBP-3 or IGFBP-2 by immunoblotting in granulosa cell-conditioned medium, while thecal cell-conditioned medium from PCOS ovaries showed the same IGFBP profile as that derived from cycling women (438).

The three groups that examined IGFBP profiles by ligand blotting in FF from cycling women (see Section IV.B.1) also examined FF from women with PCOS (460, 533, 579). They all found FF IGFBP profiles in PCOS similar to those in the androgen-dominant follicles of cycling women: levels of IGFBP-2 and IGFBP-4 are markedly elevated in PCOS follicles compared with estrogen-dominant follicles. By contrast, no differences in IGFBP-3 levels were noted in FF from PCOS follicles, androgen-dominant follicles, and estrogen-dominant follicles from cycling women (459, 579). These findings indicate that the bioavailability of IGFs within the PCOS follicle, as in all androgen-dominant follicles, is likely lowered by higher IGFBP levels. Two studies have noted that a spontaneous preovulatory follicle found in a woman with PCOS had an IGFBP profile similar to that of the preovulatory follicles of cycling women (460, 690), suggesting that the expression of IGFBP-2 and IGFBP-4 can be regulated normally in some women with PCOS. FF IGFBP-1 levels have been studied in PCOS by immunoassay. Levels in size-matched PCOS follicles were 48% of those from cycling women (458), possibly reflecting a greater inhibitory effect of insulin on IGFBP-1 production.

3. Serum IGFs. Perhaps as a consequence of decreased serum IGFBP-1 levels, serum free IGF-I levels are elevated in PCOS (348, 691). This latter finding suggests that IGF-I may be more available to the theca in PCOS than in normal women and may contribute to the increased androgen production by the PCOS theca cell (692). Serum free IGF-I levels do not correlate with IGFBP-1 levels, however, arguing against a causal relationship between decreased serum IGFBP-1 and increased IGF bioavailability at the follicular level. Serum total IGF-I and IGF-II levels are not different between PCOS and normal women (348, 691).

4. Serum IGFBPs. The role of circulating IGFBPs in modulating normal ovarian function is uncertain, in view of the lack of cycle-dependent changes in serum IGFBP-1 and IGFBP-3 (348, 693) and the lack of evidence from selective venous catheterization for a significant ovarian contribution to serum levels of these IGFBPs (694). Conversely, it is likely that FF IGFBP levels can be influenced by changes in serum levels, since FF contains transudated serum proteins. Because of the availability of immunoassays, IGFBP-3 and IGFBP-1 have been most extensively studied in PCOS.

No difference has been found by immunoassay in serum IGFBP-3 levels between PCOS and ovulatory controls (168, 348, 695, 696). Similar integrated 24-h IGFBP-3 levels were found in obese and lean women with PCOS, which also did not differ from obese or lean controls (168). One study examined the effect on serum IGFBP-3 of octreotide, which decreases insulin secretion in hyperinsulinemic women with PCOS (142). In women with PCOS unselected for insulin resistance, octreotide increased serum IGFBP-3 levels by 42%, while decreasing serum IGF-I by 63%. No change in IGFBP-3 and a smaller but significant decrease in IGF-I were observed in control women (696). The effect of octreotide on insulin secretion cannot explain this decrease in serum IGFBP-3, since insulin does not modulate circulating IGFBP-3 (697). The decrease in serum IGF-I also cannot explain the increase in IGFBP-3, since IGF-I does not appear to regulate serum IGFBP-3 (698). Rather, these findings suggest a central alteration in the GH/IGF-I axis in PCOS (168, 699).

Women with PCOS, particularly if obese, have lower serum IGFBP-1 levels than their normally cycling counterparts or anovulatory women without PCOS (348, 444, 592, 691, 700, 701). Fasting serum IGFBP-1 levels are negatively correlated with serum insulin levels in all human subjects, including those with PCOS (192, 194, 198, 701). In women with PCOS, IGFBP-1 levels decline during both OGTTs and IVGTTs in a fashion mirroring the insulin response (190, 192). Weight loss increases serum IGFBP-1 (702), while ovarian electrocautery, which improves ovulatory function, and GnRHa suppression of ovarian steroid production each has no effect on serum IGFBP-1 or insulin sensitivity (546, 621, 703). Thus, serum IGFBP-1 levels reflect both short-term fluctuations in insulin levels (183) and the degree of peripheral insulin resistance. It has been proposed that IGFBP-1 levels in women with PCOS may be useful clinically as a marker for insulin resistance (621).

E. Summary
Multiple abnormalities of the components of the insulin-related ovarian regulatory system are present in PCOS. It remains to be confirmed whether any of these abnormalities are primary in the pathogenesis of PCOS and whether they play an important role in the development of hyperandrogenism and anovulation in this disorder.

	VI. The Insulin-Related Ovarian Regulatory System: Implications for Therapy

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 
If abnormalities of the insulin-related ovarian regulatory system are of clinical importance in patients with altered ovarian function, one would expect the reversal of these abnormalities to lead to clinical improvement (32). There are several types of therapeutic interventions that may influence the ovarian insulin-related regulatory system: low calorie diets and weight reduction; insulin-sensitizing agents, including metformin, troglitazone, ß₃-adrenergic receptor agonists, and vanadate; inhibitors of insulin secretion, such as octreotide and diazoxide; promoters of insulin clearance, such as the opioid antagonist naltrexone; IGF-I and IGF-II; and GH, which can act both through its own receptors and by affecting IGF-I production in the liver and IGFBP production in both the liver and the ovary.

A. Treatment of PCOS
1. Dietary modification. In numerous studies of women with PCOS, caloric restriction (even without weight loss) or weight-reducing diets have resulted in normalization of insulin sensitivity and gonadotropin and androgen metabolism (including P₄₅₀scc and 17-hydroxylase activity); improvement of acanthosis nigricans, which is commonly observed in obese insulin-resistant women (704, 705, 706); and restoration of ovulation (147, 148, 149, 150, 151, 152, 154, 155, 156, 646, 702, 707). As discussed above, mechanisms underlying such improvements may include a decline of insulin-stimulated gonadotropin secretion as well as a reduction of the direct stimulatory effect of insulin on the ovary and/or the adrenal and alleviation of insulin-induced inhibition of both SHBG and IGFBP-1. Additionally, the reduction of leptin levels observed during caloric restriction may lead to the deactivation of the hypothalamic-pituitary-ovarian axis. In practice, however, sustained long-term weight loss using dietary intervention can be accomplished only in a small number of obese individuals (636). Therefore, other therapeutic approaches are usually needed.

2. Agents that lower circulating insulin without affecting insulin sensitivity. Both diazoxide and octreotide can directly inhibit pancreatic insulin secretion; these agents also reduce androgen levels, and octreotide has been shown to restore ovulation (30, 34, 142, 221, 623). The long-term use of these agents in PCOS, however, is not desirable, since they may worsen glucose tolerance and further increase the risk of developing diabetes (224, 538, 708).

Opioid antagonists such as naltrexone can decrease the insulin response during OGTT and may do so largely by increasing the rate of insulin clearance in a subset of women with PCOS who may have defective insulin clearance (37, 223, 709, 710); they do not affect glucose utilization during a clamp study (223). Although naltrexone treatment has not been associated with lowering of LH or androgens (37, 711, 712), improvements in both spontaneous ovulation and responsiveness to clomiphene have been noted in association with the decline in circulating insulin (38).

3. Insulin-sensitizing agents. The biguanide metformin is an insulin sensitizer that can reduce hyperglycemia in type 2 diabetes. Its mechanisms of action involve suppression of hepatic glucose output and improvement in insulin sensitivity in peripheral tissues (713, 714, 715, 716, 717). Metformin has also been reported to increase insulin receptor tyrosine kinase activity in vascular smooth muscle (718). Metformin does not appear to have a direct effect on ovarian steroidogenesis (719) or on synthesis of IGFBP-1 (720).

The effects of metformin on circulating levels of insulin, androgens, and gonadotropins and on ovulatory function have been examined in PCOS. In a dose of 500 mg three times daily for 4–8 weeks, metformin improved insulin sensitivity and decreased hyperinsulinemia, with integrated insulin secretion during OGTT decreasing by 35–40% (31). Along with the reduction of circulating insulin, SHBG was increased and serum LH and androgens, as well as the exaggerated 17-OHP secretory response to GnRHa, were reduced (29, 31, 108, 143, 145, 624). These improvements occurred in both obese and lean subjects and were noted in placebo-controlled studies (29, 108). In one of these, reduction in serum free T and LH was accompanied by restoration of menstrual cyclicity in 21 of 22 subjects, associated in most with ovulatory P levels (143). A recent report also suggests that metformin can improve the ovulatory response to clomiphene in PCOS (721). Metformin appears to exert its inhibitory effect on androgens by reducing hyperinsulinemia, which in turn leads to decreases in pituitary LH secretion, thecal androgen secretion, and an increase in SHBG. Several studies, however, have not found an improvement in insulin sensitivity or androgen metabolism in PCOS with metformin (153, 722, 723, 724). In one of these, weight was deliberately maintained at a controlled level (723). In another study, obese, hirsute women were treated with a low calorie (1500 kcal/day) diet and in a randomized fashion with either placebo or 850 mg metformin/day. Diet led to a reduction in insulin levels, a rise of SHBG, and a fall in free androgen levels, but metformin had no additional effect (153).

Troglitazone, a thiazolidinedione, decreases peripheral insulin resistance and is useful in the treatment of type 2 diabetes (725, 726, 727, 728). Thiazolidinediones are high-affinity ligands for PPAR- (729, 730), a member of the steroid nuclear receptor superfamily, and are believed to exert their effect on insulin sensitivity by activating this receptor. Activation of PPAR- in adipocytes promotes their differentiation and increases the expression of the fatty acid binding protein aP2 (731, 732, 733), as well as uncoupling proteins (734, 735, 736), which act in mitochondria to uncouple oxidation and phosphorylation. PPAR- activation is promoted by insulin (733). It is not known how thiazolidinediones mediate insulin sensitivity. It has been suggested that they may act in part by antagonizing TNF--induced insulin resistance (660, 673, 733) or by leading to a decreased production of leptin (737, 738). In subjects with impaired glucose tolerance or frank diabetes, troglitazone improves glycemic control and decreases circulating insulin concentration (725, 727, 739). In obese nondiabetic humans, troglitazone increases glucose disposal rate and improves insulin sensitivity (740). It has been proposed that troglitazone can delay or prevent the development of type 2 diabetes in insulin-resistant individuals, including women with a history of gestational diabetes (740, 741).

In two studies of obese women with PCOS, defined by hyperandrogenemia and oligomenorrhea or amenorrhea, troglitazone decreased circulating insulin levels and increased insulin sensitivity (35, 36). Notably, troglitazone also decreased serum free T and increased SHBG levels, the latter apparently a direct result of the decline in circulating insulin. In a study in which subjects were not selected for glucose intolerance (35), serum LH also declined. This study noted that 2 of 21 women (9%) ovulated spontaneously on troglitazone, based on serum P elevation. When subjects were selected for impaired glucose tolerance (36), serum total T declined and the 17-OHP response to leuprolide was also decreased, but LH levels were unchanged. The return of ovulation was not reported. In this group of subjects, characterized by abnormal pancreatic ß-cell entrainment of insulin secretion to an oscillatory glucose infusion, troglitazone normalized the insulin-secretory response (36, 742). Although the effect of troglitazone to lower circulating androgens is thought to be mediated by a reduction in plasma insulin, troglitazone has recently been reported to inhibit 3ß-HSD, and thus P production, in cultured porcine granulosa-luteal cells (743). The relevance of this finding to troglitazone treatment of women with PCOS remains to be determined.

Taken together, studies of metformin and troglitazone in PCOS suggest that reduction of insulin resistance and hyperinsulinemia leads to a decline in ovarian androgen hypersecretion, lending further support to the hypothesis that insulin resistance and hyperinsulinemia are indeed instrumental in the development of hyperandrogenism in PCOS.

Three other insulin-sensitizing agents are of potential use in PCOS: D-chiro-inositol (also called INS-1), ß₃-adrenergic receptor agonists, and vanadate (744, 745). D-chiro-Inositol, which may serve as a precursor for inositolglycan mediators of insulin signal transduction, has been shown to lower circulating insulin and improve insulin action in spontaneously insulin-resistant primates (744). A recent study suggests that inositolglycans mediate the stimulation of thecal steroidogenesis by insulin (79), and another report suggests that D-chiro-inositol, given to women with PCOS in a placebo-controlled trial, decreases insulin secretion during OGTT and increases plasma SHBG. Accompanying these changes was a significant restoration of spontaneous ovulation (746).

ß₃-Adrenergic receptors are located in brown fat, a tissue responsible for nonshivering thermogenesis and weight regulation. Ablation of brown adipose tissue in transgenic animals induces insulin resistance (747). When given to obese rodents, ß₃-adrenergic receptor agonists can produce weight loss and a reduction in insulin resistance (745).

Vanadate appears to improve insulin action through mechanisms distal to insulin-receptor kinase activation (748, 749, 750, 751, 752). Vanadium may activate cytosolic protein tyrosine kinase and thus may mimic the effects of insulin (748, 753). Both in insulin-resistant animals and in those with streptozotocin-induced diabetes, vanadate reduces blood glucose concentration and, in the former group, it reduces circulating insulin levels (745). Vanadyl sulfate can reduce insulin resistance in patients with type 2 diabetes (754).

It remains to be established whether D-chiro-inositol, ß₃-adrenergic receptor agonists, or vanadate are clinically useful in women with insulin resistance and hyperandrogenism.

B. Therapeutic use of IGF-I and IGF-II
IGF-I has been used therapeutically in several studies in patients with type 1 or type 2 diabetes (755, 756, 757, 758, 759), in syndromes of extreme insulin resistance (329, 745, 760), and in myotonic dystrophy and other diseases (761, 762). IGF-I appears to be effective in enhancing the sensitivity of tissues to insulin and in directly inhibiting insulin secretion by pancreatic ß-cells (763, 764). Its side effects include symptomatic hypophosphatemia, seen mainly with intravenous administration; arthropathy; and occasional cranial nerve palsies (745).

In patients with syndromes of extreme insulin resistance, injections of IGF-I result in a decline of plasma glucose concomitant with a decrease in insulin and C-peptide (745, 763, 765, 766). The mechanisms of these effects of IGF-I are not well understood. It is possible that in addition to having insulin-like actions of its own mediated by the type I IGF receptor, IGF-I can also indirectly activate the insulin receptor, possibly by initiating insulin receptor phosphorylation (type I IGF receptor/insulin receptor "cross-talk"). Long-term (2-yr) administration of IGF-I to a patient with extreme insulin resistance (type A syndrome) led to a reduction of glucose levels but was associated with worsening of her hyperandrogenism (767). Similarly, prolonged administration of IGF-I to women with GH receptor deficiency is associated with the development of hyperandrogenism (768). To our knowledge, there are no clinical trials of IGF-II in patients with insulin resistance and/or anovulation. When overexpressed in transgenic mice, IGF-II produces improvement in insulin sensitivity and increases in lean body mass without affecting body size (769). The reproductive function of mice overproducing IGF-II has not been examined in detail. Whether IGFs can be safely used in humans with insulin-resistant states, and whether their use will affect ovarian function, awaits further study.

C. Use of GH in ovulation induction
1. GH effects on ovarian function. Another manipulation of the components of the insulin-related ovarian regulatory system that may have therapeutic implications is the use of GH along with gonadotropins in ovulation induction. GH can potentially influence follicular function in four ways: direct action on follicular cells through GH receptors; direct action to increase ovarian IGF production; action on the liver to increase circulating IGF-I; and modulation of intrafollicular and hepatic IGFBP production and/or IGFBP levels in FF and in the circulation.

There is evidence for direct effects of GH on human granulosa cells, which express GH receptors (770, 771, 772). GH treatment of granulosa cells in vitro stimulates both steroidogenesis and mitogenesis (773, 774, 775, 776). Since evidence points against production of IGF-I by human granulosa cells, it is questionable whether GH actions on the human ovary are mediated through ovarian IGF-I. Only the theca layer expresses IGF-I mRNA, but it does not appear to express GH receptors (777). In one study, GH actions on human granulosa cells could be blocked by antibodies to IGF peptides or the type I IGF receptor (775), but in two others (773, 776), IGF-I production by granulosa cells was not detected, even with GH treatment. The latter studies, in addition to those showing that the human ovary, unlike its rodent counterpart, does not produce IGF-I (13, 14, 88, 89), suggest that GH can act directly on granulosa cells through its own receptor. At least one study, however, found no effect of GH on granulosa cell steroidogenesis (778).

GH increases hepatic production of IGF-I, and IGF-I mediates many of the effects of GH. When GH is given on an alternate-day schedule in ovulation induction protocols, both circulating and FF IGF-I levels rise (464, 779, 780, 781, 782, 783, 784, 785, 786). The increase of intrafollicular IGF-I most likely mediates the adjuvant effect of GH seen in ovulation induction of some anovulatory patients (see below).

GH treatment added to ovulation induction protocols may also influence intrafollicular IGFBP levels. GH is the principal stimulator of hepatic IGFBP-3 production and hence a major regulator of serum IGFBP-3 (429). In studies of poor responders to conventional gonadotropin ovulation-induction regimens, GH did not affect levels of IGFBP-1 or IGFBP-3 in FF, but it did raise serum IGFBP-3 (461, 464, 784). In a study of unselected women undergoing IVF (786), however, GH raised FF levels of IGFBP-1, -3, and -4, as well as serum levels of IGFBP-3, compared with matched placebo cycles in each subject. The increase in IGFBP-1 likely arises within the follicle, since serum IGFBP-1 is transiently reduced by GH (787). Differences in the IGFBP-3 response among patients suggest that the beneficial effect of increased IGF-I with GH may be blunted by stimulation of IGFBP-3 in some women (786).

The effects of GH treatment on follicular steroidogenesis have also been examined. In COH cycles, FF levels of E₂ and P did not differ between GH- and placebo-treated groups (406, 464, 774, 783, 788, 789). In one study, E₂ and P production by granulosa cells in culture was unaltered by in vivo GH exposure (788), but in another, levels of mRNA expression for 3ß-HSD and aromatase in freshly harvested granulosa cells were increased by GH (774). Supporting the latter result, a third study found that steroidogenesis by cultured granulosa cells immediately after harvest was increased by in vivo GH exposure (775).

There is evidence that endogenous GH secretion may affect ovarian function, particularly in response to gonadotropin stimulation. Women with higher basal GH levels had greater E₂ and oocyte number than those with lower GH levels in one study (790), and in another, serum IGFBP-3 level before stimulation, presumably reflecting integrated GH secretion, was positively correlated with serum E₂ and follicular response to gonadotropins (791). GH reserve, measured by response to a clonidine-provocative test, was lower in poor responders in two studies (792, 793), but not different in a third (794). The GH rise in response to gonadotropin stimulation (795) was predictive of pregnancy, but not of the degree of ovarian stimulation (796). Age and weight may be important confounders, however, as GH levels are lower in women of advanced reproductive age and in obese women (168, 797, 798).

2. Clinical trials of GH in ovulation induction. Given the potential physiological involvement of the GH/IGF-I axis in ovulation, many investigators have attempted to use exogenous GH as an adjuvant for ovulation induction. A preliminary trial by Homburg et al. (799) found that GH, in a dose of 20 IU on alternate days, significantly augmented the ovarian response to human menopausal gonadotropins (hMG) in four of seven patients undergoing ovulation induction. The four patients who showed improvement all had hypogonadotropic anovulation. The same group of investigators then undertook a randomized, double-blind, placebo-controlled trial, in which 16 women with hypogonadotropic anovulation were treated with placebo or GH, 24 IU every other day, in addition to hMG. The duration of treatment and the total number of ampules of hMG needed were reduced in the GH group, compared with the placebo group (800). Another report noted a similarly decreased hMG requirement in three anovulatory women, which persisted in the subsequent cycle (801). A study in IVF patients, who were not selected for anovulation but included a majority with ultrasound-demonstrated polycystic ovaries, also found that GH reduced hMG requirement (779). Two other studies, in hypogonadotropic women with polycystic ovaries (802) or clomiphene-resistant women with PCOS (803) undergoing ovulation induction with GnRHa and hMG, found an improvement in hMG response with GH cotreatment. A large, multicenter placebo-controlled study of 64 hypogonadotropic, anovulatory women confirmed that GH decreases the total hMG requirement in a fashion dependent on GH dose, but GH lowered pregnancy rates (804).

In contrast to these results in anovulatory women, studies in ovulatory women undergoing hyperstimulation for IVF have largely failed to show a benefit of adjunctive GH. The largest group of these studies examined poor responders to GnRHa-down-regulated hMG stimulation cycles (779, 784, 789, 805, 806, 807, 808). These studies administered GH, typically in a dose of 12 IU on alternate days, concurrently with hMG until adequate follicular maturation was achieved. None demonstrated a statistically significant improvement in pregnancy rate with GH. Of the four studies that employed a double-blind, placebo-controlled design, two (789, 808) found no benefit of GH on cycle stimulation parameters, while two others (779, 784) found a significant improvement with GH only in the fertilization rate. Two studies, however, did note a decreased hMG requirement with GH (779, 805).

GH has also been studied as an adjunct to short (flare) GnRHa-hMG regimens for stimulation before IVF. In a placebo-controlled study of poor responders receiving conventional leuprolide doses, no benefit of GH was found (785). In an open-label study of GH in a microdose (40 µg twice daily) leuprolide flare regimen, follicular development was found to be superior with GH and cycle cancellation avoided in patients whose previous long GnRHa cycles had been canceled (809). In normal responders to hMG or unselected women, no effect of GH was seen on follicular response, oocyte or embryo quality or number, or pregnancy rate in four studies of women undergoing GnRHa-down-regulated hMG treatment for IVF (783, 786, 788, 806).

