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Division of Endocrinology, Diabetes and Metabolism (A.D.M., M.J.H.), Department of Internal Medicine, St. Louis University School of Medicine, St. Louis, Missouri 63104; and Department of Medicine and Biochemistry & Molecular Biology (N.C.W.W.), University of Calgary, Alberta, Canada T2N 1N4
Correspondence: Address all correspondence and requests for reprints to: Arshag D. Mooradian, M.D., Division of Endocrinology, St. Louis University, 1402 South Grand Boulevard, St. Louis, Missouri 63104. E-mail: mooradad{at}slu.edu
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Abstract |
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 Top Abstract I. IntroductionII. Biological Functions of...III. Effects of Various...IV. ConclusionsReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Biological Functions of...III. Effects of Various...IV. ConclusionsReferences |
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The changes in plasma HDL in response to alterations in dietary intake of fat have been largely attributed to changes in fractional clearance of HDL. Thus, reducing dietary intake of saturated fat and cholesterol is associated with reduced plasma cholesterol content of various lipoproteins including low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and HDL (3, 13, 14, 15). The reduced cholesterol content of HDL accelerates its clearance. The lack of compensatory increase in HDL production in the face of increasing HDL clearance causes decreased plasma levels of HDL. In contrast, the increased HDL clearance in subjects with familial hypercholesterolemia is associated with compensatory increase in HDL production (16). Thus, it is possible that low-fat diets may also interfere with HDL production.
Several studies have documented major in vivo and in vitro interactions between various nutrients and the expression of lipoprotein genes (17). In the present communication, we will briefly review the potential mechanisms for altered apolipoprotein A-I (apo A-I) expression in response to changes in nutrient flux.
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II. Biological Functions of HDL and Its Apolipoproteins |
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TopAbstractI. IntroductionII. Biological Functions of...III. Effects of Various...IV. ConclusionsReferences |
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The cardioprotective advantage of HDL2 relative to HDL3 is controversial (18, 19). Similarly, the role of apo A-II in promoting or inhibiting the cholesterol efflux is not clear (20). Apo A-II inhibits LCAT and impairs hepatic cholesterol uptake through its effects on SR-B1 receptor. These are potentially deleterious effects. However, apo A-II also may have beneficial effects because it inhibits cholesteryl-ester transfer protein (CETP) and increases the hepatic lipase activity (20).
There are multiple mechanisms of atheroprotective effects of HDL and apo A-I (Table 1
). These mechanisms will be discussed briefly.
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. The effects of various nutrients on these individual steps of RCT remain to be elucidated.
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III. Effects of Various Nutrients on HDLc and apo A-I |
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TopAbstractI. IntroductionII. Biological Functions of...III. Effects of Various...IV. ConclusionsReferences |
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. There are multiple mechanisms by which various nutrients alter HDLc or apo A-I levels. It is likely that the cardioprotective properties of HDLc or apo A-I may depend on the underlying mechanisms responsible for increasing their plasma levels. In this communication the effects of nutrients on apo A-I gene expression will be discussed in the context of overall changes in apo A-I or HDLc levels.
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). Various transcription factors assemble into multiprotein complexes on key regulatory regions of the gene (22). General transcription factors (TFs) associated with polymerase II (Pol II), such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH are recognized by core promoter sequences and are responsible for the basal expression of the gene. The multistep assembly of these factors involved in apo AI gene modulation is shown in Fig. 3. The silent apo AI promoter contains closely spaced nucleosomes with site A located on the surface of the histone octamer. A nuclear receptor-retinoid X receptor heterodimer binds to site A. This step is accompanied by a loosening of chromatin structure, a process that is accelerated by the recruitment of one or more nuclear transcriptional coactivator(s) such as FoxA/HNF-3 (hepatocyte nuclear factor 3) and HNF-4 (hepatocyte nuclear factor 4) that bind on the chromatin and recruit chromatin-modifying complexes such as SSC, Swi/SNF, and histone acetyl transferase (22). Acetylation of the histone tails reduces the affinity of the histones for the DNA allowing for dissociation and sliding of the nucleosomes. The region of DNA in the apo A-I promoter that is nucleosome free binds to the transcription initiation complex, which contains several TFs and RNA Pol II. This is further coupled to the upstream nuclear receptor dimer through a mediator protein constituting the active apo A-I promoter. Subsequently, Pol II will start the process of RNA synthesis.
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Bile acids also regulate apo A-I gene expression. Bile acids have been shown to suppress plasma apo A-I and hepatic mRNA levels in human apo A-I transgenic mice, and to suppress apo A-I promoter activity in primary human hepatocytes and HepG2 cells (29). Transcriptional repression requires activation of the farnesoid X receptor (FXR) and a negative FXR response element in site C of the apo A-I promoter (29). On the other hand, the orphan nuclear receptor, liver receptor homolog-1 (LRH-1), which has been shown to regulate expression of genes involved in bile acid synthesis and RCT, induces apo A-I transcription through the same region (30). Therefore, bile acids may have multiple effects on apo A-I expression depending on the levels of FXR, LRH-1, and their required coactivators.
The expression of the apo A-I gene is subject to regulation by several hormone and metabolic signaling pathways. The promoter region is endowed with a host of regulatory elements that are likely to respond to signals generated by nutrients. The myriad of changes that may occur in apo A-I gene expression related to various nutrients is summarized in Table 3.
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One of the first comprehensive demonstrations of the effect of glucose on gene regulation at a pretranslational level was a study performed in primary hepatocytes treated with various concentrations of glucose and insulin (34). Changes in the steady-state levels of many mRNA species were quantified after separation of in vitro translational products with two-dimensional gel electrophoresis (34). One of the mRNAs that was down-regulated with high concentration of glucose was spot 11. This protein spot was later determined to be identical to apo A-I (35). The fact that apo A-I is suppressed with high glucose concentrations was further supported with experiments in diabetic rats (26). Hepatic expression of apo A-I protein and its mRNA is reduced in streptozotocin-induced diabetic rats (26). However this reduction in apo A-I expression could be attributed to multiple alterations inherent to streptozotocin-induced diabetes in addition to hyperglycemia.
To further study the mechanisms by which glucose regulates apo A-I levels, we have used the human hepatoma cell line HepG2. This cell line expresses insulin receptor and glucose transporter proteins and retains the ability to synthesize and secrete apo A-I as well as other proteins produced by normal hepatocytes. Treatment of HepG2 cells with 22.4 mM dextrose for 48 h results in a 50% reduction in apo A-I mRNA levels whereas treatment with 100 µU/ml of insulin causes a 2-fold increase in apo A-I mRNA levels relative to control cells maintained in media containing 5.5 mM dextrose (26). Treatment of the cells with both high concentrations of dextrose and insulin was associated with an intermediate response of 1.3-fold induction of apo A-I mRNA. The transcriptional activity of apo A-I promoter is also suppressed by dextrose and stimulated by insulin in a dose-dependent fashion (26). A single cis-acting element within the promoter located between nucleotides –425 and –376, regulates the effects of both insulin and glucose on transcriptional activity of apo A-I gene. However, only insulin, but not dextrose, alters IRCE binding activity. This suggests that there are additional carbohydrate-responsive elements remaining to be characterized, and that the IRCE may have only a permissive role for the inhibitory effects of glucose (26). It is also possible that a unique carbohydrate-responsive element overlaps the IRCE and interferes with its ability to respond to insulin.
Although insulin sensitization with thiazolidendiones may not always be sufficient to induce apo A-I expression (36, 37), insulinomimetics, such as bisperoxo (1,10-phenathroline) oxovanadate and the protein kinase C activator phorbol-12,13-dibutyrate, up-regulate apo A-I promoter activity by increasing the binding of transcription factor Sp1 to the IRCE (38). There are at least two signaling pathways by which insulin modulates apo A-I promoter activity. One is through Ras-raf activation of MAPK and the other is phosphatidylinositol-3-kinase-dependent activation of protein kinase C kinases (38). Finally, phosphorylation of Sp1 plays a critical role in insulin or certain insulinomimetic-induced apo A-I expression by regulating the ability of Sp1 to bind the IRCE (39).
2. Effect of fructose.
The effect of monosaccharides other than dextrose on apo A-I gene expression is not well studied. Reiser et al. (40) found that a diet enriched with fructose increases the levels of risk factors associated with heart disease, especially in hyperinsulinemic men. However, 60 g fructose/d incorporated in the normal diets of 13 poorly controlled, type 2 diabetic patients did not significantly alter the fasting serum lipids, lipoproteins, and apolipoproteins A-1 and B-100 levels (41).
Feeding rats a sucrose-rich diet did not affect the abundance of cellular and nuclear apo A-I and apo C-III mRNA or the transcriptional activity of these genes in liver, although apo A-IV mRNA increased significantly (33). These results are consistent with specialization of the regulatory elements of the genes coding for apo A-I, C-III, and A-IV. These genes are encoded for at the same locus within the genome and may share some regulatory elements (33).
In HepG2 cells treated with fructose, no significant changes in apo A-I expression could be demonstrated (our unpublished data). In vivo, feeding rats a high-fructose diet (60% of diet weight as fructose) for 10 d was associated with a significant increase in hepatic apo A-I m RNA concentrations compared with rats maintained on regular chow (42). This increase could have been secondary to profound hyperinsulinemia or to increased intracellular glucosamine levels (see below). Alternatively, increased apo A-I in fructose-fed rats may have been secondary to increased hepatic triglyceride content. Indeed, it has been shown in studies of cebus monkeys that as liver lipid content increases, hepatic apo A-I mRNA concentrations increase (43).
3. Effect of glucosamine.
Conversion of fructose-6-phosphate to glucosamine-6-phosphate by the rate-limiting enzyme glutamine: fructose-6-phosphate amidotransferase has been proposed as a mechanism by which intracellular glucose levels can serve as a nutrient sensor and modulate satiety (44). Conversion of glucosamine-6-phosphate to uridine diphosphate-N-acetylglucosamine provides a substrate necessary for nearly all glycosylation pathways, including those that regulate transcriptional activity (45, 46). The intracellular glucosamine level increases in periods of hyperglycemia when glutamine: fructose-6-phosphate amidotransferase is increased. Thus, glucosamine is a potential mediator of insulin resistance (47, 48).