Given the suggestion of a beneficial effect of GH in some women, particularly those with impaired ovulation, several approaches have been taken to identify candidates for adjunctive GH. In two placebo-controlled studies, women with polycystic ovarian morphology undergoing IVF showed significant improvement with GH in numbers of follicles, oocytes collected, and oocytes fertilized (779, 780). In another approach, blunted responses to provocative tests for GH secretion, which may indicate occult or borderline GH deficiency, have been used to select patients for GH treatment. Anovulatory, nonobese women with decreased GH reserve on a clonidine provocative test showed a 30% lower hMG requirement when given adjunctive GH (792). In another study, conception rates in a mixed IVF/in vivo fertilization population were increased by GH in clonidine-nonresponsive, but not in clonidine-responsive, subjects (810). Clonidine-negative women may have dysovulatory features similar to those of PCOS, which has been associated with decreased basal GH levels as well as decreased responsiveness to clonidine and L-DOPA (168, 699, 798, 811).

The extensive literature on the use of adjunctive GH in hMG-based ovulation induction regimens has failed to demonstrate the general clinical utility of GH, despite the evidence for an involvement of the GH/IGF-I axis in ovarian follicular function. As noted above, many of the studies had small numbers of subjects, lacked placebo controls, used inconsistent protocols of GH administration, and lacked uniform definitions of "poor responders." These features make comparisons of these studies difficult. At present, given its cost, in our opinion the use of adjunctive GH in all ovulation induction protocols is not warranted. Further large-scale randomized, double-blind clinical trials, which could help determine parameters that allow selection of those patients who will benefit from GH use in ovulation reduction protocols should be conducted.

	VII. Summary and Conclusions

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 
In summary, the ovarian insulin-related regulatory system consists of insulin, insulin receptors, IGF-I, IGF-II, type I IGF receptors, type II IGF receptors, IGFBPs 1–5, and IGFBP proteases. There is evidence that the components of this system interact in a complex way (Fig. 7). The insulin-related ovarian regulatory system appears to participate in the regulation of normal ovarian function, including initiation of puberty and ovulation, and its components are altered in certain pathological states, which include type 1 and type 2 diabetes mellitus, obesity, reproductive abnormalities associated with weight loss and starvation, PCOS, and states of extreme insulin resistance. Therapeutic approaches directed toward normalization of the components of this system appear to be promising in some of these diseases.

View larger version (31K): [in this window] [in a new window]   Figure 7. The relationships among the components of the insulin-related ovarian regulatory system. Insulin, IGF-I, and IGF-II, acting through insulin receptors or type I IGF receptors, increase pituitary responsiveness to GnRH; stimulate gonadotropin secretion directly; stimulate ovarian steroidogenesis; inhibit IGFBP-1 and SHBG production; and act synergistically with gonadotropins to promote ovarian growth and cyst formation (see also Tables 3–5).

	Acknowledgments

 
The authors thank Dr. Steven Spandorfer, Ms. Ming Su, and Ms. Rita Falbel for their assistance in the preparation of the manuscript. The authors apologize to the many investigators who have contributed to the reviewed field but whose work was not cited. Because of the vast literature on the subject, review articles were sometimes quoted instead of the original reports.

	Footnotes

 
Address reprint requests to: Leonid Poretsky, MD, New York Presbyterian Hospital and Weill Medical College of Cornell University, 525 East 68th Street, New York, New York 10021 USA.

	References

Top Abstract I. Introduction II. Insulin and Insulin... III. IGFs and Their... IV. IGF-Binding Proteins... V. Polycystic Ovary Syndrome... VI. The Insulin-Related Ovarian... VII. Summary and Conclusions References

 