It has recently been demonstrated that Sp1 transcriptional activity and half-life are regulated by glycosylation on serine and threonine residues with N-acetylglucosamine, the primary end product of elevated glucosamine levels (45, 49). Because Sp1 is also involved in the transcriptional modulation of apo A-I gene by insulin (27), we hypothesized that glucosamine may alter apo A-I gene expression. This hypothesis was not supported by the experimental data. Thus, treatment of HepG2 cells with 0.1, 1, or 3 mM glucosamine increased the amount of apo A-I protein secreted into the culture media 1.8-fold, 5.5-fold, and 2.3-fold, respectively (50). The decline in apo A-I secretion at the highest glucosamine levels was attributed to cell death caused by very high concentrations of glucosamine. Apo A-I mRNA levels increased 2.4-fold in hepatocytes treated with 1 mM glucosamine for 24 h, suggesting that the increase in apo A-I protein secretion was due, at least partly, to an increase in apo A-I mRNA levels. However, glucosamine had no effect on apo A-I gene transcription rate as measured either by nuclear runoff analysis or transient transfection analysis of apo A-I promoter activity in HepG2 cells. However, apo A-I mRNA turnover studies showed that treatment of HepG2 cells with 1 mM glucosamine was associated with increased apo A-I mRNA half-life, from 7.6 to 16.6 h. These findings suggest that increases in apo A-I gene expression by glucosamine occur primarily through apo A-I mRNA stabilization whereas the changes in Sp1 were not significant enough to alter apo AI gene transcription (50).
It appears that increased intracellular content of glucosamine, a known mediator of insulin resistance, cannot account for the reduced expression of apo A-I in clinical states of insulin resistance.
C. Effects of lipids on apo A-I
1. Effect of fatty acid saturation.
Several observational and interventional trials have shown that the HDLc level as well as the apo A-I level are increased with consumption of diets enriched with saturated fat. This effect has been mostly attributed to a decrease in the clearance of HDL and possibly to other translational and posttranslational changes in apo A-I gene expression (13). Overall, dietary intake of saturated fatty acids increases plasma HDLc level (3). Compared with saturated fatty acids, dietary polyunsaturated fatty acids up-regulate hepatic SR-B1 expression and increase HDLc ester transport to the liver; as a consequence plasma HDLc level is reduced (14). It is noteworthy that diets enriched in saturated fatty acids, unlike diets enriched in unsaturated fatty acids, are associated with increased HDLc and in apo A-II levels but not in apo A-I levels (15). This effect is gender specific (51, 52). HDLc and apo A-I levels decrease more in hypercholesterolemic women than in men ingesting a National Cholesterol Education Program Step II diet (51). In another study, the effects of a diet low in total fat, saturated fat, and cholesterol were compared with an average American diet on plasma lipoprotein subspecies in men and women. The low-fat diet resulted in greater reductions in total cholesterol and plasma apo B concentrations in men than in women. Postprandial triacylglycerol and LpAI:AII concentrations were reduced in men, but not in women. Similar decreases in LpAI concentrations and LDL and HDL particle size were observed in men and women (52). These data are consistent with the concept that men may have a more favorable lipoprotein response to a low-fat, low-cholesterol diet than postmenopausal women.
The reduction in HDLc and apo A-I reported above may be due to both a reduction in total dietary fat intake and the qualitative change to more polyunsaturated fats in the diet (53). To address this in an animal model, the effects of the amount of dietary fat and saturation together with cholesterol on apo A-I gene expression were studied in male rats (54). Corn oil had a hypocholesterolemic action, whether compared with chow or to cholesterol diet, due to a reduction in HDLc as well as LDL-cholesterol (LDLc). When compared with control or cholesterol diets, plasma apo A-I concentration significantly increased in coconut and 40% olive oil diets. Coconut oil or corn oil diets did not induce any significant change in hepatic apoA-I mRNA compared with control or cholesterol diets. However, compared with the cholesterol diet, 40 and 10% olive oil diets induced a significant increase in the expression of this message. Based on the increased plasma apo A-I levels found in animals consuming the coconut oil and olive oil diets, and the differences between these two diets for mRNA expression, it appears that different fatty acid-containing diets regulate apo A-I through different mechanisms (54).
A high saturated fatty acid diet containing coconut oil elevates plasma HDLc and apo A-I through a mechanism involving increased synthesis (55). Nuclear run-on transcription assays revealed that coconut oil feeding for 4 wk caused a 220% increase in hepatic apoA-I transcription rate compared with controls. However, treatment of cultured rabbit liver cells with various saturated fatty acids and sera from chow-fed and coconut oil-fed rabbits did not alter apo A-I mRNA levels as observed in vivo (55). These data demonstrate that changes in apo A-I expression in cell cultures may not be relevant to the in vivo situation. Caution should be exercised when extrapolating the results of cell culture studies to the intact organism.
Posttranscriptional mechanisms of apo A-I gene expression play an important role in the effect of dietary fat on apo A-I levels. In mice, increased apo A-I production by increased dietary fat was not associated with any increase in hepatic or intestinal apo A-I mRNA, suggesting that the mechanism of the dietary fat effect was posttranscriptional, involving either increased translatability of the apo A-I mRNA or less intracellular apo A-I degradation (56). Another potential mechanism may be through increasing the fraction of apo A-I mRNA in the translating pool (57).
The metabolic consequences of monounsaturated fatty acids (MUFAs) are somewhat different from polyunsaturated fatty acids (PUFAs). In a crossover study, the effects of two fat-reduced diets, one rich in MUFAs, the other rich in PUFAs, on serum lipid profiles in 38 healthy young adults were tested (58). Both test diets led to significant reductions in serum cholesterol, LDLc, and HDLc. Apo A-I was significantly higher on the MUFA than on the PUFA diet. Thus, a low-fat, MUFA-rich diet is as effective as a low-fat, PUFA-rich diet in lowering total cholesterol and LDLc, but both also lowered HDLc concentrations. However, the MUFA-rich diet does not lower apo A-I concentrations as much as the PUFA-rich diet (58).
The dietary effects are species specific. In a study of cynomolgus monkeys, dietary MUFAs were comparable to PUFAs in their effects on hepatic lipid and apo A-I mRNA levels with both unsaturated fats significantly reducing only hepatic apo C-III mRNA abundance relative to saturated fat (59). Similar observations were made in studies of African green monkeys (60). In this species, isocaloric substitution of polyunsaturated fat for saturated fat reduces concentrations of total plasma cholesterol and HDLc. Plasma apo A-I concentration was also reduced by 16% by polyunsaturated fat in the high-cholesterol group. This was attributed to a reduced rate of hepatic apo A-I secretion and reduced hepatic apo A-I mRNA concentrations. In contrast, intestinal apo A-I mRNA concentrations were not altered by the type of dietary fat. Plasma apo A-II and hepatic apo A-II mRNA concentrations also were not altered by the type of dietary fat. Thus, dietary polyunsaturated fat can selectively alter the expression of the apo A-I gene in a tissue-specific manner (60).
The mechanisms by which saturated fatty acids and cholesterol raise plasma HDLc levels are unknown. In rats and mice, the high-cholesterol diet did not alter plasma HDLc levels (61). However, the high-fat diet increased HDLc levels by 20% in rats and 55% in mice. A combination diet of saturated fat and cholesterol also raised plasma HDLc levels by 36% and 67% in rats and mice, respectively. Plasma apo A-I levels increased parallel to HDLc concentrations. The rat hepatic apo A-I mRNA was decreased on high-fat and fat-cholesterol combination diets, but the translational efficiency of apo A-I on isolated polysomes was significantly increased (61). These results suggest that transcriptional regulation of the apo A-I gene was not responsible for increased plasma apo A-I levels on high-fat and fat-cholesterol combination diets (61).
Additional regulation through impaired catabolism of HDL particles by high-fat diet feeding may be another pathway by which HDL levels are increased. Unlike apo A-I mRNA, the mRNA of the other HDL apoproteins, apo A-II and apo A-IV, were increased by high-fat and combination diet feeding. These results suggest that saturated fatty acids regulate plasma HDL levels by translational and posttranslational mechanisms (61). In another study of rats, triolein, but not tripalmitin or menhaden oil, increased the secretion of HDL and its components (apo A-I and lecithin cholesterol acetyl transferase) and stimulated the production of hepatic SR-B1 receptor protein (62). Overall, these results suggest that triolein, unlike tripalmitin, may promote RCT and thus retard the development of atherosclerosis (62).
The effect of the degree of fat saturation on apo A-I transcription has not been well studied. Dietary fatty acids produce diverse effects, both at the transcriptional as well as posttranscriptional levels, and the changes differ according to species and strain (13). Previously published studies have shown that in HepG2 cells, lipid supply is an important determinant of apolipoprotein synthesis (63, 64, 65). In general, unsaturated fatty acids decrease apo A-I synthesis in HepG2 cells (64). We studied the effect of FFA on apo A-I gene transcription in HepG2 cells (65). The results of these studies show that under basal conditions various concentrations of FFA did not alter basal apo A-I mRNA levels or its protein secretion in culture media. However, treatment of cells with saturated FFA abolishes both insulin and Sp-1-stimulated activation of the apo A-I promoter (65). Saturated fatty acids such as stearic acid have an important modulating role in insulin-mediated induction of apo A-I gene expression. Treatment of cells with three saturated fatty acids, stearic acid (C18), myristic acid (C14), and palmitic acid (C16), repressed the ability of exogenous Sp1 to induce apo A-I reporter gene expression. However, the three unsaturated fatty acids, oleic (C18:1), linoleic (C18:2), or linolenic acid (C18:3), had no effect on Sp1-mediated induction of the apo A-I promoter. These results suggest that the ability of FFA to repress Sp1-mediated induction of apo A-I promoter activity is dependent on degree of saturation but not necessarily chain length. Saturated FFAs, notably stearic acid, had no appreciable effect on Sp1-DNA binding (65). Therefore, stearic acid must affect Sp1 function through a mechanism unrelated to regulation of Sp1-DNA binding, most likely by altering posttranslational modification (65).