1. Shuldiner AR, Barbetti F, Raben N, Scavo L, Serrano J 1998 Insulin. In: LeRoith D (ed) Insulin-like Growth Factors: Molecular and Cellular Aspects. CRC Press, Boca Raton, FL, pp 181–219
2. White MF, Kahn CR 1993 Mechanisms of insulin action. In: Moller DE (ed) Insulin Resistance. John Wiley & Sons, New York, pp 9–47
3. Poretsky L, Kalin M 1987 The gonadotropic function of insulin. Endocr Rev 8:132–141[Abstract/Free Full Text]
4. Joslin EP, Root HF, White P 1925 The growth, development and prognosis of diabetic children. JAMA 85:420–422[Abstract/Free Full Text]
5. Bliss M 1985 The discovery of insulin: how it really happened. In: Hollenberg MD (ed) Insulin, Its Receptor and Diabetes. Marcel Dekker, Inc., New York, pp 7–19
6. Kahn CR, Flier JS, Bar RS, Archer JA, Gorden P, Martin MM, Roth J 1976 The syndromes of insulin resistance and acanthosis nigricans: insulin-receptor disorders in man. N Engl J Med 294:739–745[Abstract]
7. Flier JS, Kahn CR, Roth J, Bar RS 1975 Antibodies that impair insulin receptor binding in an unusual diabetic syndrome with severe insulin resistance. Science 190:63–65[Abstract/Free Full Text]
8. Taylor SI, Dons RF, Hernandez E, Roth J, Gorden P 1982 Insulin resistance associated with androgen excess in women with autoantibodies to the insulin receptor. Ann Intern Med 97:851–855
9. Poretsky L 1991 On the paradox of insulin-induced hyperandrogenism in insulin-resistant states. Endocr Rev 12:3–13[Abstract/Free Full Text]
10. Barbieri RL, Makris A, Ryan KJ 1983 Effects of insulin on steroidogenesis in cultured porcine ovarian theca. Fertil Steril 40:237–241[Medline]
11. Poretsky L, Smith D, Seibel M, Pazianos A, Moses AC, Flier JS 1984 Specific insulin binding sites in the human ovary. J Clin Endocrinol Metab 59:809–811[Abstract/Free Full Text]
12. Poretsky L, Grigorescu F, Seibel M, Moses AC, Flier JS 1985 Distribution and characterization of the insulin and IGF-I receptors in normal human ovary. J Clin Endocrinol Metab 61:728–734[Abstract/Free Full Text]
13. Adashi EY, Resnick CE, D’Ercole AJ, Svoboda ME, Van Wyk JJ 1985 Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr Rev 6:400–420[Abstract/Free Full Text]
14. Giudice LC 1992 Insulin-like growth factors and ovarian follicular development. Endocr Rev 13:641–669[Abstract/Free Full Text]
15. Hammond JM, Baranao JL, Skaleris D, Knight AB, Romanus JA, Rechler MM 1985 Production of insulin-like growth factors by ovarian granulosa cells. Endocrinology 117:2553–2555[Abstract/Free Full Text]
16. Gharib SD, Wierman ME, Shupnik MA, Chin WW 1990 Molecular biology of the pituitary gonadotropins. Endocr Rev 11:177–199[Abstract/Free Full Text]
17. Richards JS 1994 Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751[Abstract/Free Full Text]
18. Adashi EY 1995 Editorial: With a little help from my friends–the evolving story of intraovarian regulation. Endocrinology 136:4161–4162[CrossRef][Medline]
19. Adashi EY 1994 Growth factors and ovarian function: the IGF-I paradigm. Horm Res 42:44–48[Medline]
20. Hammond JM, Mondschein JS, Samaras SE, Canning SF 1991 The ovarian insulin-like growth factors, a local amplification mechanism for steroidogenesis and hormone action. J Steroid Biochem Mol Biol 40:411–416[CrossRef][Medline]
21. Cara JF 1996 Mechanisms subserving the action of insulin and IGFs on androgen production by the ovary. In: LeRoith D (ed) The Role of Insulin-like Factors in Ovarian Physiology. Ares Serono Symposia, Rome, pp 153–163
22. Stewart CE, Rotwein P 1996 Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol Rev 76:1005–1026[Abstract/Free Full Text]
23. Erickson GF, Danforth DR 1995 Ovarian control of follicle development. Am J Obstet Gynecol 172:736–747[CrossRef][Medline]
24. Barbieri RL, Smith S, Ryan KJ 1988 The role of hyperinsulinemia in the pathogenesis of ovarian hyperandrogenism. Fertil Steril 50:197–212[Medline]
25. Geffner ME, Golde DW 1988 Selective insulin action on skin, ovary and heart in insulin-resistant states. Diabetes Care 11:500–505[Abstract]
26. Nestler JE 1997 Role of hyperinsulinemia in the pathogenesis of the polycystic ovary syndrome, and its clinical implications. Semin Reprod Endocrinol 15:111–122[Medline]
27. Poretsky L 1994 Role of insulin resistance in the pathogenesis of the polycystic ovaries syndrome. In: Schats R, Schoemaker J (eds) Ovarian Endocrinopathies. Parthenon Publishing, London, pp 169–177
28. Poretsky L 1994 Insulin resistance and hyperandrogenism: update 1994. In: Negro-Vilar A, Underwood LE (eds) The Endocrine Pancreas, Insulin Action and Diabetes. Endocrine Society Press, Bethesda, MD, pp 125–129
29. Nestler JE, Jakubowicz DJ 1996 Decreases in ovarian cytochrome P450c17 activity and serum free testosterone after reduction of insulin secretion in polycystic ovary syndrome. N Engl J Med 335:617–623[Abstract/Free Full Text]
30. Nestler JE, Barlascini CO, Matt DW, Steingold KA, Plymate SR, Clore JN, Blackard WG 1989 Suppression of serum insulin by diazoxide reduces serum testosterone levels in obese women with polycystic ovary syndrome. J Clin Endocrinol Metab 68:1027–1032[Abstract/Free Full Text]
31. Velazquez EM, Mendoza S, Hamer T, Sosa F, Glueck CJ 1994 Metformin therapy in polycystic ovary syndrome reduces hyperinsulinemia, insulin resistance, hyperandrogenemia, and systolic blood pressure, while facilitating normal menses and pregnancy. Metabolism 43:647–654[CrossRef][Medline]
32. Utiger RD 1996 Insulin and the polycystic ovary syndrome. N Engl J Med 335:657–658[Free Full Text]
33. van Montfrans JM, van Hooff MH, Hompes PG, Lambalk CB 1998 Treatment of hyperinsulinaemia in polycystic ovary syndrome. Hum Reprod 13:5–6[Free Full Text]
34. Prelevic GM, Wurzburger MI, Balint-Peric L, Hardiman P, Okolo S, Maletic D, Ginsburg J 1992 Effects of the somatostatin analogue, octreotide, in polycystic ovary syndrome. Metabolism 41:76–79[Medline]
35. Dunaif A, Scott D, Finegood D, Quintana B, Whitcomb R 1996 The insulin-sensitizing agent troglitazone improves metabolic and reproductive abnormalities in the polycystic ovary syndrome. J Clin Endocrinol Metab 81:3299–3306[Abstract]
36. Ehrmann DA, Schneider DJ, Sobel BE, Cavaghan MK, Imperial J, Rosenfield RL, Polonsky KS 1997 Troglitazone improves defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis in women with polycystic ovary syndrome. J Clin Endocrinol Metab 82:2108–2116[Abstract/Free Full Text]
37. Lanzone A, Fulghesu AM, Cucinelli F, Ciampelli M, Caruso A, Mancuso S 1995 Evidence of a distinct derangement of opioid tone in hyperinsulinemic patients with polycystic ovarian syndrome: relationship with insulin and luteinizing hormone secretion. J Clin Endocrinol Metab 80:3501–3506[Abstract]
38. Roozenburg BJ, Van Dessel HJ, Evers JL, Bots RS 1997 Successful induction of ovulation in normogonadotrophic clomiphene resistant anovulatory women by combined naltrexone and clomiphene citrate treatment. Hum Reprod 12:1720–1722[Abstract/Free Full Text]
39. Dumonteil E, Philippe J 1996 Insulin gene: organization, expression and regulation. Diabet Med 22:164–173
40. Draznin B 1996 Editorial: Insulin signaling network–waiting for Copernicus. Endocrinology 137:2647–2648[CrossRef][Medline]
41. Taylor SI, Najjar S, Cama A, Accili D 1991 Structure and function of the insulin receptor. In: LeRoith D (ed) Insulin-Like Growth Factors: Molecular and Cellular Aspects. CRC Press, Boca Raton, FL, pp 221–244
42. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[Abstract/Free Full Text]
43. Rosen OM 1987 After insulin binds. Science 237:1452–1458[Abstract/Free Full Text]
44. Seino S, Seino M, Bell GI 1990 Human insulin-receptor gene. Diabetes 39:129–133[Abstract]
45. Seino S, Seino M, Bell GI 1990 Human insulin-receptor gene. Partial sequence and amplification of exons by polymerase chain reaction. Diabetes 39:123–128[Abstract]
46. Huang Z, Bodkin NL, Ortmeyer HK, Zenilman ME, Webster NJG, Hansen BC, Shuldiner AR 1996 Altered insulin receptor messenger ribonucleic acid splicing in liver is associated with deterioration of glucose tolerance in the spontaneously obese and diabetic Rhesus monkey: analysis of controversy between monkey and human studies. J Clin Endocrinol Metab 81:1552–1556[Abstract]
47. Hansen T, Bjorbaek C, Vestergaard H, Gronskov K, Bak JF, Pedersen O 1993 Expression of insulin receptor spliced variants and their functional correlates in muscle from patients with non-insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 77:1500–1505[Abstract]
48. Kasuga M, Karlsson FA, Kahn CR 1982 Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. Science 215:185–187[Abstract/Free Full Text]
49. Cheatham B, Kahn CR 1996 The biochemistry of insulin action. In: LeRoith D, Taylor SI, Olefsky JM (eds) Diabetes Mellitus: A Fundamental and Clinical Text. Lippincott-Raven, Philadelphia, pp 139–147
50. White MF 1997 The insulin signalling system and the IRS proteins. Diabetologia 40[Suppl 2]:S2–17
51. Smith-Hall J, Pons S, Patti ME, Burks DJ, Yenush L, Sun XJ, Kahn CR, White MF 1997 The 60 kDa insulin receptor substrate functions like an IRS protein (pp60, IRS-3) in adipose cells. Biochemistry 36:8304–8310[CrossRef][Medline]
52. Zhou L, Chen H, Lin CH, Cong LN, McGibbon MA, Sciacchitano S, Lesniak MA, Quon MJ, Taylor SI 1997 Insulin receptor substrate-2 (IRS-2) can mediate the action of insulin to stimulate translocation of GLUT4 to the cell surface in rat adipose cells. J Biol Chem 272:29829–29833[Abstract/Free Full Text]
53. Sciacchitano S, Taylor SI 1997 Cloning, tissue expression, and chromosomal localization of the mouse IRS-3 gene. Endocrinology 138:4931–4940[Abstract/Free Full Text]
54. Lavan BE, Fantin VR, Chang ET, Lane WS, Keller SR, Lienhard GE 1997 A novel 160-kDa phosphotyrosine protein in insulin-treated embryonic kidney cells is a new member of the insulin receptor substrate family. J Biol Chem 272:21403–21407[Abstract/Free Full Text]
55. Lavan BE, Lane WS, Lienhard GE 1997 The 60-kDa phosphotyrosine protein in insulin-treated adipocytes is a new member of the insulin receptor substrate family. J Biol Chem 272:11439–11443[Abstract/Free Full Text]
56. Sun XJ, Pons S, Wang LM, Zhang Y, Yenush L, Burks D, Myers Jr MG, Glasheen E, Copeland NG, Jenkins NA, Pierce JH, White MF 1997 The IRS-2 gene on murine chromosome 8 encodes a unique signaling adapter for insulin and cytokine action. Mol Endocrinol 11:251–262[Abstract/Free Full Text]
57. Valverde AM, Lorenzo M, Pons S, White MF, Benito M 1998 Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes. Mol Endocrinol 12:688–697[Abstract/Free Full Text]
58. Ohan N, Bayaa M, Kumar P, Zhu L, Liu XJ 1998 A novel insulin receptor substrate protein, xIRS-u, potentiates insulin signaling: functional importance of its pleckstrin homology domain. Mol Endocrinol 12:1086–1098[Abstract/Free Full Text]
59. White MF 1996 The role of IRS-1 during insulin signaling. In: LeRoith D, Taylor SI, Olefsky JM (eds) Diabetes Mellitus: A Fundamental and Clinical Text. Lippincott-Raven, Philadelphia, pp 154–160
60. Imai Y, Philippe N, Sesti G, Accili D, Taylor SI 1997 Expression of variant forms of insulin receptor substrate-1 identified in patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 82:4201–4207[Abstract/Free Full Text]
61. Armstrong M, Haldane F, Taylor RW, Humphriss D, Berrish T, Stewart MW, Turnbull DM, Alberti KG, Walker M 1996 Human insulin receptor substrate-1: variant sequences in familial non-insulin-dependent diabetes mellitus. Diabet Med 13:133–138[CrossRef][Medline]
62. Withers DJ, Gutierrez JS, Towery H, Burks DJ, Ren JM, Previs S, Zhang Y, Bernal D, Pons S, Shulman GI, Bonner-Weir S, White MF 1998 Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391:900–904[CrossRef][Medline]
63. Patti M, Kahn RC 1997 Transgenic animal models: insights into the pathophysiology of NIDDM. Diabetes Rev 5:149–164
64. Grigorescu F, Macari F, Renard E, Bringer J, Jaffiol C, High frequency of Gly972 to Arg variant of IRS-1 gene in women with polycystic ovary syndrome and insulin resistance. Program of the 58th Annual Meeting of the Diabetes Association, Chicago, IL, 1998 (Abstract 969)
65. Yamada K, Yuan X, Ishiyama S, Shoji S, Kohno S, Koyama K-I, Koyanagi A, Koyama W, Nonaka K 1998 Codon 972 polymorphism of the insulin receptor substrate-1 gene in impaired glucose tolerance and late-onset NIDDM. Diabetes Care 21:753–756[Abstract]
66. Sakaue H, Ogawa W, Takata M, Kuroda S, Kotani K, Matsumoto M, Sakaue M, Nishio S, Ueno H, Kasuga M 1997 Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3–L1 adipocytes. Mol Endocrinol 11:1552–1562[Abstract/Free Full Text]
67. Lam K, Carpenter CL, Ruderman NB, Friel JC, Kelly KL 1994 The phosphatidylinositol 3-kinase serine kinase phosphorylates IRS-1: stimulation by insulin and Wortmannin. J Biol Chem 269:20648–20652[Abstract/Free Full Text]
68. Norman BH, Shih C, Toth JE, Ray JE, Dodge JA, Johnson DW, Rutherford PG, Schultz RM, Worzalla JF, Vlahos CJ 1996 Studies on the mechanism of phosphatidylinositol 3-kinase inhibition by Wortmannin and related analogs. J Med Chem 39:1106–1111[CrossRef][Medline]
69. Tartare-Deckert S, Murdaca J, Sawka-Verhelle D, Holt KH, Pessin JE, Van Obberghen E 1996 Interaction of the molecular weight 85K regulatory subunit of the phosphatidylinositol 3-kinase with the insulin receptor and the insulin-like growth factor-I (IGF-I) receptor: comparative study using the yeast two-hybrid system. Endocrinology 137:1019–1024[Abstract]
70. Antoine PJ, Bertrand F, Auclair M, Magre J, Capeau J, Cherqui G 1998 Insulin induction of protein kinase C expression is independent of insulin receptor Tyr1162/1163 residues and involves mitogen-activated protein kinase kinase 1 and sustained activation of nuclear p44MAPK. Endocrinology 139:3133–3142[Abstract/Free Full Text]
71. Mastick CC, Kato H, Roberts Jr CT, Le Roith D, Saltiel AR 1994 Insulin and insulin-like growth factor-I receptors similarly stimulate deoxyribonucleic acid synthesis despite differences in cellular protein tyrosine phosphorylation. Endocrinology 135:214–222[Abstract]
72. Suga J, Yoshimasa Y, Yamada K, Yamamoto Y, Inoue G, Okamoto M, Hayashi T, Shigemoto M, Kosaki A, Kuzuya H, Nakao K 1997 Differential activation of mitogen-activated protein kinase by insulin and epidermal growth factor in 3T3–L1 adipocytes: a possible involvement of PI3-kinase in the activation of the MAP kinase by insulin. Diabetes 46:735–741[Abstract]
73. Morgan DO, Roth RA 1987 Acute insulin action requires insulin receptor kinase activity: introduction of an inhibitory monoclonal antibody into mammalian cells blocks the rapid effects of insulin. Proc Natl Acad Sci USA 84:41–45[Abstract/Free Full Text]
74. Cama A, Sierra ML, Ottini L, Kadowaki T, Gorden P, Imperato-McGinley J, Taylor SI 1991 A mutation in the tyrosine kinase domain of the insulin receptor associated with insulin resistance in an obese woman. J Clin Endocrinol Metab 73:894–901[Abstract/Free Full Text]
75. Chang PY, Goodyear LJ, Benecke H, Markuns JS, Moller DE 1995 Impaired insulin signaling in skeletal muscles from transgenic mice expressing kinase-deficient insulin receptors. J Biol Chem 270:12593–12600[Abstract/Free Full Text]
76. Chang P-Y, Benecke H, Marchand-Brustel YL, Lawitts J, Moller DE 1994 Expression of a dominant-negative mutant human insulin receptor in the muscle of transgenic mice. J Biol Chem 269:16034–16040[Abstract/Free Full Text]
77. Saltiel AR 1990 Second messengers of insulin action. Diabetes Care 13:244–256[Abstract]
78. Galasko GTF, Abe S, Lilley K, Zhang C, Larner J 1996 Circulating factors and insulin resistance. II. The action of the novel myo-inositol cyclic 1,2-inositol phosphate phosphoglycan insulin antagonist from human plasma in regulating pyruvate dehydrogenase phosphatase. J Clin Endocrinol Metab 81:1051–1057[Abstract]
79. Nestler JE, Jakubowicz DJ, De Vargas AF, Brik C, Quintero N, Medina F 1998 Insulin stimulates testosterone biosynthesis by human thecal cells from women with polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system. J Clin Endocrinol Metab 83:2001–2005[Abstract/Free Full Text]
80. Nestler JE, Romero G, Huang LC, Zhang F, Larner J 1991 Insulin mediators are the signal transduction system responsible for insulin’s actions on human placental steroidogenesis. Endocrinology 129:2951–2956[Abstract/Free Full Text]
81. Mueckler M 1990 Family of glucose-transporter genes: implications for glucose homeostasis and diabetes. Diabetes 39:6–11[Abstract]
82. Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G 1997 Daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277:942–946[Abstract/Free Full Text]
83. Chen C, Jack J, Garofalo RS 1996 The Drosophila insulin receptor is required for normal growth. Endocrinology 137:846–856[Abstract]
84. Graf R, Neuenschwander S, Brown MR, Ackermann U 1997 Insulin-mediated secretion of ecdysteroids from mosquito ovaries and molecular cloning of the insulin receptor homologue from ovaries of bloodfed Aedes aegypti. Insect Mol Biol 6:151–163[Medline]
85. Joshi RL, Lamothe B, Cordonnier N, Mesbah K, Monthioux E, Jami J, Bucchini D 1996 Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. EMBO J 15:1542–1547[Medline]
86. Dunaif A 1992 Diabetes mellitus and polycystic ovary syndrome. In: Dunaif A, Givens JR, Haseltine FP, Merriam GR (eds) Polycystic Ovary Syndrome. Blackwell Scientific Publications, Boston, pp 347–358
87. Diamond MP, Webster BW, Carr RK, Wentz AC, Osteen KG 1985 Human follicular fluid insulin concentrations. J Clin Endocrinol Metab 61:990–992[Abstract/Free Full Text]
88. El-Roeiy A, Chen X, Roberts VJ, LeRoith D, Roberts Jr CT, Yen SS 1993 Expression of insulin-like growth factor-I (IGF-I) and IGF-II and the IGF-I, IGF-II, and insulin receptor genes and localization of the gene products in the human ovary. J Clin Endocrinol Metab 77:1411–1418[Abstract]
89. El-Roeiy A, Chen X, Roberts VJ, Shimasaki S, Ling N, LeRoith D, Roberts Jr CT, Yen SS 1994 Expression of the genes encoding the insulin-like growth factors (IGF-I and II), the IGF and insulin receptors, and IGF-binding proteins-1–6 and the localization of their gene products in normal and polycystic ovary syndrome ovaries. J Clin Endocrinol Metab 78:1488–1496[Abstract]
90. Hernandez ER, Hurwitz A, Vera A, Pellicer A, Adashi EY, LeRoith D 1992 Expression of the genes encoding the insulin-like growth factors and their receptors in the human ovary. J Clin Endocrinol Metab 74:419–425[Abstract]
91. Samoto T, Maruo T, Ladines-Llave CA, Matsuo H, Deguchi J, Barnea ER, Mochizuki M 1993 Insulin receptor expression in follicular and stromal compartments of the human ovary over the course of follicular growth, regression and atresia. Endocr J 40:715–726[Medline]
92. Poretsky L, Bhargava G, Kalin MF, Wolf SA 1988 Regulation of insulin receptors in the human ovary: in vitro studies. J Clin Endocrinol Metab 67:774–778[Abstract/Free Full Text]
93. Poretsky L, Bhargava G, Saketos M, Dunaif A 1990 Regulation of human ovarian insulin receptors in vivo. Metabolism 39:161–166[CrossRef][Medline]
94. Poretsky L, Glover B, Laumas V, Kalin M, Dunaif A 1988 The effects of experimental hyperinsulinemia on steroid secretion, ovarian [¹²⁵I] insulin binding, and ovarian [¹²⁵I] insulin-like growth factor I binding in the rat. Endocrinology 122:581–585[Abstract/Free Full Text]
95. Samoto T, Maruo T, Matsuo H, Katayama K, Barnea ER, Mochizuki M 1993 Altered expression of insulin and insulin-like growth factor-I receptors in follicular and stromal compartments of polycystic ovaries. Endocr J 40:413–424[Medline]
96. Willis D, Mason H, Gilling-Smith C, Franks S 1996 Modulation by insulin of follicle-stimulating hormone and luteinizing hormone actions in human granulosa cells of normal and polycystic ovaries. J Clin Endocrinol Metab 81:302–309[Abstract]
97. Willis D, Franks S 1995 Insulin action in human granulosa cells from normal and polycystic ovaries is mediated by the insulin receptor and not the type-I insulin-like growth factor receptor. J Clin Endocrinol Metab 80:3788–3790[Abstract]
98. Barbieri RL 1994 Insulin stimulates androgen accumulation in incubations of minced porcine theca. Gynecol Obstet Invest 37:265–269[CrossRef][Medline]
99. Andreani CL, Pierro E, Lanzone A, Lazzarin N, Capitanio G, Giannini P, Mancuso S 1994 Effect of gonadotropins, insulin and IGF I on granulosa luteal cells from polycystic ovaries. Mol Cell Endocrinol 106:91–97[CrossRef][Medline]
100. Bergh C, Carlsson B, Olsson JH, Selleskog U, Hillensjo T 1993 Regulation of androgen production in cultured human thecal cells by insulin-like growth factor I and insulin. Fertil Steril 59:323–331[Medline]
101. McGee EA, Sawetawan C, Bird I, Rainey WE, Carr BR 1996 The effect of insulin and insulin-like growth factors on the expression of steroidogenic enzymes in a human ovarian thecal-like tumor cell model. Fertil Steril 65:87–93[Medline]
102. Garzo VG, Dorrington JH 1984 Aromatase activity in human granulosa cells during follicular development and the modulation by follicle-stimulating hormone and insulin. Am J Obstet Gynecol 148:657–662[Medline]
103. Mason HD, Willis DS, Beard RW, Winston RML, Margara R, Franks S 1994 Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and concentrations of gonadotropins and sex steroids in follicular fluid. J Clin Endocrinol Metab 79:1355–1360[Abstract]
104. Andreani CL, Lazzarin N, Pierro E, Lanzone A, Mancuso S 1995 Somatostatin action on rat ovarian steroidogenesis. Hum Reprod 10:1968–1973[Abstract/Free Full Text]
105. Pierro E, Andreani CL, Lazzarin N, Cento R, Lanzone A, Caruso A, Mancuso S 1997 Further evidence of increased aromatase activity in granulosa luteal cells from polycystic ovary. Hum Reprod 12:1890–1896[Abstract/Free Full Text]
106. McGee E, Sawetawan C, Bird I, Rainey WE, Carr BR 1995 The effects of insulin on 3ß-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells. J Soc Gynecol Invest 2:535–541[CrossRef][Medline]
107. Ehrmann DA, Rosenfield RL, Barnes RB, Brigell DF, Sheikh Z 1992 Detection of functional ovarian hyperandrogenism in women with androgen excess. N Engl J Med 327:157–162[Abstract]
108. Nestler JE, Jakubowicz DJ 1997 Lean women with polycystic ovary syndrome respond to insulin reduction with decreases in ovarian P450c17 activity and serum androgens. J Clin Endocrinol Metab 82:4075–4079[Abstract/Free Full Text]