The results of studies on the effect of mixing monounsaturated or polyunsaturated FFA with saturated FFA may vary depending on the precise combinations and proportions. Nevertheless, the limited set of data we had presented previously suggests that, within the concentration ranges tested, monounsaturated FFA cannot prevent the down-regulation of apo A-I transcription by saturated FFA. It is noteworthy that oxidized fatty acids, unlike nonoxidized fatty acids, may enhance intestinal apo A-I gene expression through peroxisomal proliferation-activating receptors (PPARs) (66). The differential effects of saturated and unsaturated fatty acids on the transcriptional process have been previously recognized (67).
The increased plasma FFA concentration in insulin-resistant states may contribute to the reduced plasma apo A-I levels by blunting insulin-mediated apo A-I gene transcription. It is possible that the apo A-I-inducing effect of fibrates, nicotinic acid, and thiazolidenediones is related, at least in part, to their ability to reduce plasma FFA levels, in addition to their effects on fractional clearance of apo A-I, changes in apo A-I mRNA stability, or transcriptional modulation with PPARs (68, 69, 70, 71, 72, 73, 74).
Overall, it appears that the main effect of fatty acids on apo A-I synthesis in Hep G2 cells occurs only during times when Sp1 is activated, whereas the effects are modest at best during basal conditions when Sp1 is not overexpressed. Unlike saturated fatty acids, the unsaturated fatty acids do not appear to have this potential deleterious effect on apo A-I gene expression. These observations provide an additional rationale for the current dietary recommendations of limiting intake of saturated fat in the diet.
It is noteworthy that genetic variations influence the response of plasma lipids to dietary intervention (75, 76, 77).
2. Effect of trans-fatty acids.
Relative to saturated fatty acids, trans-fatty acids/hydrogenated fat-enriched diets have been reported to increase LDLc levels and either decrease or have no effect on HDLc levels (78). In a study of 36 subjects, three test diets were used as the major sources of fat, namely vegetable oil-based semiliquid margarine, traditional margarine, or butter, for 35-d periods (78). The dietary perturbations had only modest effects on plasma HDLc level but appeared to have a larger effect on particles containing apo A-I only. Differences in CETP, phospholipid transfer protein activity, or the fractional esterification rate of cholesterol in HDL did not account for the differences observed in HDLc levels (78). This study concluded that the saturated fatty acid component of the diet, rather than the trans- or polyunsaturated fatty acid component, was the causative factor in modulating the HDLc response (78). Other studies have found significant potential adverse effects of trans-fatty acids on HDLc levels. In a study of 80 healthy subjects randomized to consume a dairy fat-based (baseline) diet for 5 wk, followed by an experimental diet high in either trans-fatty acids (8.7% of energy; n = 40) or stearic acid (9.3% of energy; n = 40) for another 5 wk, it was found that compared with the dairy fat diet, stearic acid and trans-fatty acids decreased serum total cholesterol concentrations similarly (by 13% and 12%, respectively), but the trans-fatty acid diet decreased HDLc (17%) and apo A-I (15%) significantly more than did the stearic acid diet (11% and 12%, respectively) (79).
Trans-monounsaturated fatty acids (TFA) are hypercholesterolemic compared with oleic acid to a degree approaching or equivalent to saturated fatty acids (80). However, it is unknown to what extent these effects may be due to cholesterol lowering by oleic acid rather than elevation by saturated fatty acids and TFA. In a study of 50 normocholesterolemic men fed for 5 wk in a randomized, 6 x 6 crossover design, subjects received experimental diets in which 8% of caloric energy were replaced with the following: carbohydrate (1:1 simple to complex); oleic acid; TFA; stearic acid; TFA/stearic acid (4% of energy from each); carbon 12:0–16:0 saturated fatty acids (80). Compared with the carbohydrate control diet, TFA raised LDLc at least equivalent to saturated fatty acids but had no effect on HDLc; stearic acid had no effect on LDLc but lowered HDLc; saturated fatty acids raised both LDLc and HDLc; and oleic acid raised HDLc but had no effect on LDLc (80).
To evaluate some of the molecular pathways of the effect of trans fatty acids on apo A-I expression, HepG2 cells were treated with oleic (cis 18:1), elaidic (trans 18:1), or palmitic (16:0) acids (81). The net apo A-I accumulation in the medium was not significantly altered by fatty acids, whereas apo B increased with oleic and elaidic acids (81). We have recently confirmed the lack of effect of trans fatty acids on apo A-I secretion in Hep G2 cell (65). However, we found that trans fatty acids, like saturated fatty acids, blunt Sp1 up-regulation of apo A-I promoter activity. The reduction in apo A-I promoter activity in the presence of trans fatty acids is consistent with the literature on the effect of dietary trans fatty acids on lowering HDLc levels (82, 83).
3. Effect of medium-chain fatty acids (MCFAs).
There are limited studies on the effect of MCFAs on apo A-I. The clinical trials have suggested that MCFAs may raise LDLc concentrations slightly and affect the apo A-I to apo B ratio unfavorably compared with oleic acid (84). In a study of 37 women and 23 men, the MCFA diet significantly decreased the apo A-I to apo B ratio compared with myristic acid, or oleic acid based diets. Myristic acid was hypercholesterolemic, and as expected raised both LDLc and HDLc concentrations compared with oleic acid (84). Overall effects of the MCFAs on plasma lipids and lipoproteins were modest.
4. Effect of n-3 fatty acids.
Epidemiological studies have shown that people who consume diets containing n-3 polyunsaturated (n-3 poly) fatty acids found in fish oils have reduced mortality from heart disease (85, 86). Although this benefit could be attributed to many mechanisms, it may partially be related to the effects of n-3 poly fat on lipoprotein metabolism (87, 88). The triglyceride-lowering effect of n-3 poly fat consumption is well established (89, 90). Unfortunately n-3 fatty acid-induced triglyceride reduction is often associated with a reduction in HDLc. Because ordinarily triglyceride lowering is typically associated with an increase in HDLc (91), it is likely that n-3 fatty acids directly down-regulate HDLc levels. Thus, the mechanism by which dietary fish oil reduces atherosclerosis despite the lowering of HDLc remains to be clarified. It is possible that consuming a diet enriched with n-3 poly fat results in a relative increase in the concentration of smaller HDL3 particles that are more cardioprotective (89, 90). In African green monkeys, n-3 polyunsaturated fat reduces HDLc concentration by increasing the fractional catabolic rate of medium-sized HDL particles (92). In primary hepatocytes, addition of the n-3 PUFAs, docosanohexaenoic acid and eicosanopentaenoic acid, or the fatty acid derivative 
-bromopalmitate, decreased apo A-I and increased acyl-CoA oxidase mRNA in a dose- and time-dependent manner, whereas apo A-II mRNA did not change significantly (93). When rats were fed isocaloric diets enriched in saturated fat (hydrogenated coconut oil), n-6 PUFAs (safflower oil) or n-3 poly (fish oil), a significant decrease in liver apo A-I and apo A-II mRNA levels was only observed after fish oil feeding. Results from these studies indicate that fish oil feeding reduces rat liver apo A-I and apo A-II gene expression, through a direct transcriptional action in the hepatocyte (93).
The effect of a dietary fish oil supplementation on HDLc metabolism was studied in type 2 diabetes mellitus (94). After treatment with dietary fish oil, plasma cholesterol level remained unchanged, whereas plasma triglycerides were decreased. Plasma apo A-I, HDLc, and HDL did not markedly change although apo A-I fractional catabolic rate and absolute production rate were significantly decreased after treatment with fish oil. The effect of n-3 fatty acids on transcriptional regulation of the apo A-I gene has not been well studied.
5. Effect of fat metabolites: ketones and prostanoids.
Although ketoacidosis profoundly suppresses apo A-I gene expression, the precise molecular pathway is not known. The ketoacidosis-associated down-regulation of apo A-I gene could be correlated with the process of ketogenesis but not with the presence of ketone bodies (95). In HepG2 cells, as well as in Caco2 intestinal cells, ketones and their analogs, such as butyrate or isobutyramide, do not significantly alter apo A-I expression (96). In contrast, either acidification of the culture media or altering intracellular pH with specific ionophores and inhibitors consistently suppressed apo A-I promoter activity and apo A-I production (97). The effect of acidosis on apo A-I promoter activity occurs through a pH-responsive element (pH-RE) located within the promoter (97). Acidosis increases the specific DNA binding activity of a putative repressor protein, and the response element is either close to or overlaps a negative thyroid hormone response element that is located 3' and adjacent to and overlaps the apo A-I TATA element (96, 97). This region also contains binding sites for other transcription factors (Fig. 2
) (98) including the thyroid hormone receptor (28), 
-EF1 (99), and AP-1 (100). Repression of apo A-I promoter action by acidosis does not require de novo protein or mRNA synthesis (97). Also, inhibition of tyrosine kinase activity and diacylglycerol-stimulated protein kinase C signaling with tyrophosphotin A47 and phorbol myristate acetate, respectively, did not affect the repressive effect of acidosis on apo A-I promoter activity (97).
However, not all models of ketosis are associated with apo A-I gene suppression. For example, apo A-I mRNA and protein levels are elevated in rats placed on a high-fat ketogenic diet (96). This may reflect primarily the effect of high fat content of the diet. Overall, it appears that acidosis is a required element of apo A-I gene suppression by ketoacidosis and that ketones have a minor role, if any, on apo A-I levels.
Prostanoids are metabolites of arachidonic acid and have been found to have an important role in regulating the expression of various genes. The role of prostanoids in modulating apo A-I gene expression has not been well characterized. Some prostanoids are ligands of PPAR receptors (101, 102), and PPARs are implicated in the regulation of apo A-I expression (103, 104). In addition, prostanoids interfere with insulin action, an important modulator of apo A-I gene expression (105, 106). Thus, it is possible that prostanoids may have a role in modulating apo A-I expression. Recent work in our laboratory suggests that cyclooxygenase (COX) inhibition with indomethacin or acetyl salicylic acid down-regulates apo A-I protein and mRNA expression at the transcriptional level (107). However, treatment of cells with arachidonic acid or a select group of its prostanoid metabolites including prostaglandin I2, thromboxane B2, (±) 5-hydroxyeicosatetraenoic acid (HETE) or (±) 12-HETE, prostaglandin E1 and E2 (107) did not alter apo A-I expression. This raises the possibility that the effect of indomethacin on apo A-I gene expression is independent of COX activity. It is noteworthy that aspirin, but not indomethacin, has been shown to decrease the expression of apolipoprotein (a) (apo a) in human hepatocytes at the transcriptional level (108). This effect was also independent of COX inhibition. Deletion analysis of apo(a) gene promoter showed that promoter region extending from –30 to +138 is critical for the effect of aspirin (108). It is possible that both the apo A-I and apo a genes have aspirin-responsive regions in their promoters.