109. McAllister JM, Byrd W, Simpson ER 1994 The effects of growth factors and phorbol esters on steroid biosynthesis in isolated human theca interna and granulosa-lutein cells in long term culture. J Clin Endocrinol Metab 79:106–112[Abstract]
110. Sahin Y, Ayata D, Kelestimur F 1997 Lack of relationship between 17-hydroxyprogesterone response to buserelin testing and hyperinsulinemia in polycystic ovary syndrome. Eur J Endocrinol 136:410–415[Abstract/Free Full Text]
111. Flores JA, Garmey JC, Nestler JE, Veldhuis JD 1993 Sites of inhibition of steroidogenesis by activation of protein kinase-C in swine ovarian (granulosa) cells. Endocrinology 132:1983–1990[Abstract/Free Full Text]
112. Srivastava RK, Van der Kraak G 1994 Insulin as an amplifier of gonadotropin action on steroid production: mechanisms and sites of action in goldfish prematurational full-grown ovarian follicles. Gen Comp Endocrinol 95:60–70[CrossRef][Medline]
113. Nestler JE, Clore JN, Blackard WG 1992 Effects of insulin on steroidogenesis in vivo. In: Dunaif A, Givens JR, Haseltine FP, Merriam GR (eds) Polycystic Ovary Syndrome. Blackwell Scientific Publications, Boston, pp 265–278
114. Dunaif A, Graf M 1989 Insulin administration alters gonadal steroid metabolism independent of changes in gonadotropin secretion in insulin-resistant women with the polycystic ovary syndrome. J Clin Invest 83:23–29
115. Fulghesu AM, Villa P, Pavone V, Guido M, Apa R, Caruso A, Lanzone A, Rossodivita A, Mancuso S 1997 The impact of insulin secretion on the ovarian response to exogenous gonadotropins in polycystic ovary syndrome. J Clin Endocrinol Metab 82:644–648[Abstract/Free Full Text]
116. Stamataki KE, Spina J, Rangou DB, Chlouverakis CS, Piaditis GP 1996 Ovarian function in women with non-insulin dependent diabetes mellitus. Clin Endocrinol (Oxf) 45:615–621[CrossRef][Medline]
117. Stuart CA, Nagamani M 1992 Acute augmentation of plasma androstenedione and dehydroepiandrosterone by euglycemic insulin infusion: evidence for a direct effect of insulin on ovarian steroidogenesis. In: Dunaif A, Givens JR, Haseltine FP, Merriam GR (eds) Polycystic Ovary Syndrome. Blackwell Scientific Publications, Boston, pp 279–288
118. Stuart CA, Prince MJ, Peters EJ, Meyer WJ 1987 Hyperinsulinemia and hyperandrogenemia: in vivo androgen response to insulin infusion. Obstet Gynecol 69:921–925[Medline]
119. Burghen GA, Givens JR, Kitabchi AE 1980 Correlation of hyperandrogenism with hyperinsulinism in polycystic ovarian disease. J Clin Endocrinol Metab 50:113–116[Abstract/Free Full Text]
120. Chang RJ, Nakamura RM, Judd HL, Kaplan SA 1983 Insulin resistance in nonobese patients with polycystic ovarian disease. J Clin Endocrinol Metab 57:356–359[Abstract/Free Full Text]
121. Pasquali R, Casimirri F, Venturoli S, Paradisi R, Mattioli L, Capelli M, Melchionda N, Labo G 1983 Insulin resistance in patients with polycystic ovaries: its relationship to body weight and androgen levels. Acta Endocrinol (Copenh) 104:110–116[Abstract/Free Full Text]
122. Falcone T, Finegood DT, Fantus IG, Morris D 1990 Androgen response to endogenous insulin secretion during the frequently sampled intravenous glucose tolerance test in normal and hyperandrogenic women. J Clin Endocrinol Metab 71:1653–1657[Abstract/Free Full Text]
123. Toscano V, Bianchi P, Balducci R, Guglielmi R, Mangiantini A, Lubrano C, Sciarra F 1992 Lack of linear relationship between hyperinsulinaemia and hyperandrogenism. Clin Endocrinol (Oxf) 36:197–202[Medline]
124. Anttila L, Ding YQ, Ruutiainen K, Erkkola R, Irjala K, Huhtaniemi I 1991 Clinical features and circulating gonadotropin, insulin, and androgen interactions in women with polycystic ovarian disease. Fertil Steril 55:1057–1061[Medline]
125. Buyalos RP, Geffner ME, Watanabe RM, Bergman RN, Gornbein JA, Judd HL 1993 The influence of luteinizing hormone and insulin on sex steroids and sex hormone-binding globulin in the polycystic ovarian syndrome. Fertil Steril 60:626–633[Medline]
126. Sharp PS, Kiddy DS, Reed MJ, Anyaoku V, Johnston DG, Franks S 1991 Correlation of plasma insulin and insulin-like growth factor-I with indices of androgen transport and metabolism in women with polycystic ovary syndrome. Clin Endocrinol (Oxf) 35:253–257[Medline]
127. Vidal-Puig A, Munoz-Torres M, Jodar-Gimeno E, Garcia-Calvente C, Lardelli P, Ruiz-Requena ME, Escobar-Jimenez F 1994 Hyperinsulinemia in polycystic ovary syndrome: relationship to clinical and hormonal factors. Clin Invest 72:853–857[Medline]
128. Stuart CA, Nagamani M 1990 Insulin infusion acutely augments ovarian androgen production in normal women. Fertil Steril 54:788–792[Medline]
129. Micic D, Popovic V, Nesovic M, Sumarac M, Dragasevic M, Kendereski A, Markovic D, Djordjevic P, Manojlovic D, Micic J 1988 Androgen levels during sequential insulin euglycemic clamp studies in patients with polycystic ovary disease. J Steroid Biochem 31:995–999[CrossRef][Medline]
130. Diamond MP, Grainger DA, Laudano AJ, Starick-Zych K, DeFronzo RA 1991 Effect of acute physiological elevations of insulin on circulating androgen levels in nonobese women. J Clin Endocrinol Metab 72:883–887[Abstract/Free Full Text]
131. Nestler JE, Clore JN, Strauss III JF, Blackard WG 1987 The effects of hyperinsulinemia on serum testosterone, progesterone, dehydroepiandrosterone sulfate, and cortisol levels in normal women and in a woman with hyperandrogenism, insulin resistance, and acanthosis nigricans. J Clin Endocrinol Metab 64:180–184[Abstract/Free Full Text]
132. Parra A, Godoy H, Ayala J, Ramirez A, Coria I, Espinosa de los Monteros A 1995 Opposite effects of breakfast vs. oral glucose on circulating androgen levels in healthy women. Arch Med Res 26:379–383[Medline]
133. Elkind-Hirsch KE, Valdes CT, McConnell TG, Malinak LR 1991 Androgen responses to acutely increased endogenous insulin levels in hyperandrogenic and normal cycling women. Fertil Steril 55:486–491[Medline]
134. Smith S, Ravnikar VA, Barbieri RL 1987 Androgen and insulin response to an oral glucose challenge in hyperandrogenic women. Fertil Steril 48:72–77[Medline]
135. Tiitinen A, Pekonen F, Stenman UH, Laatikainen T 1990 Plasma androgens and oestradiol during oral glucose tolerance test in patients with polycystic ovaries. Hum Reprod 5:242–245[Abstract/Free Full Text]
136. Tropeano G, Lucisano A, Liberale I, Barini A, Vuolo IP, Martino G, Menini E, Dell’Acqua S 1994 Insulin, C-peptide, androgens, and ß-endorphin response to oral glucose in patients with polycystic ovary syndrome. J Clin Endocrinol Metab 78:305–309[Abstract]
137. Anttila L, Koskinen P, Jaatinen TA, Erkkola R, Irjala K, Ruutiainen K 1993 Insulin hypersecretion together with high luteinizing hormone concentration augments androgen secretion in oral glucose tolerance test in women with polycystic ovarian disease. Hum Reprod 8:1179–1183[Abstract/Free Full Text]
138. Fox JH, Licholai T, Green G, Dunaif A 1993 Differential effects of oral glucose-mediated vs. intravenous hyperinsulinemia on circulating androgen levels in women. Fertil Steril 60:994–1000[Medline]
139. Nestler JE 1997 Suppression of ovarian androgen biosynthesis: reduction of circulating insulin. In: Azziz R, Nestler JE, Dewailly D (eds) Androgen Excess Disorders in Women. Lippincott-Raven, Philadelphia, pp 727–735
140. Dunaif A 1997 Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 18:774–800[Abstract/Free Full Text]
141. Krassas GE, Kaltsas TT, Pontikides N, Jacobs H, Blum W, Messinis I 1998 Leptin levels in women with polycystic ovary syndrome before and after treatment with diazoxide. Eur J Endocrinol 139:184–189[Abstract]
142. Fulghesu A, Lanzone A, Andreani CL, Pierro E, Caruso A, Mancuso S 1995 Effectiveness of a somatostatin analogue in lowering luteinizing hormone and insulin-stimulated secretion in hyperinsulinemic women with polycystic ovary disease. Fertil Steril 64:703–708[Medline]
143. Velazquez E, Acosta A, Mendoza SG 1997 Menstrual cyclicity after metformin therapy in polycystic ovary syndrome. Obstet Gynecol 90:392–395[CrossRef][Medline]
144. Velazquez EM, Mendoza S, Hamer T, Sosa F, Glueck CJ 1994 Metformin therapy in polycystic ovary syndrome reduces hyperinsulinemia, insulin resistance, hyperandrogenemia, and systolic blood pressure, while facilitating normal menses and pregnancy. Metabolism 43:647–654
145. Diamanti-Kandarakis E, Kouli C, Tsianateli T, Bergiele A 1998 Therapeutic effects of metformin on insulin resistance and hyperandrogenism in polycystic ovary syndrome. Eur J Endocrinol 138:269–274[Abstract]
146. Morin-Papunen LC, Koivunen RM, Ruokonen A, Martikainen HK 1998 Metformin therapy improves the menstrual pattern with minimal endocrine and metabolic effects in women with polycystic ovary syndrome. Fertil Steril 69:691–696[CrossRef][Medline]
147. Kiddy DS, Hamilton-Fairley D, Bush A, Short F, Anyaoku V, Reed MJ, Franks S 1992 Improvement in endocrine and ovarian function during dietary treatment of obese women with polycystic ovary syndrome. Clin Endocrinol (Oxf) 36:105–111[Medline]
148. Kopelman PG, White N, Pilkington TR, Jeffcoate SL 1981 The effect of weight loss on sex steroid secretion and binding in massively obese women. Clin Endocrinol (Oxf) 15:113–116[Medline]
149. Harlass FE, Plymate SR, Fariss BL, Belts RP 1984 Weight loss is associated with correction of gonadotropin and sex steroid abnormalities in the obese anovulatory female. Fertil Steril 42:649–652[Medline]
150. Bates GW, Whitworth NS 1982 Effect of body weight reduction on plasma androgens in obese, infertile women. Fertil Steril 38:406–409[Medline]
151. Pasquali R, Antenucci D, Casimirri F, Venturoli S, Paradisi R, Fabbri R, Balestra V, Melchionda N, Barbara L 1989 Clinical and hormonal characteristics of obese amenorrheic hyperandrogenic women before and after weight loss. J Clin Endocrinol Metab 68:173–179[Abstract/Free Full Text]
152. Guzick DS, Wing R, Smith D, Berga SL, Winters SJ 1994 Endocrine consequences of weight loss in obese, hyperandrogenic, anovulatory women. Fertil Steril 61:598–604[Medline]
153. Crave JC, Fimbel S, Lejeune H, Cugnardey N, Dechaud H, Pugeat M 1995 Effects of diet and metformin administration on sex hormone-binding globulin, androgens, and insulin in hirsute and obese women. J Clin Endocrinol Metab 80:2057–2062[Abstract]
154. Clark AM, Ledger W, Galletly C, Tomlinson L, Blaney F, Wang X, Norman RJ 1995 Weight loss results in significant improvement in pregnancy and ovulation rates in anovulatory obese women. Hum Reprod 10:2705–2712[Abstract/Free Full Text]
155. Holte J, Bergh T, Berne C, Wide L, Lithell H 1995 Restored insulin sensitivity but persistently increased early insulin secretion after weight loss in obese women with polycystic ovary syndrome. J Clin Endocrinol Metab 80:2586–2593[Abstract]
156. Jakubowicz DJ, Nestler JE 1997 17-Hydroxyprogesterone responses to leuprolide and serum androgens in obese women with and without polycystic ovary syndrome after dietary weight loss. J Clin Endocrinol Metab 82:556–560[Abstract/Free Full Text]
157. Poretsky L, Piper B 1994 Insulin resistance, hypersecretion of LH, and a dual-defect hypothesis for the pathogenesis of polycystic ovary syndrome. Obstet Gynecol 84:613–621[Medline]
158. Nahum R, Thong KJ, Hillier SG 1995 Metabolic regulation of androgen production by human thecal cells in vitro. Hum Reprod 10:75–81[Abstract/Free Full Text]
159. Caubo B, De Vinna RS, Tonetta SA 1989 Regulation of steroidogenesis in cultured porcine theca cells by growth factors. Endocrinology 125:321–326[Abstract/Free Full Text]
160. Cara JF, Rosenfield RL 1988 Insulin-like growth factor I and insulin potentiate luteinizing hormone-induced androgen synthesis. Endocrinology 123:733–739[Abstract/Free Full Text]
161. Magoffin DA, Erickson GF 1988 An improved method for primary culture of ovarian androgen-producing cells in serum-free medium: effect of lipoproteins, insulin, and insulin-like growth factor-I. In Vitro Cell Dev Biol 24:862–870[Medline]
162. Simone DA, Mahesh VB 1993 An autoregulatory process for androgen production in rat thecal-interstitial cells. Biol Reprod 48:46–56[Abstract]
163. Davoren JB, Hsueh AJ 1984 Insulin enhances FSH-stimulated steroidogenesis by cultured rat granulosa cells. Mol Cell Endocrinol 35:97–105[CrossRef][Medline]
164. Davoren JB, Kasson BG, Li CH, Hsueh AJ 1986 Specific insulin-like growth factor (IGF) I-binding sites on rat granulosa cells: relation to IGF action. Endocrinology 119:2155–2162[Abstract/Free Full Text]
165. Adashi EY, Hsueh AJW, Yen SSC 1981 Insulin enhancement of luteinizing hormone and follicle-stimulating hormone release by cultured pituitary cells. Endocrinology 108:1441–1449[Abstract/Free Full Text]
166. Soldani R, Cagnacci A, Yen SS 1994 Insulin, insulin-like growth factor I (IGF-I) and IGF-II enhance basal and gonadotrophin-releasing hormone-stimulated luteinizing hormone release from rat anterior pituitary cells in vitro. Eur J Endocrinol 131:641–645[Abstract/Free Full Text]
167. Arroyo A, Laughlin GA, Morales AJ, Yen SSC 1997 Inappropriate gonadotropin secretion in polycystic ovary syndrome: influence of adiposity. J Clin Endocrinol Metab 82:3728–3733[Abstract/Free Full Text]
168. Morales AJ, Laughlin GA, Butzow T, Maheshwari H, Baumann G, Yen SS 1996 Insulin, somatotropic and luteinizing hormone axes in lean and obese women with polycystic ovary syndrome: common and distinct features. J Clin Endocrinol Metab 81:2854–2864[Abstract/Free Full Text]
169. Taylor AE, McCourt B, Martin KA, Anderson EJ, Adams JM, Schoenfeld D, Hall JE 1997 Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab 82:2248–2256[Abstract/Free Full Text]
170. Poretsky L, Clemons J, Bogovich K 1992 Hyperinsulinemia and human chorionic gonadotropin synergistically promote the growth of ovarian follicular cysts in rats. Metabolism 41:903–910[CrossRef][Medline]
171. Duleba AJ, Spaczynski RZ, Olive DL, Behrman HR 1997 Effects of insulin and insulin-like growth factors on proliferation of rat ovarian theca-interstitial cells. Biol Reprod 56:891–897[Abstract]
172. Duleba AJ, Spaczynski RZ, Olive DL 1998 Insulin and insulin-like growth factor I stimulate the proliferation of human ovarian theca-interstitial cells. Fertil Steril 69:335–340[CrossRef][Medline]
173. Watson H, Willis D, Mason H, Modgil G, Wright C, Franks S, The effects of ovarian steroids, epidermal growth factor (EGF), insulin (I), and insulin-like growth factor-1 (IGF-1) on ovarian stromal cell growth. Program of the 79th Annual Meeting of The Endocrine Society, Minneapolis, MN, 1997 (Abstract 389)
174. De Clue TJ, Shah SC, Marchese M, Malone JI 1991 Insulin resistance and hyperinsulinemia induce hyperandrogenism in a young type B insulin-resistant female. J Clin Endocrinol Metab 72:1308–1311[Abstract/Free Full Text]
175. Pache TD, De Jong FH, Hop WC, Fauser BC 1993 Association between ovarian changes assessed by transvaginal sonography and clinical and endocrine signs of the polycystic ovary syndrome. Fertil Steril 59:544–549[Medline]
176. Markussis V, Goni NH, Tolis G 1994 The role of insulin in ovarian size in patients with the polycystic ovary syndrome. Gynecol Endocrinol 8:197–202[Medline]
177. Plymate SR, Matej LA, Jones RE, Friedl KE 1988 Inhibition of sex hormone-binding globulin production in the human hepatoma (HepG2) cell line by insulin and prolactin. J Clin Endocrinol Metab 67:460–464[Abstract/Free Full Text]
178. Peiris AN, Stagner JL, Plymate SR, Vogel RL, Heck M, Samols E 1993 Relationship of insulin secretory pulses to sex hormone-binding globulin in normal men. J Clin Endocrinol Metab 76:279–282[Abstract]
179. Fendri S, Arlot S, Marcelli JM, Dubreuil A, Lalau JD 1994 Relationship between insulin sensitivity and circulating sex hormone-binding globulin levels in hyperandrogenic obese women. Int J Obes Relat Metab Disord 18:755–759[Medline]
180. Nestler JE, Powers LP, Matt DW, Steingold KA, Plymate SR, Rittmaster RS, Clore JN, Blackard WG 1991 A direct effect of hyperinsulinemia on serum sex hormone-binding globulin levels in obese women with the polycystic ovary syndrome. J Clin Endocrinol Metab 72:83–89[Abstract/Free Full Text]
181. Nestler JE 1992 Editorial: Sex hormone-binding globulin: a marker for hyperinsulinemia and/or insulin resistance. J Clin Endocrinol Metab 76:273–274[CrossRef][Medline]
182. Preziosi P, Barrett-Connor E, Papoz L, Roger M, Saint-Paul M, Nahoul K, Simon D 1993 Interrelation between plasma sex hormone-binding globulin and plasma insulin in healthy adult women: the Telecom study. J Clin Endocrinol Metab 76:283–287[Abstract]
183. Hamilton-Fairley D, White D, Griffiths M, Anyaoku V, Koistinen R, Seppala M, Franks S 1995 Diurnal variation of sex hormone binding globulin and insulin-like growth factor binding protein-I in women with polycystic ovary syndrome. Clin Endocrinol (Oxf) 43:159–165[Medline]
184. Lindstedt G, Lundberg P, Lapidus L, Lundgren H, Bengtsson C, Bjorntorp P 1991 Low sex-hormone-binding globulin concentration as independent risk factor for development of NIDDM. Diabetes 40:123–128[Abstract]
185. Mogul HR, Marshall M, Frey M, Burke HB, Wynn PES, Wilker S, Southren AL, Gambert SR 1996 Insulin like growth factor-binding protein-1 as a marker for hyperinsulinemia in obese menopausal women. J Clin Endocrinol Metab 81:4492–4495[Abstract]
186. Travers SH, Labarta JI, Gargosky SE, Rosenfeld RG, Jeffers BW, Eckel RH 1998 Insulin-like growth factor binding protein-I levels are strongly associated with insulin sensitivity and obesity in early pubertal children. J Clin Endocrinol Metab 83:1935–1939[Abstract/Free Full Text]
187. Attia N, Tamborlane WV, Heptulla R, Maggs D, Grozman A, Sherwin RS, Caprio S 1998 The metabolic syndrome and insulin-like growth factor I regulation in adolescent obesity. J Clin Endocrinol Metab 83:1467–1471[Abstract/Free Full Text]
188. Suikkari AM, Ruutiainen K, Erkkola R, Seppala M 1989 Low levels of low molecular weight insulin-like growth factor-binding protein in patients with polycystic ovarian disease. Hum Reprod 4:136–139[Abstract/Free Full Text]
189. Suikkari AM, Koivisto VA, Rutanen EM, Yki-Jarvinen H, Karonen SL, Seppala M 1988 Insulin regulates the serum levels of low molecular weight insulin-like growth factor-binding protein. J Clin Endocrinol Metab 66:266–272[Abstract/Free Full Text]
190. Hamilton-Fairley D, Kiddy D, Anyaoku V, Koistinen R, Seppala M, Franks S 1993 Response of sex hormone binding globulin and insulin-like growth factor binding protein-1 to an oral glucose tolerance test in obese women with polycystic ovary syndrome before and after calorie restriction. Clin Endocrinol (Oxf) 39:363–367[Medline]
191. Apter D, Butzow T, Laughlin GA, Yen SS 1995 Metabolic features of polycystic ovary syndrome are found in adolescent girls with hyperandrogenism. J Clin Endocrinol Metab 80:2966–2973[Abstract/Free Full Text]
192. Buyalos RP, Pekonen F, Halme JK, Judd HL, Rutanen EM 1995 The relationship between circulating androgens, obesity, and hyperinsulinemia on serum insulin-like growth factor binding protein-1 in the polycystic ovarian syndrome. Am J Obstet Gynecol 172:932–939[CrossRef][Medline]
193. Pao CI, Farmer PK, Begovic S, Villafuerte BC, Wu G, Robertson DG, Phillips LS 1993 Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein I gene transcription by hormones and provision of amino acids in rat hepatocytes. Mol Endocrinol 7:1561–1568[Abstract/Free Full Text]
194. Lee PDK, Jensen MD, Divertie GD, Heiling VJ, Katz HH, Conover CA 1993 Insulin-like growth factor-binding protein-1 response to insulin during suppression of endogenous insulin secretion. Metabolism 42:409–414[CrossRef][Medline]
195. Orskov H, Wolthers T, Grofte T, Flyvbjerg A, Vilstrup H, Hamberg O 1994 Somatostatin-stimulated insulin-like growth factor binding protein-1 release is abolished by hyperinsulinemia. J Clin Endocrinol Metab 78:138–140[Abstract]
196. Singh A, Hamilton-Fairley D, Koistinen R, Seppala M, James VHT, Franks S, Reed MJ 1990 Effect of insulin-like growth factor-type I (IGF-I) and insulin on the secretion of sex hormone binding globulin and IGF binding protein-1 (IGFBP-1) by human hepatoma cells. J Endocrinol 124:R1–R3
197. Brismar K, Fernqvist-Forbes E, Wahren J, Hall K 1994 Effect of insulin on the hepatic production of insulin-like growth factor-binding protein-1 (IGFBP-1), IGFBP-3 and IGF-1 in insulin-dependent diabetes. J Clin Endocrinol Metab 79:872–878[Abstract]
198. Lee PD, Giudice LC, Conover CA, Powell DR 1997 Insulin-like growth factor binding protein-1: recent findings and new directions. Proc Soc Exp Biol Med 216:319–357[CrossRef][Medline]
199. Poretsky L, Chandrasekher YA, Bai C, Liu HC, Rosenwaks Z, Giudice L 1996 Insulin receptor mediates inhibitory effect of insulin, but not of insulin-like growth factor (IGF)-I, on IGF binding protein 1 (IGFBP-1) production in human granulosa cells. J Clin Endocrinol Metab 81:493–496[Abstract]
200. Chun SY, Billig H, Tilly JL, Furuta I, Tsafriri A, Hsueh AJW 1994 Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor-I. Endocrinology 135:1845–1853[Abstract]
201. Hsueh AJW, Billig H, Tsafriri A 1994 Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 15:707–724[Abstract/Free Full Text]
202. Morley JE 1998 Sex hormones and diabetes. Diabetes Rev 6:6–15
203. Rogers DG, Valdes CT, Elkind-Hirsch KE 1990 The effect of ovarian function on insulin-like growth factor I plasma levels and hepatic IGF-I mRNA. Diabetes Res Clin Pract 8:235–242[CrossRef][Medline]
204. Powers RW, Chambers C, Larsen WJ 1996 Diabetes-mediated decreases in ovarian superoxide dismutase activity are related to blood-follicle barrier and ovulation defects. Endocrinology 137:3101–3110[Abstract]
205. Griffin ML, South SA, Yankov VI, Booth Jr RA, Asplin CM, Veldhuis JD, Evans WS 1994 Insulin-dependent diabetes mellitus and menstrual dysfunction. Ann Med 25:331–340
206. Steger RW, Rabe MB 1997 The effect of diabetes mellitus on endocrine and reproductive function. Proc Soc Exp Biol Med 214:1–11[CrossRef][Medline]
207. Djursing H, Hagen C, Nyboe Andersen A, Svenstrup B, Bennett P, Molsted Pedersen L 1985 Serum sex hormone concentrations in insulin dependent diabetic women with and without amenorrhoea. Clin Endocrinol (Oxf) 23:147–154[Medline]
208. Zumoff B, Miller L, Poretsky L, Levit C, Miller E, Heinz U, Denman H, Jandorek R, Rosenfeld R 1990 Subnormal follicular-phase serum progesterone levels and elevated follicular-phase serum estradiol levels in young women with insulin-dependent diabetes. Steroids 55:560–564[CrossRef][Medline]
209. Adcock CJ, Perry LA, Lindsell DR, Taylor AM, Holly JMP, Jones J, Dunger DB 1994 Menstrual irregularities are more common in adolescents with type I diabetes: association with poor glycaemic control and weight gain. Diabetic Med 11:465–470[Medline]
210. Virdis R, Zampolli M, Street ME, Vanelli M, Potau N, Terzi C, Ghizzoni L, Ibanez L 1997 Ovarian 17-hydroxyprogesterone responses to GnRH analog testing in oligomenorrheic insulin-dependent diabetic adolescents. Eur J Endocrinol 136:624–629[Abstract/Free Full Text]
211. Andersson B, Marin P, Lissner L, Vermeulen A, Bjorntorp P 1994 Testosterone concentrations in women and men with NIDDM. Diabetes Care 17:405–411[Abstract]
212. Roumain J, Charles MA, De Courten MP, Hanson RL, Brodie TD, Pettitt DJ, Knowler WC 1998 The relationship of menstrual irregularity to type 2 diabetes in Pima Indian women. Diabetes Care 21:346–349[Abstract]
213. Adashi EY 1991 Insulin and related peptides in hyperandrogenism. Clin Obstet Gynecol 34:872–881[Medline]
214. Taylor SI, Moller DE 1993 Mutations of the insulin receptor gene. In: Moller DE (ed) Insulin Resistance. John Wiley & Sons, New York, pp 83–121
215. Krook A, Kumar S, Laing I, Boulton AJM, Wass JAH, O’Rahilly S 1994 Molecular scanning of the insulin receptor gene in syndromes of insulin resistance. Diabetes 43:357–367[Abstract]
216. Moller DE, Cohen O, Yamaguchi Y, Assiz R, Grigorescu F, Eberle A, Morrow LA, Moses AC, Flier JS 1994 Prevalence of mutations in the insulin receptor gene in subjects with features of the type A syndrome of insulin resistance. Diabetes 43:247–255[Abstract]
217. Desbois-Mouthon C, Sert-Langeron C, Magre J, Oreal E, Blivet MJ, Flori E, Besmond C, Capeau J, Caron M 1996 Deletion of Asn281 in the -subunit of the human insulin receptor causes constitutive activation of the receptor and insulin desensitization. J Clin Endocrinol Metab 81:719–727[Abstract]
218. Longo N, Langley SD, Griffin LD, Elsas LJ 1992 Reduced mRNA and a nonsense mutation in the insulin-receptor gene produce heritable severe insulin resistance. Am J Hum Genet 50:998–1007[Medline]
219. Flier JS, Moller DE, Moses AC, O’Rahilly S, Chaiken RL, Grigorescu F, Elahi D, Kahn BB, Weinreb JE, Eastman R 1993 Insulin-mediated pseudoacromegaly: clinical and biochemical characterization of a syndrome of selective insulin resistance. J Clin Endocrinol Metab 76:1533–1541[Abstract]
220. Grigorescu F, Flier JS, Kahn CR 1984 Defect in insulin receptor phosphorylation in erythrocytes and fibroblasts associated with severe insulin resistance. J Biol Chem 259:15003–15006[Abstract/Free Full Text]
221. Prelevic GM, Ginsburg J, Maletic D, Hardiman P, Okolo S, Balint-Peric L, Thomas M, Orskov H 1995 The effects of the somatostatin analogue octreotide on ovulatory performance in women with polycystic ovaries. Hum Reprod 10:28–32[Abstract/Free Full Text]
222. van der Meer M, Hompes PG, Scheele F, Schoute E, Veersema S, Schoemaker J 1994 Follicle stimulating hormone (FSH) dynamics of low dose step-up ovulation induction with FSH in patients with polycystic ovary syndrome. Hum Reprod 9:1612–1617[Abstract/Free Full Text]
223. Fulghesu AM, Ciampelli M, Guido M, Murgia F, Caruso A, Mancuso S, Lanzone A 1998 Role of opioid tone in the pathophysiology of hyperinsulinemia and insulin resistance in polycystic ovarian disease. Metabolism 47:158–162[CrossRef][Medline]
224. Chen Y-DI, Reaven GM 1997 Insulin resistance and atherosclerosis. Diabetes Rev 5:331–342
225. Genuth S, Brownlee MA, Kuller LH, Samols E, Saudek CD, Sherwin R 1998 Consensus development conference on insulin resistance. Diabetes Care 21:310–314[Medline]
226. Stevenson RW, Kreutter DK, Andrews KM, Genereux PE, Gibbs EM 1998 Possibility of distinct insulin-signaling pathways beyond phosphatidylinositol 3-kinase-mediating glucose transport and lipogenesis. Diabetes 47:179–185[Abstract]
227. Sasaoka T, Ishiki M, Sawa T, Ishihara H, Takata Y, Imamura T, Usui I, Olefsky JM, Kobayashi M 1996 Comparison of the insulin and insulin-like growth factor I mitogenic intracellular signaling pathways. Endocrinology 137:4427–4434[Abstract]
228. Federici M, Lauro D, D’Adamo M, Giovannone B, Porzio O, Mellozzi M, Tamburrano G, Sbraccia P, Sesti G 1998 Expression of insulin/IGF-I hybrid receptors is increased in skeletal muscle of patients with chronic primary hyperinsulinemia. Diabetes 47:87–92[Abstract]
229. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM 1994 Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432[CrossRef][Medline]
230. Zhang F, Basinski MB, Beals JM, Briggs SL, Churgay LM, Clawson DK, DiMarchi RD, Furman TC, Hale JE, Hsiung HM, Schoner BE, Smith DP, Zhang XY, Wery JP, Schevitz RW 1997 Crystal structure of the obese protein leptin-E100. Nature 387:206–209[CrossRef][Medline]
231. Grasso P, Leinung MC, Ingher SP, Lee DW 1997 In vivo effects of leptin-related synthetic peptides on body weight and food intake in female ob/ob mice: localization of leptin activity to domains between amino acid residues 106–140. Endocrinology 138:1413–1418[Abstract/Free Full Text]
232. Samson WK, Murphy TC, Robison D, Vargas T, Tau E, Chang JK 1996 A 35 amino acid fragment of leptin inhibits feeding in the rat. Endocrinology 137:5182–5185[Abstract]
233. Flier JS 1998 Clinical review 94: What’s in a name? In search of leptin’s physiologic role. J Clin Endocrinol Metab 83:1407–1413[Free Full Text]
234. Blum WF, Englaro P, Hanitsch S, Juul A, Hertel NT, Muller J, Skakkebaek NE, Heiman ML, Birkett M, Attanasio AM, Keiss W, Rascher W 1997 Plasma leptin levels in healthy children and adolescents: dependence on body mass index, body fat mass, gender, pubertal stage, and testosterone. J Clin Endocrinol Metab 82:2904–2910[Abstract/Free Full Text]
235. Lahlou N, Landais P, De Boissieu D, Bougneres PF 1997 Circulating leptin in normal children and during the dynamic phase of juvenile obesity. Diabetes 46:989–994[Abstract]
236. Saad MF, Riad-Gabriel MG, Khan A, Sharma A, Michael R, Jinagouda SD, Boyadjian R, Steil GM 1998 Diurnal and ultradian rhythmicity of plasma leptin: effects of gender and adiposity. J Clin Endocrinol Metab 83:453–459[Abstract/Free Full Text]
237. Considine RV 1997 Leptin and obesity in humans. Eating and Weight Disorders 2:61–66
238. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, Mckee LJ, Bauer TL, Caro JF 1996 Serum immunoreactive-leptin concentration in normal-weight and obese humans. N Engl J Med 334:292–295[Abstract/Free Full Text]
239. Chessler SD, Fujimoto WY, Shofer JB, Boyko EJ, Weigle DS 1998 Increased plasma leptin levels are associated with fat accumulation in Japanese Americans. Diabetes 47:239–243[Abstract]
240. Pardini VC, Victoria IM, Rocha SMV, Andrade DG, Rocha AM, Pieroni FB, Milagres G, Purisch S, Velho G 1998 Leptin levels, ß-cell function and insulin sensitivity in families with congenital and acquired generalized lipoatropic diabetes. J Clin Endocrinol Metab 83:503–508[Abstract/Free Full Text]
241. Mantzoros CS, Moschos S, Avramopoulos I, Kaklamani V, Liolios A, Doulgerakis DE, Griveas I, Katsilambros N, Flier JS 1997 Leptin concentrations in relation to body mass index and the tumor necrosis factor- system in humans. J Clin Endocrinol Metab 82:3408–3413[Abstract/Free Full Text]
242. Flier JS, Maratos-Flier E 1998 Obesity and the hypothalamus: novel peptides for new pathways. Cell 92:437–440[CrossRef][Medline]
243. Boston BA, Blaydon KM, Varnerin J, Cone RD 1997 Indepedent and additive effects of central POMC and leptin pathways on murine obesity. Science 278:1641–1644[Abstract/Free Full Text]
244. Rosenbaum M, Leibel R, Hirsch J 1997 Obesity. N Engl J Med 337:396–406[Free Full Text]
245. Weigle DS, Kuijpers JL 1997 Mouse models of human obesity. Science Med 4:38–45
246. Chehab FF, Lim ME, Lu R 1996 Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12:318–320[CrossRef][Medline]
247. Harris RBS, Zhou J, Redmann SM, Smagin GN, Smith SR, Rodgers E, Zachwieja JJ 1998 A leptin dose-response study in obese (ob/ob) and lean(+/?) mice. Endocrinology 139:8–19[Abstract/Free Full Text]
248. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP 1996 Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84:491–495[CrossRef][Medline]
249. Montague CT, Farooqi IS, Whitehead JP, Soos MA, Rau H, Wareham NJ, Sewter CP, Digby JE, Mohammed SN, Hurst JA, Cheetham CH, Earley AR, Barnett AH, Prins JB, O’Rahilly S 1997 Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 387:903–908[CrossRef][Medline]
250. Clement K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, Cassuto D, Gourmelen M, Dina C, Chambaz J, Lacorte JM, Basdevant A, Bougneres P, Lebouc Y, Froguel P, Guy-Grand B 1998 A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392:398–401[CrossRef][Medline]
251. Mantzoros CS, Flier JS, Rogol AD 1997 A longitudinal assessment of hormonal and physical alterations during normal puberty in boys. V. Rising leptin levels may signal the onset of puberty. J Clin Endocrinol Metab 82:1066–1070[Abstract/Free Full Text]
252. Barash IA, Cheung CC, Weigle DS, Ren H, Kabigting EB, Kuijper JL, Clifton DK, Steiner RA 1996 Leptin is a metabolic signal to the reproductive system. Endocrinology 137:3144–3147[Abstract]
253. Matkovic V, Ilich JZ, Skugor M, Badenhop NE, Goel P, Clairmont A, Klisovic D, Nahhas RW, Landoll JD 1997 Leptin is inversely related to age at menarche in human females. J Clin Endocrinol Metab 82:3239–3245[Abstract/Free Full Text]
254. Cheung CC, Thorton JE, Kuijper JL, Weigle DS, Clifton DK, Steiner RA 1997 Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology 138:855–858[Abstract/Free Full Text]
255. Pombo M, Herrera-Justiniano E, Considine RV, Hermida RC, Galvez MJ, Martin T, Barreiro J, Casanueva FF, Dieguez C 1997 Nocturnal rise of leptin in normal prepubertal and pubertal children and in patients with perinatal stalk-transection syndrome. J Clin Endocrinol Metab 82:2751–2754[Abstract/Free Full Text]
256. Ahima RS, Dushay J, Flier SN, Prabakaran D, Flier JS 1997 Leptin accelerates the onset of puberty in normal female mice. J Clin Invest 99:391–395[Medline]
257. Chehab FF, Mounzih K, Lu R, Lim ME 1997 Early onset of reproductive function in normal female mice treated with leptin. Science 275:88–90[Abstract/Free Full Text]
258. Wadden TA, Considine RV, Foster FD, Anderson DA, Sarwer DB, Caro JS 1998 Short and long-term changes in serum leptin in dieting obese women: effects of caloric restriction and weight loss. J Clin Endocrinol Metab 83:214–218[Abstract/Free Full Text]
259. Schwartz MW, Seeley RJ 1997 Neuroendocrine responses to starvation and weight loss. N Engl J Med 336:1802–1811[Free Full Text]
260. Weigle DS, Duell PB, Connor WE, Steiner RA, Soules MR, Kuijper JL 1997 Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels. J Clin Endocrinol Metab 82:561–565[Abstract/Free Full Text]
261. Loucks AB, Heath EM, Law T, Sr. Verdun M, Watts JR 1994 Dietary restriction reduces luteinizing hormone (LH) pulse frequency during waking hours and increases LH pulse amplitude during sleep in young menstruating women. J Clin Endocrinol Metab 78:910–915[Abstract]
262. Loucks AB, Mortola JF, Girton L, Yen SSC 1989 Alterations in the hypothalamic-pituitary-ovarian and the hypothalamic-pituitary-adrenal axes in athletic women. J Clin Endocrinol Metab 68:402–411[Abstract/Free Full Text]
263. Heiman ML, Ahima RS, Craft LS, Schoner B, Stephens TW, Flier JS 1997 Leptin inhibition of the hypothalamic-pituitary-adrenal axis in response to stress. Endocrinology 138:3859–3863[Abstract/Free Full Text]
264. Spicer LJ, Francisco CC 1997 The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function. Endocrinology 138:3374–3379[Abstract/Free Full Text]
265. Zamorano PL, Mahesh VB, De Sevilla LM, Chorich LP, Bhat GK, Brann DW 1997 Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 65:223–228[CrossRef][Medline]
266. Karlsson C, Lindell K, Svensson E, Bergh C, Lind P, Billig H, Carlsson LM, Carlsson B 1997 Expression of functional leptin receptors in the human ovary. J Clin Endocrinol Metab 82:4144–4148[Abstract/Free Full Text]
267. McClain D 1998 Editorial: Further insights into leptin action. Endocrinology 139:3679–3680[Free Full Text]
268. Zachow RJ, Magoffin DA 1997 Direct intraovarian effects of leptin: impairment of the synergistic action of insulin-like growth factor-I on follicle-stimulating hormone-dependent estradiol-17ß production by rat ovarian granulosa cells. Endocrinology 138:847–850[Abstract/Free Full Text]
269. Barr VA, Malide D, Zarnowski MJ, Taylor S, Cushman SW 1997 Insulin stimulates both leptin secretion and production by rat white adipose tissue. Endocrinology 138:4463–4472[Abstract/Free Full Text]
270. Mueller WM, Gregoire FM, Stanhope KL, Mobbs CV, Mizuno TM, Warden CH, Stern JS, Havel PJ 1998 Evidence that glucose metabolism regulates leptin secretion from cultured rat adipocytes. Endocrinology 139:551–558[Abstract/Free Full Text]
271. Kolaczynski JW, Nyce MR, Considine RV, Boden G, Nolan JJ, Henry R, Mudaliar SR, Olefsky J, Caro JF 1996 Acute and chronic effects of insulin on leptin production in humans: studies in vivo and in vitro. Diabetes 45:699–701[Abstract]
272. Saad MF, Khan A, Sharma A, Michael R, Riad-Gabriel MG, Boyadjian R, Jinagouda SD, Steil GM, Kamdar V 1998 Physiological insulinemia acutely modulates plasma leptin. Diabetes 47:544–549[Abstract]
273. Clapham JC, Smith SA, Moore GB, Hughes MG, Azam H, Scott A, Jung RT 1997 Plasma leptin concentrations and OB gene expression in subcutaneous adipose tissue are not regulated acutely by physiological hyperinsulinaemia in lean and obese humans. Int J Obes Relat Metab Disord 21:179–183[CrossRef][Medline]
274. Poretsky L, Brillon DJ 1998 Circulating leptin levels do not change postprandially in patients with type 2 diabetes mellitus who are not on insulin therapy. Program of the 80th Annual Meeting of The Endocrine Society, New Orleans, LA, 1998 (Abstract P3–389), p 466
275. Kolaczynski JW, Ohannesian JP, Considine RV, Marco CC, Caro JF 1996 Response of leptin to short-term and prolonged overfeeding in humans. J Clin Endocrinol Metab 81:4162–4165[Abstract/Free Full Text]
276. Ookuma M, Ookuma K, York DA 1998 Effects of leptin on insulin secretion from isolated rat pancreatic islets. Diabetes 47:219–223[Abstract]
277. Poitout V, Rouault C, Guerre-Millo M, Briaud I, Reach G 1998 Inhibition of insulin secretion by leptin in normal rodent islets of Langerhans. Endocrinology 139:822–826[Abstract/Free Full Text]
278. Tanizawa Y, Okuya S, Ishihara H, Asano T, Yada T, Oka Y 1997 Direct stimulation of basal insulin secretion by physiological concentrations of leptin in pancreatic ß cells. Endocrinology 138:4513–4516[Abstract/Free Full Text]
279. Quintela M, Senaris R, Heiman ML, Casanueva FF, Dieguez C 1997 Leptin inhibits in vitro hypothalamic somatostatin secretion and somatostatin mRNA levels. Endocrinology 138:5641–5644[Abstract/Free Full Text]
280. Mizuno A, Murakami T, Otani S, Kuwajima M, Shima K 1998 Leptin affects pancreatic endocrine functions through the sympathetic nervous system. Endocrinology 139:3863–3870[Abstract/Free Full Text]
281. Sivitz WI, Walsh SA, Morgan DA, Thomas MJ, Haynes WG 1997 Effects of leptin on insulin sensitivity in normal rats. Endocrinology 138:3395–3401[Abstract/Free Full Text]
282. Cusin I, Zakrzewska KE, Boss O, Muzzin P, Giacobino J-P, Ricquier D, Jeanrenaud B, Rohner-Jeanrenaud F 1998 Chronic central leptin infusion enhances insulin-stimulated glucose metabolism and favors the expression of uncoupling proteins. Diabetes 47:1014–1019[Abstract]
283. Shimabukuro M, Koyama K, Chen G, Wang MY, Trieu F, Lee Y, Newgard CB, Unger RH 1997 Direct antidiabetic effect of leptin through triglyceride depletion of tissues. Proc Natl Acad Sci USA 94:4637–4641[Abstract/Free Full Text]
284. Kamohara S, Burcelin R, Halaas JL, Friedman JM, Charron MJ 1997 Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389:374–377[CrossRef][Medline]
285. Ranganathan S, Ciaraldi TP, Henry RR, Mudaliar S, Kern PA 1998 Lack of effect of leptin on glucose transport, lipoprotein lipase, and insulin action in adipose and muscle cells. Endocrinology 139:2509–2513[Abstract/Free Full Text]
286. Carantoni M, Abbasi F, Azhar S, Chen Y-DI, Klebanov M, Wang P-W, Warmerdam F, Reaven GM 1998 Plasma leptin concentrations do not appear to decrease insulin-mediated glucose disposal or glucose-stimulated insulin secretion in women with normal glucose tolerance. Diabetes 47:244–247[Abstract]
287. Taylor SI, Barr V, Reitman M 1996 Does leptin contribute to diabetes caused by obesity? Science 274:1151–1152[Free Full Text]
288. Muller G, Ertl J, Gerl M, Preibisch G 1997 Leptin impairs metabolic actions of insulin in isolated rat adipocytes. J Biol Chem 272:10585–10593[Abstract/Free Full Text]
289. Anai M, Funaki M, Ogihara T, Terasaki J, Inukai K, Katagiri H, Fukushima Y, Yazaki Y, Kikuchi M, Oka Y, Asano T 1998 Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes 47:13–23[Abstract]
290. Holmang A, Mimura K, Bjorntorp P, Lonnroth P 1997 Interstitial muscle insulin and glucose levels in normal and insulin-resistant Zucker rats. Diabetes 46:1799–1804[Abstract]
291. Frisch RE 1990 Body fat, menarche, fitness and fertility. In: Frisch RE (ed) Adipose Tissue and Reproduction. Karger, Basel, pp 1–26
292. Grinspoon S, Corcoran C, Miller K, Biller BMK, Askari H, Wang E, Hubbard J, Anderson EJ, Basgoz N, Heller HM, Klibanski A 1997 Body composition and endocrine function in women with acquired immunodeficiency syndrome wasting. J Clin Endocrinol Metab 82:1332–1337[Abstract/Free Full Text]
293. Laughlin GA, Dominguez CE, Yen SS 1998 Nutritional and endocrine-metabolic aberrations in women with functional hypothalamic amenorrhea. J Clin Endocrinol 83:25–32[Abstract/Free Full Text]
294. Laughlin GA, Yen SS 1997 Hypoleptinemia in women athletes: absence of a diurnal rhythm with amenorrhea. J Clin Endocrinol Metab 82:318–321[Abstract/Free Full Text]
295. Olson BR, Cartledge T, Sebring N, Defensor R, Nieman L 1995 Short-term fasting affects luteinizing hormone secretory dynamics but not reproductive function in normal-weight sedentary women. J Clin Endocrinol Metab 80:1187–1193[Abstract]
296. Nagamani M, Stuart CA 1990 Specific binding sites for insulin-like growth factor I in the ovarian stroma of women with polycystic ovarian disease and stromal hyperthecosis. Am J Obstet Gynecol 163:1992–1997[Medline]
297. Pepper GM, Poretsky L, Gabrilove JL, Aritone MM 1987 Ketoconazole reverses hyperandrogenism in a patient with insulin resistance. J Clin Endocrinol Metab 65:1047–1052[Abstract/Free Full Text]
298. Poretsky L, Bhargava G, Levitan E 1990 Type I insulin-like growth factor receptors in human ovarian stroma. Horm Res 33:22–26[CrossRef][Medline]
299. Gdansky E, Diamant YZ, Laron Z, Silbergeld A, Kaplan B, Eshet R 1997 Increased number of IGF-I receptors on erythrocytes of women with polycystic ovarian syndrome. Clin Endocrinol (Oxf) 47:185–190[CrossRef][Medline]
300. Federici M, Porzio O, Lauro D, Borboni P, Giovannone B, Zucaro L, Hribal ML, Sesti G 1998 Increased abundance of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of obese subjects is correlated with in vivo insulin sensitivity. J Clin Endocrinol Metab 83:2911–2915[Abstract/Free Full Text]
301. Leitner JW, Kline T, Carel K, Goalstone M, Draznin B 1997 Hyperinsulinemia potentiates activation of p21Ras by growth factors. Endocrinology 138:2211–2214[Abstract/Free Full Text]
302. Fradkin JE, Eastman RC, Lesniak MA, Roth J 1989 Specificity spillover at the hormone receptor: exploring its role in human disease. N Engl J Med 320:640–645[Medline]
303. Daughaday WH, Rotwein P 1989 Insulin-like growth factors I and II: peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 10:68–91[Abstract/Free Full Text]
304. Salmon WD, Daughaday WH 1957 A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825–836[Medline]
305. Thomas MJ, Kikuchi K, Bichell DP, Rotwein P 1994 Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no alterations in deoxyribonucleic acid-protein interactions within the major promoter. Endocrinology 135:1584–1592[Abstract]
306. Adamo ML, Neuenschwander S, LeRoith D, Roberts Jr CT 1993 Structure, expression, and regulation of the IGF-I gene. Adv Exp Med Biol 343:1–11[Medline]
307. Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellve AR, Efstratiadis A 1996 Effects of an IGF-1 gene null mutation on mouse reproduction. Mol Endocrinol 10:903–918[Abstract/Free Full Text]
308. Bellve AR 1996 Differential effects of IGF null mutations in male and female mice. In: LeRoith D (ed) The Role of Insulin-like Growth Factors in Ovarian Physiology. Ares Serono Symposia, Rome, pp 35–46
309. Nielsen FC 1992 The molecular and cellular biology of insulin-like growth factor II. Prog Growth Factor Res 4:257–290[CrossRef][Medline]
310. Humbel RE 1990 Insulin-like growth factors I and II. Eur J Biochem 190:445–462[Medline]
311. LeRoith D, Clemmons D, Nissley P, Rechler MM 1992 Insulin-like growth factors in health and disease. Ann Intern Med 116:854–862
312. Buyalos R 1995 Insulin-like growth factors: clinical experience in ovarian function. Am J Med [Suppl 1A]98:55S–66S
313. DeChiara TM, Efstratiadis A, Robertson EJ 1990 A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345:78–80[CrossRef][Medline]
314. Morrione A, Valentinis B, Xu SQ, Yumet G, Louvi A, Efstratiadis A, Baserga R 1997 Insulin-like growth factor II stimulates cell proliferation through the insulin receptor. Proc Natl Acad Sci USA 94:3777–3782[Abstract/Free Full Text]
315. LeRoith D, Werner H, Beitner-Johnson D, Roberts CT 1995 Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 16:143–163[Abstract/Free Full Text]
316. Feld S, Hirschberg R 1996 Growth hormone, the insulin-like growth factor system and the kidney. Endocr Rev 17:423–480[Abstract/Free Full Text]
317. Mohseni-Zadeh S, Binoux M 1997 Insulin-like growth factor (IGF) binding protein-3 interacts with the type 1 IGF receptor, reducing the affinity of the receptor for its ligand: an alternative mechanism in the regulation of IGF action. Endocrinology 138:5645–5648[Abstract/Free Full Text]