D. Effects of micronutrients on apo A-I
With increasing consumption of vitamin and mineral supplements, the potential clinical and metabolic consequences on HDLc metabolism must be clarified. In a randomized trial of 69 type 2 diabetic subjects, supplementation with magnesium, zinc, and vitamins C and E significantly increased HDLc and apo A-I levels (109). However, large interventional trials have found that supplementation with antioxidants does not protect against cardiovascular disease. Indeed some studies have found potential adverse effects. One small interventional trial found that antioxidants may partially blunt HDLc induction by simvastatin and niacin combination therapy (110). Vitamins, notably those with antioxidative potential, may affect apo A-I gene expression. The apo A-I promoter is sensitive to the oxidative state of the cell, and some antioxidants, at high concentrations, can suppress apo A-I promoter activity in Hep G2 cells (111). However, high dose (1 g/d) vitamin C supplementation in vivo does not have consistent effects on plasma lipids or lipoprotein levels (112, 113, 114, 115). Thus, it appears that an optimal concentration of these vitamins is needed for apo A-I gene expression. However, supplementation with excessive doses may aggravate the lowered plasma HDLc concentration.
Like vitamins C and E, optimal vitamin D levels are probably necessary to support adequate apo A-I levels. In a normal Belgian population study, those with high vitamin D levels also had the highest plasma apo A-I levels (116). However, vitamin D supplementation in postmenopausal women has been recently shown to be associated with adverse changes in serum lipoproteins and aortic calcification (117, 118). In a recent study, when Hep G2 cells were treated with 1,25-(OH)2D3, apo A-I secretion and mRNA levels were suppressed in a dose-dependent manner (119). This was accompanied by a similar decrease in apo A-I promoter activity (119). This suppression required a region within site A of the apo A-I gene promoter. However, vitamin D treatment did not alter factor binding to site A, and therefore the effect was attributed to secondary alterations in coactivator/corepressor activities. Treatment with the vitamin D receptor antagonist ZK-191784 inhibited the ability of 1,25-(OH)2 D3 to repress apo A-I promoter activity, whereas higher doses of ZK-191784 actually increased apo A-I promoter activity (119).
Finally, vitamin A appears to have significant effects on hepatic, but not intestinal, apo A-I gene transcription, although apo A-I protein content does not change, suggesting additional regulation of posttranscriptional events (120, 121, 122, 123). The abundance of hepatic apo A-I mRNA of vitamin A-deficient rats was 2.2- to 6-fold that of vitamin A-sufficient rats. These data indicate that apo A-I gene expression in vivo is sensitive to retinoid status (120). Treatment of cocultured rat hepatocytes with retinoic acid resulted in a specific decrease in apo A-I mRNA levels whereas no marked difference in apo A-II mRNA levels was observed (122). These results show that, contrary to primary pure hepatocyte cultures and hepatoma cell lines (123), cocultures of rat hepatocytes with rat liver epithelial cells reproduce the in vivo results in which retinoic acid appears to suppress the expression of apo A-I (122).
Some minerals have been found to have an important modulatory role in apo A-I gene expression (124, 125, 126, 127, 128, 129). In addition, chromium, vanadium, magnesium, and zinc have either insulinomimetic effects or permissive effects on insulin action. Thus it is not surprising that minerals can alter apo A-I expression. In particular, zinc deficiency causes down-regulation of apo A-I expression (124, 125) whereas supraphysiological concentrations of zinc as well as chromium or vanadium down-regulate apo A-I promoter activity (128). Some of the effects of zinc deficiency may be explained by the dependence of zinc finger-containing transcription factors, such as Sp1, for the coordinating effect of zinc ions.
Low cellular copper status can enhance apo A-I mRNA production and increase apo A-I synthesis (126, 129). The increase in apo A-I gene transcription was attributed to an elevated level of the regulatory factor, HNF-4 (126). Copper depletion did not alter apo B synthesis. In rats, copper deficiency increases hepatic apo A-I synthesis and secretion but does not alter hepatic total cellular apo A-I mRNA abundance (129). Thus, the enhanced hepatic apo A-I synthesis observed in copper-deficient cells may have resulted from alterations in posttranscriptional and translational processes (129).
Until more clinical data are available for the safety and efficacy of supplementation of micronutrients at pharmacological doses, it is best if their consumption, with few possible exceptions, is limited to the Recommended Daily Allowances.
E. Other nutritional elements modulating apo A-I
1. Cholic acid.
Dietary cholic acid lowers plasma levels of apo A-I primarily via a transcriptional mechanism (130). A putative cis-acting element responsible for the cholic acid-mediated down-regulation of the apo A-I promoter activity has been identified, but its trans-binding factors are not completely defined. Cholic acid increased mRNA levels of the ARP-1, a negative regulator of the apo A-I gene in the mouse. However, this magnitude of change in apo A-I gene transcription alone is not sufficient to account for the lowering of plasma HDLc levels (130).