318. Dey BR, Frick K, Lopaczynski W, Nissley SP, Furlanetto RW 1996 Evidence for the direct interaction of the insulin-like growth factor 1 receptor with IRS-1, Shc, and Grb 10. Mol Endocrinol 10:631–641[Abstract/Free Full Text]
319. Kaliman P, Canicio J, Shepherd P, Beeton CA, Xavier T, Palacin M, Zorzano A 1998 Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells. Mol Endocrinol 12:85–110
320. D’Ercole AJ, Ye P, Gutierrez-Ospina G 1996 Use of transgenic mice for understanding the physiology of insulin-like growth factors. Horm Res 45 [Suppl 1]:5–7
321. Siebler T, Lopaczynski W, Terry CL, Casella SJ, Munson P, DeLeon DD, Phang L, Blakemore KJ, McEvoy RC, Kelley R 1995 Insulin-like growth factor I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15. J Clin Endocrinol Metab 80:3447–3457[Abstract]
322. Jain S, Golde DW, Bailey R, Geffner ME 1998 Insulin-like growth factor-I resistance. Endocr Rev 19:625–646[Abstract/Free Full Text]
323. Werner W, Woloschak M, Stannard B, Shen-Orr Z, Roberts Jr CT, LeRoith D 1991 The insulin-like growth factor I receptor: molecular biology, heterogeneity, and regulation. In: LeRoith D (ed) Insulin-like Growth Factors: Molecular and Cellular Aspects. CRC Press, Boca Raton, FL, pp 17–47
324. Kiess W, Blickenstaff GD, Sklar MM, Thomas CL, Nissley SP, Sahagian GG 1988 Biochemical evidence that the type II insulin-like growth factor receptor is identical to the cation-independent mannose 6-phosphate receptor. J Biol Chem 263:9339–9344[Abstract/Free Full Text]
325. Nissley P, Wieland K, Sklar MM 1991 The insulin-like growth factor-II/mannose 6-phosphate receptor. In: LeRoith D (ed) Insulin-Like Growth Factors: Molecular and Cellular Aspects. CRC Press, Boca Raton, FL, pp 111–150
326. Roth RA 1988 Structure of the receptor for insulin-like growth factor II: the puzzle amplified. Science 239:1269–1271[Abstract/Free Full Text]
327. Schmidt B, Kiecke-Siemsen C, Waheed A, Braulke T, Von Figura K 1995 Localization of the insulin-like growth factor II binding site to amino acids 1508–1566 in repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor. J Biol Chem 270:14975–14982[Abstract/Free Full Text]
328. Ludwig T, Eggenschwiler J, Fisher P, D’Ercole AJ, Davenport ML, Efstratiadis A 1996 Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds. Dev Biol 177:527–535
329. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[Abstract/Free Full Text]
330. Scott CD, Baxter RC 1996 Regulation of soluble insulin-like growth factor-II mannose 6-phosphate receptor in hepatocytes from intact and regenerating rat liver. Endocrinology 137:3864–3870[Abstract]
331. Gelato MC, Kiess W, Lee L, Malozowski S, Rechler MM, Nissley P 1988 The insulin-like growth factor II/mannose-6-phosphate receptor is present in monkey serum. J Clin Endocrinol Metab 67:669–675[Abstract/Free Full Text]
332. Xu Y, Papageorgiou A, Polychronakos C 1998 Developmental regulation of the soluble form of insulin-like growth factor-II/mannose 6-phosphate receptor in human serum and amniotic fluid. J Clin Endocrinol Metab 83:437–442[Abstract/Free Full Text]
333. Clairmont KB, Czech MP 1991 Extracellular release as the major degradative pathway of the insulin-like growth factor II/mannose 6-phosphate receptor. J Biol Chem 266:1231–1234
334. Nissley P, Lopaczynski W 1991 Insulin-like growth factor receptors. Growth Factors 5:29–43[Medline]
335. Zaina S, Newton RVS, Paul MR, Graham CF 1998 Local reduction of organ size in transgenic mice expressing a soluble insulin-like growth factor II/mannose-6-phosphate receptor. Endocrinology 139:3886–3895[Abstract/Free Full Text]
336. Scott CD, Ballesteros M, Madrid J, Baxter RC 1996 Soluble insulin-like growth factor-II/mannose 6-P receptor inhibits deoxyribonucleic acid synthesis in cultured rat hepatocytes. Endocrinology 137:873–878[Abstract]
337. Okamoto T, Nishimoto I, Murayama Y, Ohkuni Y, Ogata E 1990 Insulin-like growth factor-II/mannose 6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose 6-phosphate, but capable in response to insulin-like growth factor-II. Biochem Biophys Res Commun 168:1201–1210[CrossRef][Medline]
338. Ikezu T, Okamoto T, Giambarella U, Yokota T, Nishimoto I 1995 In vivo coupling of insulin-like growth factor II/mannose 6-phosphate receptor to heteromeric G proteins. Distinct roles of cytoplasmic domains and signal sequestration by the receptor. J Biol Chem 270:29224–29228[Abstract/Free Full Text]
339. Willis DS, Mason HD, Watson H, Franks S 1998 Developmentally regulated responses of human granulosa cells to insulin-like growth factors (IGFs): IGF-I and IGF-II action mediated via the type-I IGF receptor. J Clin Endocrinol Metab 83:1256–1259[Abstract/Free Full Text]
340. Volpert O, Jackson D, Bouck N, Linzer DI 1996 The insulin-like growth factor II/mannose 6-phosphate receptor is required for proliferin-induced angiogenesis. Endocrinology 137:3871–3876[Abstract]
341. Blanchard F, Raher S, Duplomb L, Vusio P, Pitard V, Taupin JL, Moreau JF, Hoflack B, Minvielle S, Jacques Y, Godard A 1998 The mannose 6-phosphate/insulin-like growth factor II receptor is a nanomolar affinity receptor for glycosylated human leukemia inhibitory factor. J Biol Chem 273:20886–20893[Abstract/Free Full Text]
342. Bondy CA, Zhou J 1996 Functional correlates of IGF system gene expression in the murine and primate ovary. In: LeRoith D (ed) The Role of Insulin-like Growth Factors in Ovarian Physiology. Ares-Serono Symposia, Rome, pp 59–69
343. Zhou J, Bondy C 1993 Anatomy of the human ovarian insulin-like growth factor system. Biol Reprod 48:467–482[Abstract]
344. Ramasharma K, Li CH 1987 Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells. Proc Natl Acad Sci USA 84:2643–2647[Abstract/Free Full Text]
345. Voutilainen T, Miller WL 1987 Coordinate tropic hormone regulation of mRNAs for insulin-like growth factor II and the cholesterol side-chain-cleavage enzyme, P450scc in human steroidogenic tissues. Proc Natl Acad Sci USA 84:1590–1594[Abstract/Free Full Text]
346. Geisthovel F, Moretti-Rojas I, Asch RH, Rojas F 1989 Expression of insulin-like growth factor-II (IGF-II) messenger ribonucleic acid (mRNA), but not IGF-I mRNA, in human preovulatory granulosa cells. Hum Reprod 4:899–902[Abstract/Free Full Text]
347. Voutilainen R, Franks S, Mason HD, Martikainen H 1996 Expression of insulin-like growth factor (IGF), IGF-binding protein, and IGF receptor messenger ribonucleic acids in normal and polycystic ovaries. J Clin Endocrinol Metab 81:1003–1008[Abstract]
348. Van Dessel THJHM, Chandrasekher Y, Yap OWS, Lee PDK, Hintz RL, Faessen GHJ, Braat DDM, Fauser BCJM 1996 Serum and follicular fluid levels of insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-1 and -3 during the normal menstrual cycle. J Clin Endocrinol Metab 81:1224–1231[Abstract]
349. Jesionowska H, Hemmings R, Guyda HJ, Posner BI 1990 Determination of insulin and insulin-like growth factors in the ovarian circulation. Fertil Steril 53:88–91[Medline]
350. Geisthoevel F, Moretti-Rojas IM, Rojas FJ, Asch RH 1989 Immunoreactive insulin-like growth factor I in human follicular fluid. Hum Reprod 4:35–38[Abstract/Free Full Text]
351. Rabinovici J, Dandekar P, Angle MJ, Rosenthal S, Martin MC 1990 Insulin-like growth factor I (IGF-I) levels in follicular fluid from human preovulatory follicles: correlation with serum IGF-I levels. Fertil Steril 54:428–433[Medline]
352. Giudice LC, Farrell EM, Pham H, Rosenfeld RG 1990 Identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and IGFBP-2 in human follicular fluid. J Clin Endocrinol Metab 71:1330–1338[Abstract/Free Full Text]
353. Kubota T, Kamada S, Ohara M, Taguchi M, Sakamoto S, Shimizu Y, Aso T 1993 Insulin-like growth factor II in follicular fluid of the patients with in vitro fertilization and embryo transfer. Fertil Steril 59:844–849[Medline]
354. Seifer DB, Giudice LC, Dsupin BA, Haning Jr RV, Frishman GN, Burger HG 1995 Follicular fluid insulin-like growth factor-I and insulin-like growth factor-II concentrations vary as a function of day 3 serum follicle stimulating hormone. Hum Reprod 10:804–806[Abstract/Free Full Text]
355. Menashe Y, Sack J, Mashiach S 1991 Spontaneous pregnancies in two women with Laron-type dwarfism: are growth hormone and circulating insulin-like growth factor mandatory for induction of ovulation? Hum Reprod 6:670–671[Abstract/Free Full Text]
356. Dor J, Ben-Shlomo I, Lunenfeld B, Pariente C, Levran D, Karasik A, Seppala M, Mashiach S 1992 Insulin-like growth factor-I (IGF-I) may not be essential for ovarian follicular development: evidence from IGF-I deficiency. J Clin Endocrinol Metab 74:539–542[Abstract]
357. Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J 1994 Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr Rev 15:369–390[Abstract/Free Full Text]
358. Laron Z 1995 Prismatic cases: Laron syndrome (primary growth hormone resistance) from patient to laboratory to patient. J Clin Endocrinol Metab 80:1526–1531[Abstract/Free Full Text]
359. Gates GS, Bayer S, Seibel M, Poretsky L, Flier JS, Moses AC 1987 Characterization of insulin-like growth factor binding to human granulosa cells obtained during in vitro fertilization. J Recept Res 7:885–902[Medline]
360. Oliver JE, Aitman TJ, Powell JF, Wilson CA, Clayton RN 1989 Insulin-like growth factor-I gene expression in the rat ovary is confined to the granulosa cells of developing follicles. Endocrinology 124:2671–2679[Abstract/Free Full Text]
361. Hansson HA, Nilsson A, Isgaard J, Billig H, Isaksson O, Skottner A, Andersson IK, Rozell B 1988 Immunohistochemical localization of insulin-like growth factor I in the adult rat. Histochemistry 89:403–410[CrossRef][Medline]
362. Hernandez ER, Roberts Jr CT, Le Roith D, Adashi EY 1989 Rat ovarian insulin-like growth factor (IGF-I) gene expression is granulosa cell-selective: 5'-untranslated mRNA variant representation and hormonal regulation. Endocrinology 125:572–574[Abstract/Free Full Text]
363. Zhou J, Chin E, Bondy C 1991 Cellular pattern of insulin-like growth factor-I (IGF-I) and IGF-I receptor gene expression in the developing and mature ovarian follicle. Endocrinology 129:3281–3288[Abstract/Free Full Text]
364. Adashi EY, Resnick CE, Payne DW, Rosenfeld RG, Matsumoto T, Hunter MK, Gargosky SE, Zhou J, Bondy CA 1997 The mouse intraovarian insulin-like growth factor I system: departures from the rat paradigm. Endocrinology 138:3881–3890[Abstract/Free Full Text]
365. Levy MJ, Hernandez ER, Adashi EY, Stillman RJ, Roberts Jr CT, LeRoith D 1992 Expression of the insulin-like growth factor (IGF)-I and II and the IGF-I and II receptor genes during postnatal development of the rat ovary. Endocrinology 131:1202–1206[Abstract/Free Full Text]
366. Adashi EY 1996 Regulation of intrafollicular IGFBPs: possible relevance to ovarian follicular selection. In: LeRoith D (ed) The Role of Insulin-Like Growth Factors in Ovarian Physiology. Ares-Serono Symposia, Rome, pp 25–34
367. Wandji S-A, Wood TL, Crawford J, Levison SW, Hammond JM 1998 Expression of mouse ovarian insulin growth factor system components during follicular development and atresia. Endocrinology 139:5205–5214[Abstract/Free Full Text]
368. Zhou J, Kumar TR, Matzuk MM, Bondy C 1997 Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary. Mol Endocrinol 11:1924–1933[Abstract/Free Full Text]
369. Bryan KA, Hammond JM, Canning S, Mondschein J, Carbaugh DE, Clark AM, Hagen DR 1989 Reproductive and growth responses of gilts to exogenous porcine pituitary growth hormone. J Anim Sci 67:196–205
370. Samaras SE, Hagen DR, Bryan KA, Mondschein JS, Canning SF, Hammond JM 1994 Effects of growth hormone and gonadotropin on the insulin-like growth factor system in the porcine ovary. Biol Reprod 50:178–186[Abstract]
371. Hammond JM, Hsu CJ, Klindt J, Tsang BK, Downey BR 1988 Gonadotropins increase concentrations of immunoreactive insulin-like growth factor-I in porcine follicular fluid in vivo. Biol Reprod 38:304–308[Abstract]
372. Spicer LJ, Klindt J, Buonomo FC, Maurer R, Yen JT, Echternkamp SE 1992 Effect of porcine somatotropin on number of granulosa cell luteinizing hormone/human chorionic gonadotropin receptors, oocyte viability, and concentrations of steroids and insulin-like growth factors I and II in follicular fluid of lean and obese gilts. J Anim Sci 70:3149–3157[Abstract]
373. Bryan KA, Hagen DR, Hammond JM 1992 Effect of frequency of administration of exogenous porcine growth hormone on growth and carcass traits and ovarian function of prepubertal gilts. J Anim Sci 70:1454–1463[Abstract]
374. Monget P, Besnard N, Huet C, Pisselet C, Monniaux D 1996 Insulin-like growth factor-binding proteins and ovarian folliculogenesis. Horm Res 45:211–217[Medline]
375. Ord R, Ledgard A, Berg D, Peterson J 1993 Ovine IGF antisense RNAs? Proc N Z Soc Anim Prod 53:449–452
376. Tisdall DJ, Smith P, Leeuwenberg B, McNatty KP 1995 FSH-receptor, ß-B inhibin subunit, follistatin, ß-A and inhibin subunits and IGF-I genes are expressed sequentially in ovine granulosa cells during early follicular development. J Reprod Fertil 15:12 (Abstract)
377. Perks CM, Denning-Kendall PA, Gilmour RS, Wathes DC 1995 Localization of messenger ribonucleic acids for insulin-like growth factor I (IGF-I), IGF-II, and the type I IGF receptor in the ovine ovary throughout the estrous cycle. Endocrinology 136:5266–5273[Abstract]
378. Monget P, Monniaux D, Pisselet C, Durand P 1993 Changes in insulin-like growth factor-I (IGF-I), IGF-II, and their binding proteins during growth and atresia of ovine ovarian follicles. Endocrinology 132:1438–1446[Abstract/Free Full Text]
379. Spicer LJ, Alpizar E, Echtemkamp SE 1993 Effects of insulin-like growth factor I, and gonadotropins on bovine granulosa cell proliferation, progesterone production, estradiol production, and(or) insulin-like growth factor I production in vitro. J Anim Sci 71:1232–1241[Abstract]
380. Spicer LJ, Echternkamp SE 1995 The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals. Domest Anim Endocrinol 12:223–245[CrossRef][Medline]
381. Spicer LJ, Echternkamp SE, Canning SF, Hammond JM 1988 Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles. Biol Reprod 39:573–580[Abstract]
382. Spicer LJ, Enright WJ 1991 Concentrations of insulin-like growth factor I and steroids in follicular fluid of preovulatory bovine ovarian follicles: effect of daily injections of a growth hormone-releasing factor analog and(or) thyrotropin-releasing hormone. J Anim Sci 69:1133–1139[Abstract]
383. Echternkamp SE, Spicer LJ, Gregory KE, Canning SF, Hammond JM 1990 Concentrations of insulin-like growth factor-I in blood and ovarian follicular fluid of cattle selected for twins. Biol Reprod 43:8–14[Abstract]
384. Echternkamp SE, Howard HJ, Roberts AJ, Grizzle J, Wise T 1994 Relationships among concentrations of steroids, insulin-like growth factor-I, and insulin-like growth factor binding proteins in ovarian follicular fluid of beef cattle. Biol Reprod 51:971–981[Abstract]
385. Badinga L, Driancourt MA, Savio JD, Wolfenson D, Drost M, de la Sota RL, Thatcher WW 1992 Endocrine and ovarian responses associated with the first-wave dominant follicle in cattle. Biol Reprod 47:871–883[Abstract]
386. Stanko RL, Cohick WS, Shaw DW, Harvey RW, Clemmons DR, Whitacre MD, Armstrong JD 1994 Effect of somatotropin and/or equine chorionic gonadotropin on serum and follicular insulin-like growth factor I and insulin-like growth factor binding proteins in cattle. Biol Reprod 50:290–300[Abstract]
387. de la Sota RL, Simmen FA, Diaz T, Thatcher WW 1996 Insulin-like growth factor system in bovine first-wave dominant and subordinate follicles. Biol Reprod 55:803–812[Abstract]
388. Yuan W, Bao B, Garverick HA, Youngquist RS, Lucy MC 1998 Follicular dominance in cattle is associated with divergent patterns of ovarian gene expression for insulin-like growth factor I (IGF-I), IGF-II, and IGF binding protein-2 in dominant and subordinate follicles. Domest Anim Endocrinol 15:55–63[CrossRef][Medline]
389. Olsson JH, Carlsson B, Hillensjo T 1990 Effect of insulin-like growth factor I on deoxyribonucleic acid synthesis in cultured human granulosa cells. Fertil Steril 54:1052–1057[Medline]
390. Angervo M, Koistinen R, Suikkari AM, Seppala M 1991 Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor 1 in human granulosa-luteal cells. Hum Reprod 6:770–773[Abstract/Free Full Text]
391. Yong EL, Baird DT, Yates R, Reichert Jr LE, Hillier SG 1992 Hormonal regulation of the growth and steroidogenic function of human granulosa cells. J Clin Endocrinol Metab 74:842–849[Abstract]
392. Wood AM, Lambert A, Hooper MA, Mitchell GG, Robertson WR 1994 Exogenous steroids and the control of oestradiol secretion by human granulosa-lutein cells by follicle stimulating hormone and insulin-like growth factor-I. Hum Reprod 9:19–23[Abstract/Free Full Text]
393. Erickson GF, Garzo VG, Magoffin DA 1989 Insulin-like growth factor-I regulates aromatase activity in human granulosa and granulosa luteal cells. J Clin Endocrinol Metab 69:716–724[Abstract/Free Full Text]
394. Erickson GF, Magoffin DA, Cragun JR, Chang RJ 1990 The effects of insulin and insulin-like growth factors-I and -II on estradiol production by granulosa cells of polycystic ovaries. J Clin Endocrinol Metab 70:894–902[Abstract/Free Full Text]
395. Tapanainen J, Leinonen PJ, Tapanainen P, Yamamoto M, Jaffe RB 1987 Regulation of human granulosa-luteal cell progesterone production and proliferation by gonadotropins and growth factors. Fertil Steril 48:576–580[Medline]
396. Poretsky L, Chun B, Liu HC, Rosenwaks Z 1996 Insulin-like growth factor II (IGF-II) inhibits insulin-like growth factor binding protein I (IGFBP-1) production in luteinized human granulosa cells with a potency similar to insulin-like growth factor I (IGF-I). J Clin Endocrinol Metab 81:4312–3414
397. Bergh C, Olsson JH, Hillensjo T 1991 Effect of insulin-like growth factor I on steroidogenesis in cultured human granulosa cells. Acta Endocrinol (Copenh) 125:177–185[Abstract/Free Full Text]
398. Christman GM, Randolph Jr JF, Peegel H, Menon KM 1991 Differential responsiveness of luteinized human granulosa cells to gonadotropins and insulin-like growth factor I for induction of aromatase activity. Fertil Steril 55:1099–1105[Medline]
399. Mason HD, Willis D, Holly JMP, Cwyfan-Hughes SC, Seppala M, Franks S 1992 Inhibitory effects of insulin-like growth factor-binding proteins on steroidogenesis by human granulosa cells in culture. Mol Cell Endocrinol 89:R1–R4
400. Erickson GF, Garzo VG, Magoffin DA 1991 Progesterone production by human granulosa cells cultured in serum free medium: effects of gonadotrophins and insulin-like growth factor I (IGF-I). Hum Reprod 6:1074–1081[Abstract/Free Full Text]
401. Kamada S, Kubota T, Taguchi M, Ho WR, Sakamoto S, Aso T 1992 Effects of insulin-like growth factor-II on proliferation and differentiation of ovarian granulosa cells. Horm Res 37:141–149[Medline]
402. Mason HD, Willis DS, Holly JMP, Franks S 1994 Insulin preincubation enhances insulin-like growth factor-II (IGF-II) action on steroidogenesis in human granulosa cells. J Clin Endocrinol Metab 78:1265–1267[Abstract]
403. Cataldo NA, Woodruff TK, Giudice LC 1993 Regulation of insulin-like growth factor binding protein production by human luteinizing granulosa cells cultured in defined medium. J Clin Endocrinol Metab 76:207–215[Abstract]
404. Di Blasio AM, Vigano P, Ferrari A 1994 Insulin-like growth factor-II stimulates human granulosa-luteal cell proliferation in vitro. Fertil Steril 61:483–487[Medline]
405. Steinkampf MP, Mendelson CR, Simpson ER 1988 Effects of epidermal growth factor and insulin-like growth factor I on the levels of mRNA encoding aromatase cytochrome P-450 of human ovarian granulosa cells. Mol Cell Endocrinol 59:93–99[CrossRef][Medline]
406. Hillier SG, Yong EL, Illingworth PJ, Baird DT, Schwall RH, Mason AJ 1991 Effect of recombinant activin on androgen synthesis in cultured human thecal cells. J Clin Endocrinol Metab 72:1206–1211[Abstract/Free Full Text]
407. Fiad TM, Smith TP, Cunningham SK, McKenna TJ 1998 Decline in insulin-like growth factor I levels after clomiphene citrate does not correct hyperandrogenemia in polycystic ovary syndrome. J Clin Endocrinol Metab 83:2394–2398[Abstract/Free Full Text]
408. Gomez E, Tarin JJ, Pellicer A 1993 Oocyte maturation in humans: the role of gonadotropins and growth factors. Fertil Steril 60:40–46[Medline]
409. Adashi EY 1995 Insulin-like growth factors as determinants of follicular fate. J Soc Gynecol Invest 2:721–726[CrossRef][Medline]
410. Erickson GF, Nakatani A, Liu XJ, Shimasaki S, Ling N 1994 The role of IGF-I and IGFBPs in folliculogenesis. In: Findlay JK (ed) Molecular Biology of the Female Reproductive System. Academic Press, New York, pp 101–127
411. Magoffin DA, Weitsman SR 1994 Insulin-like growth factor-I regulation of luteinizing hormone (LH) receptor messenger ribonucleic acid expression and LH-stimulated signal transduction in rat ovarian theca-interstitial cells. Biol Reprod 51:766–775[Abstract]
412. Cara JF, Fan J, Azzarello J, Rosenfield RL 1990 Insulin-like growth factor-I enhances luteinizing hormone binding to rat ovarian theca-interstitial cells. J Clin Invest 86:560–565
413. Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W, Hsueh AJ 1986 Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119:2711–2719[Abstract/Free Full Text]
414. Aloi JA, Dalkin AC, Schwartz NB, Yasin M, Mann B, Haisenleder DJ, Marshall JC 1995 Ovarian inhibin subunit gene expression: regulation by gonadotropins and estradiol. Endocrinology 136:1227–1232[Abstract]
415. Li D, Kubo T, Kim H, Shimasaki S, Erickson GF 1998 Endogenous insulin-like growth factor-I is obligatory for stimulation of rat inhibin -subunit expression by follicle-stimulating hormone. Biol Reprod 58:219–225[Abstract/Free Full Text]
416. Bley MA, Simon JC, Estevez AG, de Asua LJ, Baranao JL 1992 Effect of follicle-stimulating hormone on insulin-like growth factor-I-stimulated rat granulosa cell deoxyribonucleic acid synthesis. Endocrinology 131:1223–1229[Abstract/Free Full Text]
417. Tilly JL, Kowalski KI, Schomberg DW, Hsueh AJ 1992 Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase. Endocrinology 131:1670–1676[Abstract/Free Full Text]
418. Yuan W, Giudice LC 1997 Programmed cell death in human ovary is a function of follicle and corpus luteum status. J Clin Endocrinol Metab 82:3148–3155[Abstract/Free Full Text]