2. Soy proteins.
Soy is a commonly consumed plant protein that reduces LDLc concentrations. It has been suggested that specific soy components, including isoflavones, may contribute to the hypocholesterolemic effect of soy (131, 132, 133). Isoflavones have structural similarities with estrogen, which has hypocholesterolemic properties and may affect the metabolism of apoproteins. However, in a study of gerbils, consumption of isoflavone did not contribute to the hypocholesterolemic effect of soy (134). To examine the effect of isoflavones on cholesterol metabolism in these animals, isoflavone-free and isoflavone-supplemented soy protein diets were compared. The addition of the isoflavone-enriched alcohol extract to isolated soy protein did not reduce serum cholesterol concentrations any further but reduced hepatic apo A-I mRNA levels compared with casein- and isolated soy protein-fed groups (134). Levels of apo E mRNA were not affected by the diet. These data suggest that, in gerbils, consumption of an isoflavone-containing extract does not contribute to the hypocholesterolemic effect of soy but may influence lipid metabolism by altering the expression of lipid-related genes (134). It should be borne in mind that these effects may be species specific and gender specific.
Soy phytoestrogens have been shown to increase plasma levels of HDLc and apo A-I in animal studies and in some human studies. In Hep G2 cells, both genistein and daidzein increased apo A-I secretion in a dose-dependent fashion (132). The effect of genistein on apo A-I secretion was similar to that observed with 17ß-estradiol. Treatment of cells with genistein for 24 h increased transcriptional activity of the apo A-I gene as measured by nuclear run-on assay. It appears that the MAPK pathway is involved in the regulation of apo A-I gene expression by genistein and E2, possibly through downstream regulation of transcription factors binding to the promoter region (133).
3. Alcohol.
In an observational study, alcohol consumption was associated with an increase of serum levels of both LpA-I and LpA-I:A-II particles, and this may explain, at least in part, the reduced cardiovascular morbidity observed in subjects drinking moderate amounts of alcoholic beverages (135). Moderate alcohol consumption (one to two drinks per day) may decrease cardiovascular disease risk by improving lipid profiles. In postmenopausal women, plasma HDLc increased significantly after daily consumption of 30 g alcohol. In addition, plasma apo A-I increased significantly and apo B decreased significantly (136). Exposure of HepG2 and Hep3B cells to ethanol resulted in an increase of accumulation of apo A-I by 15%–45% in a dose-dependent manner (137). All other major apolipoproteins, which included apo C-II, apo C-III, and apo E, with the exception of apo B, were not affected by these treatments. At a concentration of ethanol of 25 mM or greater, accumulation of apo B, VLDL, and LDL triglyceride was increased by 20–25% over the control level. Elevation of HDLc by 40–70% was observed when the cells were exposed to an ethanol concentration of 10 mM or more. This effect was attributed to a metabolite of ethanol generated by the microsomal ethanol-oxidizing system (137).
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IV. Conclusions |
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The reduced plasma apo A-I levels related to consumption of a low-fat diet are mostly attributed to increased fractional clearance of HDLc (3). However, it is possible that reduced apo A-I expression, due to changes in apo A-I gene expression induced by various nutrients, may contribute to the reduced plasma concentrations of HDLc. Studies elucidating nutrient effects on apo A-I gene expression should help in the rational design of dietary recommendations to increase HDLc level and reduce cardiovascular disease.
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Abbreviations: apo A-I, Apolipoprotein A-I; ARP-1, apo A-I regulatory protein 1; CETP, cholesteryl-ester transfer protein; COX, cyclooxygenase; FXR, farnesoid X receptor; HDL, high-density lipoprotein; HDLc, HDL cholesterol; HNF, hepatocyte nuclear factor; IRCE, insulin response core element; LCAT, lecithin-cholesterol acyltransferase; LDL, low-density lipoprotein; LDLc, LDL cholesterol; Lp, lipoprotein; MCFA, medium-chain fatty acid; MUFA, monounsaturated fatty acid; Pol II, polymerase II; poly, polyunsaturated; PPAR, peroxisomal proliferation-activating receptor; PUFA, polyunsaturated fatty acid; RCT, reverse cholesterol transport; Sp1, specific protein 1; TF, transcription factor; TFA, trans-monounsaturated fatty acid; VLDL, very low-density lipoprotein.
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-3 fatty acids on high-density lipoprotein apolipoprotein AI kinetics in type II diabetes mellitus. Atherosclerosis 157:131–135[CrossRef][Medline]-crystalline enhancer-binding protein -EF1 is a repressor of E2-box-mediated gene activation. Mol Cell Biol 14:5692–5700 and Rev-erb mediate the species-specific regulation of apolipoprotein A1 expression by fibrates. J Biol Chem 273:25713–25720-tocopherol down regulate apolipoprotein AI gene expression in HepG2 and Caco-2 cell lines. Metabolism, in press-3 fatty acids on lipoproteins, apo A-I, apo B, and malondialdehyde in hyperlipidemic patients. Int J Vitam Nutr Res 73:163–170[CrossRef][Medline]This article has been cited by other articles:
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, Increases promoter activity; 
, decreases promoter activity.