419. Baranao JL, Hammond JM 1984 Comparative effects of insulin and insulin-like growth factors on DNA synthesis and differentiation of porcine granulosa cells. Biochem Biophys Res Commun 124:484–490[CrossRef][Medline]
420. Singh B, Armstrong DT 1997 Insulin-like growth factor-1, a component of serum that enables porcine cumulus cells to expand in response to follicle-stimulating hormone in vitro. Biol Reprod 56:1370–1375[Abstract]
421. Garmey JC, Day RN, Veldhuis JD 1993 Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II: in vitro studies with swine granulosa cells. Endocrinology 133:800–808[Abstract/Free Full Text]
422. Rohan RM, Ricciarelli E, Kiefer MC, Resnick CE, Adashi EY 1993 Rat ovarian insulin-like growth factor-binding protein-6: a hormonally regulated theca-interstitial-selective species with limited antigonadotropic activity. Endocrinology 132:2507–2512[Abstract/Free Full Text]
423. Monget P, Monniaux D, Durand P 1989 Localization, characterization, and quantification of insulin-like growth factor-I-binding sites in the ewe ovary. Endocrinology 125:2486–2493[Abstract/Free Full Text]
424. Armstrong DG, Hogg CO, Campbell BK, Webb R 1996 Insulin-like growth factor (IGF)-binding protein production by primary cultures of ovine granulosa and theca cells: the effects of IGF-I, gonadotropin, and follicle size. Biol Reprod 55:1163–1171[Abstract]
425. Ballard FJ, Baxter RC, Binoux M, Clemmons DR, Drop SLS, Hall K, Hintz RL, Rechler MM, Rutanen EM, Schwander J 1990 Report on the nomenclature of the IGF binding proteins. J Clin Endocrinol Metab 70:817–818[Abstract/Free Full Text]
426. Ballard FJ, Baxter RC, Binoux M, Clemmons DR, Drop SLS, Hall K, Hintz RL, Rechler MM, Rutanen EM, Schwander J, Ling N, Mohan S, Spencer EM, Zapf J 1992 Report on the nomenclature of the IGF-binding proteins. J Clin Endocrinol Metab 74:1215–1216
427. Baxter RC, Binoux MA, Clemmons DR, Conover CA, Drop SL, Holly JM, Mohan S, Oh Y, Rosenfeld RG 1998 Recommendations for nomenclature of the insulin-like growth factor binding protein superfamily. Endocrinology 139:4036[Free Full Text]
428. Rosenfeld R 1998 Editorial: The blind men and the elephant—a parable for the study of insulin-like growth factor binding proteins. Endocrinology 139:5–7[Free Full Text]
429. Rajaram S, Baylink DJ, Mohan S 1997 Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocr Rev 18:801–831[Abstract/Free Full Text]
430. Collett-Solberg PF, Cohen P 1996 The role of the insulin-like growth factor binding proteins and the IGFBP proteases in modulating IGF action. Endocrinol Metab Clin North Am 25:591–614[CrossRef][Medline]
431. Mason HD, Willis DS, Watson H, Galea R, Brincat M, Franks S, Holly JMP, Inhibition of human granulosa cell steroidogenesis by IGF binding protein-4 (IGFBP-4) is independent of its effect on endogenous insulin-like growth factor (IGF). Program of the 80th Annual Meeting of The Endocrine Society, New Orleans, LA, 1998 (Abstract P1–302)
432. Mohan S, Nakao Y, Honda Y, Landale E, Leser U, Dony C, Lang K, Baylink DJ 1995 Studies on the mechanisms by which insulin-like growth factor IGF binding protein-4 (IGFBP-4) and IGFBP-5 modulate IGF actions in bone cells. J Biol Chem 270:20424–20431[Abstract/Free Full Text]
433. Peng X, Maruo T, Samoto T, Mochizuki M 1996 Comparison of immunocytologic localization of insulin-like growth factor binding protein-4 in normal and polycystic ovary syndrome human ovaries. Endocr J 43:269–278[Medline]
434. Seppala M, Wahlstrom T, Koskimies AI, Tenhunen A, Rutanen EM, Koistinen R, Huhtaniemi I, Bohn H, Stenman UH 1984 Human preovulatory follicular fluid, luteinized cells of hyperstimulated preovulatory follicles, and corpus luteum contain placental protein 12. J Clin Endocrinol Metab 58:505–510[Abstract/Free Full Text]
435. Chandrasekher YA, Clark CR, Faessen GH, Giudice LC Role of insulin-like growth factor binding protein-4 (IGFBP-4) and IGFBP-4 protease in human granulosa cell function. Proceedings of the 28th Annual Meeting of the Society for the Study of Reproduction, 1995, Davis, CA (Abstract 102)
436. Cwyfan-Hughes S, Mason HD, Franks S, Holly JMP 1997 Modulation of the insulin-like growth factor-binding proteins by follicle size in the human ovary. J Endocrinol 154:35–43[Abstract/Free Full Text]
437. Chandrasekher YA, Clark CR, Faessen GH, Giudice LC 1995 Insulin-like growth factor binding protein profile in theca explant cultures from normal human ovaries. Program of the 77th Annual Meeting of The Endocrine Society, Washington DC, 1995 (Abstract 166)
438. Mason HD, Cwyfan-Hughes SC, Heinrich G, Franks S, Holly JMP 1996 Insulin-like growth factor (IGF) I and II, IGF-binding proteins, and IGF-binding protein proteases are produced by theca and stroma of normal and polycystic human ovaries. J Clin Endocrinol Metab 81:276–284[Abstract]
439. Jalkanen J, Suikkari AM, Koistinen R, Butzow R, Ritvos O, Seppala M, Ranta T 1989 Regulation of insulin-like growth factor-binding protein-1 production in human granulosa-luteal cells. J Clin Endocrinol Metab 69:1174–1179[Abstract/Free Full Text]
440. Koistinen R, Suikkari AM, Tiitinen A, Kontula K, Seppala M 1990 Human granulosa cells contain insulin-like growth factor-binding protein (IGFBP-1) mRNA. Clin Endocrinol (Oxf) 32:635–640[Medline]
441. Giudice LC, Milki AA, Milkowski DA, el-Danasouri I 1991 Human granulosa contain messenger ribonucleic acids encoding insulin-like growth factor-binding proteins (IGFBPs) and secrete IGFBPs in culture. Fertil Steril 56:475–480[Medline]
442. Cataldo NA, Fujimoto VY, Jaffe RB 1995 Activin-A stimulates the expression of insulin-like growth factor binding protein-5 messenger RNA in human luteinizing granulosa cells. Recent Prog Horm Res 50:437–442
443. Cataldo NA, Fujimoto VY, Jaffe RB 1998 Interferon- and activin-A promote insulin-like growth factor-binding protein-2 and -4 accumulation by human luteinizing granulosa cells, and interferon- promotes their apoptosis. J Clin Endocrinol Metab 83:179–186[Abstract/Free Full Text]
444. Suikkari AM, Jalkanen J, Koistinen R, Butzow R, Ritvos O, Ranta T, Seppala M 1989 Human granulosa cells synthesize low molecular weight insulin-like growth factor-binding protein. Endocrinology 124:1088–1090[Abstract/Free Full Text]
445. Sarvas K, Angervo M, Koistinen R, Tiitinen A, Seppala M 1994 Prostaglandin F2 stimulates release of insulin-like growth factor binding protein-3 from cultured human granulosa-luteal cells. Hum Reprod 9:1643–1646[Abstract/Free Full Text]
446. San Roman GA, Magoffin DA 1993 Insulin-like growth factor-binding proteins in healthy and atretic follicles during natural menstrual cycles. J Clin Endocrinol Metab 76:625–632[Abstract]
447. Fielder PJ, Pham H, Adashi EY, Rosenfeld RG 1993 Insulin-like growth factors (IGFs) block FSH-induced proteolysis of IGF-binding protein-5 in cultured rat granulosa cells. Endocrinology 133:415–418[Abstract/Free Full Text]
448. Liu XJ, Malkowski M, Guo Y, Erickson GF, Shimasaki S, Ling N 1993 Development of specific antibodies to rat insulin-like growth factor-binding proteins (IGFBP’s) 2 to -6: analysis of IGFBP production by rat granulosa cells. Endocrinology 132:1176–1183[Abstract/Free Full Text]
449. Dor J, Costritsci N, Pariente C, Rabinovici J, Mashiach S, Lunenfeld B, Kaneti H, Seppala M, Koistinen R, Karasik A 1992 Insulin-like growth factor-I and follicle-stimulating hormone suppress insulin-like growth factor binding protein-I secretion by human granulosa-luteal cells. J Clin Endocrinol Metab 75:969–971[Abstract]
450. Angervo M, Koistinen R, Seppala M 1992 Epidermal growth factor stimulates production of insulin-like growth factor-binding protein-1 in human granulosa-luteal cells. J Endocrinol 134:127–131[Abstract/Free Full Text]
451. Adachi T, Iwashita M, Kuroshima A, Takeda Y 1995 Regulation of IGF binding proteins by FSH in human luteinizing granulosa cells. J Assist Reprod Genet 12:639–643[CrossRef][Medline]
452. Holst N, Kierulf KH, Seppala M, Koistinen R, Jacobsen MB 1997 Regulation of insulin-like growth factor-binding protein-1 and progesterone secretion from human granulosa-luteal cells: effects of octreotide and insulin. Fertil Steril 68:478–482[CrossRef][Medline]
453. Yap OWS, Chandrasekher YA, Giudice LC 1998 Growth factor regulation of insulin-like growth factor binding protein secretion by cultured human granulosa-luteal cells. Fertil Steril 70:535–540[CrossRef][Medline]
454. Mimuro T, Smith H, Iwashita M, Illingworth PJ 1998 The somatostatin analogue, octreotide, modifies both steroidogenesis and IGFBP-1 secretion in human luteinizing granulosa cells. Hum Reprod 13:150–153[Abstract/Free Full Text]
455. Mason HD, Margara R, Winston RML, Seppala M, Koistinen R, Franks S 1993 Insulin-like growth factor-1 (IGF-1) inhibits production of IGF-binding protein-1 while stimulating estradiol secretion in granulosa cells from normal and polycystic human ovaries. J Clin Endocrinol Metab 76:1275–1279[Abstract]
456. Hamori M, Blum WF, Stehle R, Waibel E, Cledon P, Ranke MB 1991 Immunoreactive insulin-like growth factor binding protein-3 in the culture of human luteinized granulosa cells. Acta Endocrinol (Copenh) 124:685–691[Abstract/Free Full Text]
457. Chandrasekher YA, Van Dessel HJ, Fauser BC, Giudice LC 1995 Estrogen- but not androgen-dominant human ovarian follicular fluid contains an insulin-like growth factor binding protein-4 protease. J Clin Endocrinol Metab 80:2734–2739[Abstract]
458. Holly JMP, Eden JA, Alaghband-Zadeh J, Carter GD, Jemmott RC, Cianfarani S, Chard T, Wass JA 1990 Insulin-like growth factor binding proteins in follicular fluid from normal dominant and cohort follicles, polycystic and multicystic ovaries. Clin Endocrinol (Oxf) 33:53–64[Medline]
459. Cataldo NA, Giudice LC 1992 Insulin-like growth factor binding protein profiles in human ovarian follicular fluid correlate with follicular functional status. J Clin Endocrinol Metab 74:821–829[Abstract]
460. Schuller AG, Lindenbergh-Kortleve DJ, Pache TD, Zwarthoff EC, Fauser BC, Drop SL 1993 Insulin-like growth factor binding protein-2, 28 kDa and 24 kDa insulin-like growth factor binding protein levels are decreased in fluid of dominant follicles, obtained from normal and polycystic ovaries. Regul Pept 48:157–163[CrossRef][Medline]
461. Huang ZH, Matson P, Lieberman BA, Morris ID 1994 Insulin-like growth factor binding proteins in serum and follicular fluid from women undergoing ovarian stimulation with and without growth hormone. Hum Reprod 9:1421–1426[Abstract/Free Full Text]
462. Hartshorne GM, Bell SC, Waites GT 1990 Binding proteins for insulin-like growth factors in the human ovary: identification, follicular fluid levels and immunohistological localization of the 29–32 kd type 1 binding protein, IGF-bp1. Hum Reprod 5:649–660[Abstract/Free Full Text]
463. Chang SY, Hsieh KC, Wang HS, Soong YK 1994 Follicular fluid levels of insulin-like growth factor I, insulin-like growth factor binding protein I, and ovarian steroids collected during ovum pick-up. Fertil Steril 62:1162–1167[Medline]
464. Huang ZH, Baxter RC, Hughes SM, Matson PL, Lieberman BA, Morris ID 1993 Supplementary growth hormone treatment of women with poor ovarian response to exogenous gonadotrophins: changes in serum and follicular fluid insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 (IGFBP-3). Hum Reprod 8:850–857[Abstract/Free Full Text]
465. Gargosky SE, Pham HM, Wilson KF, Liu F, Giudice LC, Rosenfeld RG 1992 Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: discrepancies between radioimmunoassay and ligand blotting. Endocrinology 131:3051–3060[Abstract/Free Full Text]
466. Baxter RC, Saunders H 1992 Radioimmunoassay of insulin-like growth factor-binding protein-6 in human serum and other body fluids. J Endocrinol 134:133–139[Abstract/Free Full Text]
467. Hamori M, Blum WF, Torok A, Stehle R, Waibel E, Cledon P, Ranke MB 1991 Insulin-like growth factors and their binding proteins in human follicular fluid. Hum Reprod 6:313–318[Abstract/Free Full Text]
468. Nakatani A, Shimasaki S, Erickson GF, Ling N 1991 Tissue-specific expression of four insulin-like growth factor-binding proteins (1, 2, 3, and 4) in the rat ovary. Endocrinology 129:1521–1529[Abstract/Free Full Text]
469. Erickson GF, Nakatani A, Ling N, Shimasaki S 1992 Localization of insulin-like growth factor-binding protein-5 messenger ribonucleic acid in rat ovaries during the estrous cycle. Endocrinology 130:1867–1878[Abstract/Free Full Text]
470. Erickson GF, Nakatani A, Ling N, Shimasaki S 1992 Cyclic changes in insulin-like growth factor-binding protein-4 messenger ribonucleic acid in the rat ovary. Endocrinology 130:625–636[Abstract/Free Full Text]
471. Putowski L, Rohan RM, Choi DS, Scherzer WJ, Ricciarelli E, Mordacq J, Mayo KE, Adashi EY 1997 Rat ovarian insulin-like growth factor binding protein-4: a hormone-dependent granulosa cell-derived antigonadotropin. J Soc Gynecol Invest 4:144–151[CrossRef][Medline]
472. Liu XJ, Ling N 1993 Regulation of IGFBP-4 and -5 expression in rat granulosa cells. Adv Exp Med Biol 343:367–376[Medline]
473. Erickson GF, Li D, Sadrkhanloo R, Liu XJ, Shimasaki S, Ling N 1994 Extrapituitary actions of gonadotropin-releasing hormone: stimulation of insulin-like growth factor-binding protein-4 and atresia. Endocrinology 134:1365–1372[Abstract/Free Full Text]
474. Billig H, Furuta I, Hsueh AJ 1994 Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinology 134:245–252[Abstract/Free Full Text]
475. Adashi EY, Resnick CE, Rosenfeld RG 1994 IGF-I stimulates granulosa cell-derived insulin-like growth factor binding protein-5: evidence for mediation via type IGF-I receptors. Mol Cell Endocrinol 99:279–284[CrossRef][Medline]
476. Onoda N, Li D, Mickey G, Erickson G, Shimasaki S 1995 Gonadotropin-releasing hormone overcomes follicle-stimulating hormone’s inhibition of insulin-like growth factor-5 synthesis and promotion of its degradation in rat granulosa cells. Mol Cell Endocrinol 110:17–25[CrossRef][Medline]
477. Erickson GF, Li DM, Ling N, Shimasaki S 1996 Growth factor regulation of insulin-like growth factor binding protein-4 in rat granulosa cells. In: LeRoith D (ed) The Role of Insulin-Like Growth Factors in Ovarian Physiology. Ares-Serono Symposia, Rome, pp 145–151
478. Choi D, Rohan RM, Rosenfeld RG, Matsumoto T, Gargosky SE, Adashi EY 1997 Activin-attenuated expression of transcripts encoding granulosa cell-derived insulin-like growth factor binding proteins 4 and 5 in the rat: a putative antiatretic effect. Biol Reprod 56:508–515[Abstract]
479. Ricciarelli E, Hernandez ER, Hurwitz A, Kokia E, Rosenfeld RG, Schwander J, Adashi EY 1991 The ovarian expression of the antigonadotropic insulin-like growth factor binding protein-2 is theca-interstitial cell-selective: evidence for hormonal regulation. Endocrinology 129:2266–2268[Abstract/Free Full Text]
480. Ricciarelli E, Hernandez ER, Tedeschi C, Botero LF, Kokia E, Rohan RM, Rosenfeld RG, Albiston AL, Herington AC, Adashi EY 1992 Rat ovarian insulin-like growth factor binding protein-3: a growth hormone-dependent theca-interstitial cell-derived antigonadotropin. Endocrinology 130:3092–3094[Abstract/Free Full Text]
481. Erickson GF, Nakatani A, Ling N, Shimasaki S 1993 Insulin-like growth factor binding protein-3 gene expression is restricted to involuting corpora lutea in rat ovaries. Endocrinology 133:1147–1157[Abstract/Free Full Text]
482. Zhou J, Adesanya OO, Vatzias G, Hammond JM, Bondy CA 1996 Selective expression of insulin-like growth factor system components during porcine ovary follicular selection. Endocrinology 137:4893–4901[Abstract]
483. Samaras SE, Guthrie HD, Barber JA, Hammond JM 1993 Expression of the mRNAs for the insulin-like growth factors and their binding proteins during development of porcine ovarian follicles. Endocrinology 133:2395–2398[Abstract/Free Full Text]
484. Grimes RW, Hammond JM 1992 Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells. Endocrinology 131:553–558[Abstract/Free Full Text]
485. Grimes RW, Samaras SE, Barber JA, Shimasaki S, Ling N, Hammond JM 1992 Gonadotropin and cAMP modulation of IGF binding protein production in ovarian granulosa cells. Am J Physiol 262:E497–503
486. Grimes RW, Barber JA, Shimasaki S, Ling N, Hammond JM 1994 Porcine ovarian granulosa cells secrete insulin-like growth factor-binding proteins-4 and -5 and express their messenger ribonucleic acids: regulation by follicle-stimulating hormone and insulin-like growth factor-1. Biol Reprod 50:695–701[Abstract]
487. Grimes RW, Guthrie HD, Hammond JM 1994 Insulin-like growth factor-binding protein-2 and -3 are correlated with atresia and preovulatory maturation in the porcine ovary. Endocrinology 135:1996–2000[Abstract]
488. Mondschein JS, Etherton TD, Hammond JM 1991 Characterization of insulin-like growth factor-binding proteins of porcine ovarian follicular fluid. Biol Reprod 44:315–320[Abstract]
489. Howard HJ, Ford JJ 1992 Relationships among concentrations of steroids, inhibin, insulin-like growth factor-I (IGF-I), and IGF-binding proteins during follicular development in weaned sows. Biol Reprod 47:193–201[Abstract]
490. Guthrie HD, Grimes RW, Hammond JM 1995 Changes in insulin-like growth factor-binding protein-2 and -3 in follicular fluid during atresia of follicles grown after ovulation in pigs. J Reprod Fertil 104:225–230[Abstract/Free Full Text]
491. Teissier MP, Monget P, Monniaux D, Durand P 1994 Changes in insulin-like growth factor-II/mannose-6-phosphate receptor during growth and atresia of ovine ovarian follicles. Biol Reprod 50:111–119[Abstract]
492. Perks CM, Wathes DC 1996 Expression of mRNA’s for insulin-like growth factor binding proteins-2, -3 and -4 in the ovine ovary throughout the oestrous cycle. J Endocrinol 151:241–249[Abstract/Free Full Text]
493. Besnard N, Pisselet C, Monniaux D, Locatelli A, Benne F, Gasser F, Hatey F, Monget P 1996 Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles. Biol Reprod 55:1356–1367[Abstract]
494. Manikkam M, Rajamahendran R 1997 Progesterone-induced atresia of the proestrous dominant follicle in the bovine ovary: changes in diameter, insulin-like growth factor system, aromatase activity, steroid hormones, and apoptotic index. Biol Reprod 57:580–587[Abstract]
495. Funston RN, Seidel Jr GE, Klindt J, Roberts AJ 1996 Insulin-like growth factor I and insulin-like growth factor-binding proteins in bovine serum and follicular fluid before and after the preovulatory surge of luteinizing hormone. Biol Reprod 55:1390–1396[Abstract]
496. Stewart RE, Spicer LJ, Hamilton TD, Keefer BE, Dawson LJ, Morgan GL, Echternkamp SE 1996 Levels of insulin-like growth factor (IGF) binding proteins, luteinizing hormone and IGF-I receptors, and steroids in dominant follicles during the first follicular wave in cattle exhibiting regular estrous cycles. Endocrinology 137:2842–2850[Abstract]
497. Armstrong DG, Baxter G, Gutierrez CG, Hogg CO, Glazyrin AL, Campbell BK, Bramley TA, Webb R 1998 Insulin-like growth factor binding protein -2 and -4 messenger ribonucleic acid expression in bovine ovarian follicles: effect of gonadotropins and developmental status. Endocrinology 139:2146–2154[Abstract/Free Full Text]
498. Giudice LC, Farrell EM, Pham H, Lamson G, Rosenfeld RG 1990 Insulin-like growth factor binding proteins in maternal serum throughout gestation and in the puerperium: effects of a pregnancy-associated serum protease activity. J Clin Endocrinol Metab 71:806–816[Abstract/Free Full Text]
499. Hossenlopp P, Segovia B, Lassarre C, Roghani M, Bredon M, Binoux M 1990 Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150 k complex during pregnancy. J Clin Endocrinol Metab 71:797–805[Abstract/Free Full Text]
500. Fielder PJ, Thordarson G, Talamantes F, Rosenfeld RG 1990 Characterization of insulin-like growth factor binding proteins (IGFBPs) during gestation in mice: effects of hypophysectomy and an IGFBP-specific serum protease activity. Endocrinology 127:2270–2280[Abstract/Free Full Text]
501. Davenport ML, Pucilowska J, Clemmons DR, Lundblad R, Spencer JA, Underwood LE 1992 Tissue-specific expression of insulin-like growth factor binding protein-3 protease activity during rat pregnancy. Endocrinology 130:2505–2512[Abstract/Free Full Text]
502. Binoux M, Lalou C, Lassarre C, Blat C, Hossenlopp P 1993 Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3): a physiological mechanism in the regulation of IGF bioavailability. Adv Exp Med Biol 343:293–300[Medline]
503. Kuebler B, Cowell S, Zapf J, Braulke T 1998 Proteolysis of insulin-like growth factor binding proteins by a novel 50-kilodalton metalloproteinase in human pregnancy serum. Endocrinology 139:1556–1563[Abstract/Free Full Text]
504. Lamson G, Giudice LC, Cohen P, Liu F, Gargosky S, Mueller HL, Oh Y, Wilson KF, Hintz RL, Rosenfeld RG 1993 Proteolysis of IGFBP-3 may be a common regulatory mechanism of IGF action in vivo. Growth Regul 3:91–95[Medline]
505. Chernausek SD, Smith CE, Duffin KL, Busby WH, Wright G, Clemmons DR 1995 Proteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4): localization of cleavage site to non-homologous region of native IGFBP-4. J Biol Chem 270:11377–11382[Abstract/Free Full Text]
506. Blat C, Villaudy J, Binoux M 1994 In vivo proteolysis of serum insulin-like growth factor IGF binding protein-3 results in increased availability of IGF to target cells. J Clin Invest 93:2286–2290
507. Cohen P, Peehl DM, Graves HC, Rosenfeld RG 1994 Biological effects of prostate specific antigen as an insulin-like growth factor binding protein-3 protease. J Endocrinol 142:407–415[Abstract/Free Full Text]
508. Conover CA 1992 Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: studies of underlying mechanism. Endocrinology 130:3191–3199[Abstract/Free Full Text]
509. Besnard N, Pisselet C, Monniaux D, Monget P 1997 Proteolytic activity degrading insulin-like growth factor-binding protein-2, -3, -4, and -5 in healthy growing and atretic follicles in the pig ovary. Biol Reprod 56:1050–1058[Abstract]
510. Besnard N, Pisselet C, Zapf J, Hornebeck W, Monniaux D, Monget P 1996 Proteolytic activity is involved in changes in intrafollicular insulin-like growth factor-binding protein levels during growth and atresia of ovine ovarian follicles. Endocrinology 137:1599–1607[Abstract]
511. Monget P, Monniaux D 1995 Growth factors and the control of folliculogenesis. J Reprod Fertil Suppl 49:321–333
512. Mason HD, Cwyfan-Hughes S, Holly JMP, Franks S 1998 Potent inhibition of human ovarian steroidogenesis by insulin-like growth factor binding protein-4 (IGFBP-4). J Clin Endocrinol Metab 83:284–287[Abstract/Free Full Text]
513. Iwashita M, Kudo Y, Yoshimura Y, Adachi T, Katayama E, Takeda Y 1996 Physiological role of insulin-like-growth-factor-binding protein-4 in human folliculogenesis. Horm Res 46 [Suppl 1]:31–36
514. Donnelly MJ, Holly JMP 1996 The role of IGFBP-3 in the regulation of IGFBP-4 proteolysis. J Endocrinol 149:R1–R7
515. Iwashita M, Adachi T, Kudo Y, Takeda Y 1995 Gonadotropins regulate insulin-like growth factor binding proteins in human luteinizing granulosa cells. In: Fujimoto S, Hsueh AJ, Strauss III JF, Tanaka T (eds) New Achievements in Research of Ovarian Function. Ares-Serono Symposia, Rome, pp 141–146
516. Mason HD, Cwyfan-Hughes S, Voutilainen R, Martikainen H, Franks S, Holly J 1996 Insulin-like growth factors (IGF) and IGF binding proteins (IGFBP) in normal and polycystic ovaries (PCO). In: LeRoith D (ed) The Role of Insulin-Like Growth Factors in Ovarian Physiology. Ares-Serono Symposia, Rome, pp 207–217
517. Shimasaki S, Murakami K, Morita Y, Erickson GF 1996 Regulation of IGFBP-4 proteolysis by FSH in rat granulosa cells. In: LeRoith D (ed) The Role of Insulin-Like Growth Factors in Ovarian Physiology. Ares-Serono Symposia, Rome, pp 133–143
518. Resnick CE, Fielder PJ, Rosenfeld RG, Adashi EY 1998 Characterization and hormonal regulation of a rat ovarian insulin-like growth factor binding protein-5 endopeptidase: an FSH-inducible granulosa cell-derived metalloprotease. Endocrinology 139:1249–1257[Abstract/Free Full Text]
519. Bicsak TA, Shimonaka M, Malkowski M, Ling N 1990 Insulin-like growth factor-binding protein (IGF-BP) inhibition of granulosa cell function: effect on cyclic adenosine 3',5'-monophosphate, deoxyribonucleic acid synthesis, and comparison with the effect of an IGF-I antibody. Endocrinology 126:2184–2189[Abstract/Free Full Text]
520. Adashi EY, Resnick CE, Ricciarelli E, Hurwitz A, Hernandez ER Insulin-like growth factor (IGF) binding protein-1 is an antigonadotropin: evidence that optimal FSH action is contingent upon amplification by endogenously-derived IGFs. Program of the 38th Annual Meeting of the Society for Gynecologic Investigation, San Antonio, TX, 1991 (Abstract 429)
521. Ui M, Shimonaka M, Shimasaki S, Ling N 1989 An insulin-like growth factor-binding protein in ovarian follicular fluid blocks follicle-stimulating hormone-stimulated steroid production by ovarian granulosa cells. Endocrinology 125:912–916[Abstract/Free Full Text]
522. Adashi EY, Resnick CE, Ricciarelli E, Hurwitz A, Kokia E, Tedeschi C, Botero L, Hernandez ER, Rosenfeld RG, Carlsson-Skwirut C 1992 Granulosa cell-derived insulin-like growth factor (IGF) binding proteins are inhibitory to IGF-I hormonal action: evidence derived from the use of a truncated IGF-I analogue. J Clin Invest 90:1593–1599
523. Bicsak TA, Ling N, DePaolo LV 1991 Ovarian intrabursal administration of insulin-like growth factor-binding protein inhibits follicle rupture in gonadotropin-treated immature female rats. Biol Reprod 44:599–603[Abstract]
524. Yoshimura Y, Nagamatsu S, Ando M, Iwashita M, Oda T, Katsumata Y, Shiokawa S, Nakamura Y 1996 Insulin-like growth factor binding protein-3 inhibits gonadotropin-induced ovulation, oocyte maturation, and steroidogenesis in rabbit ovary. Endocrinology 137:438–446[Abstract]
525. Huang H, Rajkumar K, Murphy LJ 1997 Reduced fecundity in insulin-like growth factor-binding protein-1 transgenic mice. Biol Reprod 56:284–289[Abstract]
526. Iwashita M, Adachi T, Katayama E, Kudo Y, Takeda Y 1994 Regulation and physiological role of insulin-like-growth-factor-binding protein-I in human granulosa cells. Horm Res 41:22–28
527. Barreca A, Artini PG, Cesarone A, Arvigo M, D’Ambrogio G, Genazzani AR, Giordano G, Minuto F 1996 Interrelationships between follicle stimulating hormone and the growth hormone-insulin-like growth factor (IGF)-binding proteins axes in human granulosa cells in culture. J Endocrinol Invest 19:35–42[Medline]
528. Giudice LC 1995 The insulin-like growth factor system in normal and abnormal human ovarian follicle development. Am J Med [Suppl]98:48S–54S
529. Eisenhauer KM, Chun SY, Billig H, Hsueh AJ 1995 Growth hormone suppression of apoptosis in preovulatory rat follicles and partial neutralization by insulin-like growth factor binding protein. Biol Reprod 53:13–20[Abstract]
530. Adashi EY, Resnick CE, Hernandez ER, Svoboda ME, Van Wyk JJ 1988 In vivo regulation of granulosa cell somatomedin-C/insulin-like growth factor I receptors. Endocrinology 122:1383–1389[Abstract/Free Full Text]
531. Hernandez ER, Hurwitz A, Botero L, Ricciarelli E, Werner H, Roberts Jr CT, LeRoith D, Adashi EY 1991 Insulin-like growth factor receptor gene expression in the rat ovary: divergent regulation of distinct receptor species. Mol Endocrinol 5:1799–1805[Abstract/Free Full Text]
532. Morton-Bours E, Batey D, Suen LF, Yuan W, Giudice LC, Regulation of insulin-like growth factor-II expression in human granulosa-luteal cells by human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). Proceedings of the 16th World Congress on Fertility and Sterility, San Francisco, CA, 1998 (Abstract P-873)
533. Cataldo NA, Giudice LC 1992 Follicular fluid insulin-like growth factor binding protein profiles in polycystic ovary syndrome. J Clin Endocrinol Metab 74:695–697[Abstract]
534. Magoffin DA 1992 Insulin-like growth factor binding proteins in ovarian follicles from women with polycystic ovarian disease: cellular source and levels in follicular fluid. J Clin Endocrinol Metab 75:1010–1016[Abstract]
535. Franks S 1995 Polycystic ovary syndrome. N Engl J Med 333:853–861[Free Full Text]
536. Goldzieher JW, Young RL 1992 Selected aspects of polycystic ovarian disease. Endocrinol Metab Clin North Am 21:141–171[Medline]
537. Stein IF, Leventhal ML 1935 Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynecol 29:181–191
538. Dunaif A 1995 Hyperandrogenic anovulation (PCOS): a unique disorder of insulin action associated with an increased risk of non-insulin-depedent diabetes mellitus. Am J Med [Suppl] 98:33S–39S[CrossRef]
539. Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R 1998 Prevalence of the polycystic ovary syndrome in unselected black and white women of the Southeastern United States: a prospective study. J Clin Endocrinol Metab 83:3078–3082[Abstract/Free Full Text]
540. Lobo RA 1995 A disorder without identity: "HCA," "PCO," "PCOD," "PCOS," "SLS". What are we to call it? Fertil Steril 63:1158–1160[Medline]
541. Zawadzki JK, Dunaif A 1995 Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In: Dunaif A (ed) Polycystic Ovary Syndrome. Blackwell Scientific, Boston, pp 377–384
542. Carmina E, Koyama T, Chang L, Stanczyk FZ, Lobo RA 1992 Does ethnicity influence the prevalence of adrenal hyperandrogenism and insulin resistance in polycystic ovary syndrome? Am J Obstet Gynecol 167:1807–1812[Medline]
543. Dunaif A, Sorbara L, Delson R, Green G 1993 Ethnicity and polycystic ovary syndrome are associated with independent and additive decreases in insulin action in Caribbean-Hispanic women. Diabetes 42:1462–1468[Abstract]
544. Yen SS 1980 The polycystic ovary syndrome. Clin Endocrinol (Oxf) 12:177–207[Medline]
545. Judd HL, Rigg LA, Anderson DC, Yen SS 1976 The effects of ovarian wedge resection on circulating gonadotropin and ovarian steroid levels in patients with polycystic ovary syndrome. J Clin Endocrinol Metab 43:347–355[Abstract/Free Full Text]
546. Greenblatt E, Casper RF 1987 Endocrine changes after laparoscopic ovarian cautery in polycystic ovarian syndrome. Am J Obstet Gynecol 156:279–285[Medline]
547. Macleod AF, Wheeler MJ, Gordon P, Lowy C, Sonksen PH, Conaglen JV 1990 Effect of long-term inhibition of gonadotrophin secretion by the gonadotrophin-releasing hormone agonist, buserelin, on sex steroid secretion and ovarian morphology in polycystic ovary syndrome. J Endocrinol 125:317–325[Abstract/Free Full Text]
548. Genazzani AD, Petraglia F, Battaglia C, Gamba O, Volpe A, Genazzani AR 1997 A long-term treatment with gonadotropin-releasing hormone agonist plus a low-dose oral contraceptive improves the recovery of the ovulatory function in patients with polycystic ovary syndrome. Fertil Steril 67:463–468[CrossRef][Medline]
549. Ghadirian AM, Chouinard G, Annable L 1982 Sexual dysfunction and plasma prolactin levels in neuroleptic-treated schizophrenic outpatients. J Nerv Ment Dis 170:463–467[Medline]
550. Lobo RA, Kletzky OA 1983 Normalization of androgen and sex hormone-binding globulin levels after treatment of hyperprolactinemia. J Clin Endocrinol Metab 56:562–566[Abstract/Free Full Text]
551. New MI 1993 Nonclassical congenital adrenal hyperplasia and the polycystic ovarian syndrome. Ann NY Acad Sci 687:193–205[Medline]
552. Barnes RB, Rosenfield RL, Ehrmann DA, Cara JF, Cuttler L, Levitsky LL, Rosenthal IM 1994 Ovarian hyperandrogenism as a result of congenital adrenal virilizing disorders: evidence for perinatal masculinization of neuroendocrine function in women. J Clin Endocrinol Metab 79:1328–1333[Abstract]
553. Kazer RR, Kessel B, Yen SS 1987 Circulating luteinizing hormone pulse frequency in women with polycystic ovary syndrome. J Clin Endocrinol Metab 65:233–236[Abstract/Free Full Text]
554. Berga SL, Guzick DS, Winters SJ 1993 Increased luteinizing hormone and -subunit secretion in women with hyperandrogenic anovulation. J Clin Endocrinol Metab 77:895–901[Abstract]
555. Berga SL, Daniels TL 1997 Can polycystic ovary syndrome exist without concomitant hypothalamic dysfunction? Semin Reprod Endocrinol 15:169–175[Medline]
556. Cheung AP, Lu JK, Chang RJ 1997 Pulsatile gonadotrophin secretion in women with polycystic ovary syndrome after gonadotrophin-releasing hormone agonist treatment. Hum Reprod 12:1156–1164
557. Daniels TL, Berga SL 1997 Resistance of gonadotropin releasing hormone drive to sex steroid-induced suppression in hyperandrogenic anovulation. J Clin Endocrinol Metab 82:4179–4183[Abstract/Free Full Text]
558. Pastor CL, Griffin-Korf ML, Aloi JA, Evans WS, Marshall JC 1998 Polycystic ovary syndrome: evidence for reduced sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone. J Clin Endocrinol Metab 83:582–590[Abstract/Free Full Text]
559. Zumoff B, Freeman R, Coupey S, Saenger P, Marrowitz M, Kream J 1983 A chronobiologic abnormality in luteinizing hormone secretion in teenage girls with the polycystic-ovary syndrome. N Engl J Med 309:1206–1209[Abstract]
560. Lanzone A, Apa R, Fulghesu AM, Cutillo G, Caruso A, Mancuso S 1993 Long-term naltrexone treatment normalizes the pituitary response to gonadotropin-releasing hormone in polycystic ovarian syndrome. Fertil Steril 59:734–737[Medline]
561. Cumming DC, Reid RL, Quigley ME, Rebar RW, Yen SS 1984 Evidence for decreased endogenous dopamine and opioid inhibitory influences on LH secretion in polycystic ovary syndrome. Clin Endocrinol (Oxf) 20:643–648[Medline]
562. Legro RS, Muhleman DR, Comings DE, Lobo RA, Kovacs BW 1995 A dopamine D3 receptor genotype is associated with hyperandrogenic chronic anovulation and resistant to ovulation induction with clomiphene citrate in female Hispanics. Fertil Steril 63:779–784[Medline]
563. Brzechffa PR, Jakimiuk AJ, Agarwal SK, Weitsman SR, Buyalos RP, Magoffin DA 1996 Serum immunoreactive leptin concentrations in women with polycystic ovary syndrome. J Clin Endocrinol Metab 81:4166–4169[Abstract/Free Full Text]
564. Mantzoros CS, Dunaif A, Flier JS 1997 Leptin concentrations in the polycystic ovary syndrome. J Clin Endocrinol Metab 82:1687–1691[Abstract/Free Full Text]
565. Laughlin GA, Morales AJ, Yen SS 1997 Serum leptin levels in women with polycystic ovary syndrome: the role of insulin resistance/hyperinsulinemia. J Clin Endocrinol Metab 82:1692–1696[Abstract/Free Full Text]
566. Rouru J, Anttila L, Koskinen P, Penttila TA, Irjala K, Huupponen R, Koulu M 1997 Serum leptin concentrations in women with polycystic ovary syndrome. J Clin Endocrinol Metab 82:1685–1686[Free Full Text]
567. Chapman IM, Wittert GA, Norman RJ 1997 Circulating leptin concentrations in polycystic ovary syndrome: relation to anthropometric and metabolic parameters. Clin Endocrinol (Oxf) 46:175–181[CrossRef][Medline]
568. Caro JF 1997 Leptin is normal in PCOS: an editorial about three "negative" papers. J Clin Endocrinol Metab 82:1685
569. Vicennati V, Gambineri A, Calzoni F, Casimirri F, Macor C, Vettor R, Pasquali R 1998 Serum leptin in obese women with polycystic ovary syndrome is correlated with body weight and fat distribution but not with androgen and insulin levels. Metabolism 47:988–992[CrossRef][Medline]
570. Taylor AE, Martin KA, Hall JE, Evidence for a reproductive role of leptin: correlation with LH pulse amplitude but not frequency in normal women and polycystic ovary syndrome. Program of the 79th Annual Meeting of The Endocrine Society, Minneapolis, MN, 1997 (Abstract OR38–6)
571. Ibanez L, Hall JE, Potau N, Carrascosa A, Prat N, Taylor AE 1996 Ovarian 17-hydroxyprogesterone hyperresponsiveness to gonadotropin-releasing hormone (GnRH) agonist challenge in women with polycystic ovary syndrome is not mediated by luteinizing hormone hypersecretion: evidence from GnRH agonist and human chorionic gonadotropin stimulation testing. J Clin Endocrinol Metab 81:4103–4107[Abstract/Free Full Text]
572. Gilling-Smith C, Story H, Rogers V, Franks S 1997 Evidence for a primary abnormality of thecal cell steroidogenesis in the polycystic ovary syndrome. Clin Endocrinol (Oxf) 47:93–99[CrossRef][Medline]
573. Barnes RB, Rosenfield RL, Burstein S, Ehrmann DA 1989 Pituitary-ovarian responses to nafarelin testing in the polycystic ovary syndrome. N Engl J Med 320:559–565[Abstract]
574. White D, Leigh A, Wilson C, Donaldson A, Franks S 1995 Gonadotrophin and gonadal steroid response to a single dose of a long-acting agonist of gonadotrophin-releasing hormone in ovulatory and anovulatory women with polycystic ovary syndrome. Clin Endocrinol (Oxf) 42:953–954
575. Ehrmann DA, Barnes RB, Rosenfield RL 1995 Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev 16:322–353[Abstract/Free Full Text]
576. Zhang LH, Rodriguez H, Ohno S, Miller WL 1995 Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome. Proc Natl Acad Sci USA 92:10619–10623[Abstract/Free Full Text]
577. Dunaif A, Book CB, Schenker E, Tang Z 1995 Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle: a potential mechanism for insulin resistance in the polycystic ovary syndrome. J Clin Invest 96:801–810
578. Erickson GF, Magoffin DA, Garzo VG, Cheung AP, Chang RJ 1992 Granulosa cells of polycystic ovaries: are they normal or abnormal? Hum Reprod 7:293–299[Abstract/Free Full Text]
579. San Roman GA, Magoffin DA 1992 Insulin-like growth factor binding proteins in ovarian follicles from women with polycystic ovarian disease: cellular source and levels in follicular fluid. J Clin Endocrinol Metab 75:1010–1016
580. Schneyer AL, Sluss PM, Whitcomb RW, Martin KA, Sprengel R, Crowley Jr WF 1991 Precursors of -inhibin modulate follicle-stimulating hormone receptor binding and biological activity. Endocrinology 129:1987–1999[Abstract/Free Full Text]
581. Lambert-Messerlian GM, Isaacson K, Crowley Jr WF, Sluss P, Schneyer AL 1994 Human follicular fluid contains pro- and C-terminal immunoreactive -inhibin precursor proteins. J Clin Endocrinol Metab 78:433–439[Abstract]
582. Erickson GF, Yen SSC 1984 New data on follicle cells in polycystic ovaries: a proposed mechanism for the generation of cystic follicles. Semin Reprod Endocrinol 2:231–243
583. Ibanez L, Potau N, Virdis R, Zampolli M, Terzi C, Gussinye M, Carrascosa A, Vicens-Calvet E 1993 Postpubertal outcome in girls diagnosed of premature pubarche during childhood: increased frequency of functional ovarian hyperandrogenism. J Clin Endocrinol Metab 76:1599–1603[Abstract]
584. Ibanez L, Potau N, Zampolli M, Street ME, Carrascosa A 1997 Girls diagnosed with premature pubarche show an exaggerated ovarian androgen synthesis from the early stages of puberty: evidence from gonadotropin-releasing hormone agonist testing. Fertil Steril 67:849–855[CrossRef][Medline]
585. Oppenheimer E, Linder B, DiMartino-Nardi J 1995 Decreased insulin sensitivity in prepubertal girls with premature adrenarche and acanthosis nigricans. J Clin Endocrinol Metab 80:614–618[Abstract]
586. Ibanez L, Potau N, Zampolli M, Prat N, Virdis R, Vicens-Calvet E, Carrascosa A 1996 Hyperinsulinemia in postpubertal girls with a history of premature pubarche and functional ovarian hyperandrogenism. J Clin Endocrinol Metab 81:1237–1243[Abstract]
587. Moghetti P, Castello R, Negri C, Tosi F, Spiazzi GG, Brun E, Balducci R, Toscano V, Muggeo M 1996 Insulin infusion amplifies 17-hydroxycorticosteroid intermediates response to adrenocorticotropin in hyperandrogenic women: apparent relative impairment of 17,20-lyase activity. J Clin Endocrinol Metab 81:881–886[Abstract]
588. Bergman RN, Finegood DT, Ader M 1985 Assessment of insulin sensitivity in vivo. Endocr Rev 6:45–86[Abstract/Free Full Text]
589. Dunaif A, Segal KR, Futterweit W, Dobrjansky A 1989 Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 38:1165–1174[Abstract]
590. Dunaif A 1993 Insulin resistance and ovarian dysfunction. In: Moller DE (ed) Insulin Resistance. John Wiley & Sons, New York, pp 301–325
591. Rajkhowa M, Bicknell J, Jones M, Clayton RN 1994 Insulin sensitivity in women with polycystic ovary syndrome: relationship to hyperandrogenemia. Fertil Steril 61:605–612[Medline]
592. Conway GS, Jacobs HS, Holly JMP, Wass JA 1990 Effects of luteinizing hormone, insulin, insulin-like growth factor-I and insulin-like growth factor small binding protein 1 in the polycystic ovary syndrome. Clin Endocrinol (Oxf) 33:593–603[Medline]
593. Ovesen P, Moller J, Ingerslev HJ, Jorgensen JO, Mengel A, Schmitz O, Alberti KG, Moller N 1993 Normal basal and insulin-stimulated fuel metabolism in lean women with the polycystic ovary syndrome. J Clin Endocrinol Metab 77:1636–1640[Abstract]
594. Franks S, Gharani N, Waterworth D, Batty S, White D, Williamson R, McCarthy M 1997 The genetic basis of polycystic ovary syndrome. Hum Reprod 12:2641–2648[Abstract/Free Full Text]
595. Carey AH, Chan KL, Short F, White D, Williamson R, Franks S 1993 Evidence for a single gene effect causing polycystic ovaries and male pattern baldness. Clin Endocrinol (Oxf) 38:653–658[Medline]
596. Waterworth DM, Bennett ST, Gharani N, McCarthy MI, Hague S, Batty S, Conway GS, White D, Todd JA, Franks S, Williamson R 1997 Linkage and association of insulin gene VNTR regulatory polymorphism with polycystic ovary syndrome. Lancet 349:986–990[CrossRef][Medline]
597. Talbot JA, Bicknell EJ, Rajkhowa M, Krook A, O’Rahilly S, Clayton RN 1996 Molecular scanning of the insulin receptor gene in women with polycystic ovarian syndrome. J Clin Endocrinol Metab 81:1979–1983[Abstract]
598. Sorbara LR, Tang Z, Cama A, Xia J, Schenker E, Kohanski RA, Poretsky L, Koller E, Taylor S, Dunaif A 1994 Absence of insulin receptor gene mutations in three insulin-resistant women with the polycystic ovary syndrome. Metabolism 43:1568–1574[CrossRef][Medline]
599. Conway GS, Avey C, Rumsby G 1994 The tyrosine kinase domain of the insulin receptor gene is normal in women with hyperinsulinaemia and polycystic ovary syndrome. Hum Reprod 9:1681–1683[Abstract/Free Full Text]
600. Taylor SI, Kadowaki T, Accili D, Cama A, Kadowaki H, McKeon C, Moncada V, Marcus-Samuels B, Bevins C, Ojamaa K, Frapier C, Beitz L, Perrotti N, Rees-Jones R, Margolis R, Imano E, Najjar S, Courtney F, Araraki R, Gorden P, Roth J 1990 Mutations in the insulin receptor gene in genetic forms of insulin resistance. Recent Prog Horm Res 46:185–213
601. Cama A, Sierra ML, Kadowaki T, Kadowaki H, Quon MJ, Rudiger HW, Dreyer M, Taylor SI 1995 Two mutant alleles of the insulin receptor gene in a family with a genetic form of insulin resistance: a 10 base pair deletion in exon 1 and a mutation substituting serine for asparagine-462. Hum Genet 95:174–182[Medline]
602. Harrison LC, Dean B, Peluso I, Clark S, Ward G 1985 Insulin resistance, acanthosis nigricans, and polycystic ovaries associated with a circulating inhibitor of postbinding insulin action. J Clin Endocrinol Metab 60:1047–1052[Abstract/Free Full Text]
603. Ciaraldi TP, El-Roeiy A, Madar Z, Reichart D, Olefsky JM, Yen SS 1992 Cellular mechanisms of insulin resistance in polycystic ovarian syndrome. J Clin Endocrinol Metab 65:577–583
604. Dunaif A, Segal KR, Shelley DR, Green G, Dobrjansky A, Licholai T 1992 Evidence for distinctive and intrinsic defects in insulin action in polycystic ovary syndrome. Diabetes 41:1257–1266[Abstract]
605. Marsden PJ, Murdoch A, Taylor R 1994 Severe impairment of insulin action in adipocytes from amenorrheic subjects with polycystic ovary syndrome. Metabolism 43:1536–1542[CrossRef][Medline]
606. Ciaraldi TP, Morales AJ, Hickman MG, Odom-Ford R, Yen SSC, Olefsky JM 1998 Lack of insulin resistance in fibroblasts from subjects with polycystic ovary syndrome. Metabolism 47:940–946[CrossRef][Medline]
607. Ek I, Arner P, Bergqvist A, Carlstrom K, Wahrenberg H 1997 Impaired adipocyte lipolysis in nonobese women with the polycystic ovary syndrome: a possible link to insulin resistance? J Clin Endocrinol Metab 82:1147–1153[Abstract/Free Full Text]
608. Rosenbaum D, Haber RS, Dunaif A 1993 Insulin resistance in polycystic ovary syndrome: decreased expression of GLUT-4 glucose transporters in adipocytes. Am J Physiol 264:E197–202
609. Ciaraldi TP, Morales AJ, Hickman MG, Odom-Ford R, Olefsky JM, Yen SSC 1997 Cellular insulin resistance in adipocytes from obese polycystic ovary syndrome subjects involves adenosine modulation of insulin sensitivity. J Clin Endocrinol Metab 82:1421–1425[Abstract/Free Full Text]
610. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM 1995 15-Deoxy-delta 12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR-. Cell 83:803–812[CrossRef][Medline]
611. Lambe KG, Tugwood JD 1996 A human peroxisome-proliferator-activated receptor- is activated by inducers of adipogenesis, including thiazolidinedione drugs. Eur J Biochem 239:1–7