The PPARy Gene and Sensitivity to Thiazolidinediones

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Thiazolidinediones are a novel class of antidiabetic medication that exert multiple effects beyond glycemic control and may have beneficial effects on cardiovascular risk factors. They decrease insulin resistance and reduce cardiovascular risk by improving various aspects of the cardiovascular dysmetabolic syndrome. They may reduce accelerated atherosclerosis associated with type 2 DM not only by improving glycemia and decreasing plasma insulin levels but also by increasing high-density lipoprotein (HDL) and decreasing triglyceride levels, improving blood pressure, improving fibrinolysis, and reducing vessel wall abnormalities.

Although discovered more than two decades ago, it was not until the mid-1990s that their molecular mechanism of action was elucidated. Thiazolidinediones are synthetic ligands that activate nuclear receptors called peroxisome proliferator-activated receptors (PPARs). Once activated, the PPARs form heterodimers with another nuclear receptor, the 9-cis-retinoic acid receptor (RXR). By binding to specific DNA sequences, these PPAR/RXR heterodimers regulate transcription and translation of proteins involved in glucose and lipid metabolism (70,71). There are three subtypes of PPARs currently identified: PPARa, PPAR^ (also known as PPAR5, NUC-1, and FAAR), and PPARy. The antidiabetic actions of thiazolidinediones correspond to their ability to activate PPARy receptors found in key target tissues

THE BETA CELL

!. Glucose transported into beta eell by GLUT-2 transporter molecule1

4. ATP acts lot K-ATP channel in a rise in the t potential of the

3. Pyruvate end citric acid cyck mitochondria.

producing ATP

2. Glucose en glycolytic pal metabolizes ti

THE BETA CELL

!. Glucose transported into beta eell by GLUT-2 transporter molecule1

4. ATP acts lot K-ATP channel in a rise in the t potential of the

3. Pyruvate end citric acid cyck mitochondria.

producing ATP

2. Glucose en glycolytic pal metabolizes ti

\[/ SULP1IONYLUREAS bind to the su I phony I urea stihunit of ATP-sensitivc potassium channel, causing channel closure.

/ 5. Rise in membrane potential « xv «x*^ causes opening of voltage galed calcium channels and net influx of calcium inlo beta cell.

\[/ SULP1IONYLUREAS bind to the su I phony I urea stihunit of ATP-sensitivc potassium channel, causing channel closure.

!1HNF-la mutations:

1. Dccrcascd transcription of glut2.

2. Decreased transcription o!" L-type pyruvate kinase,

3. Decreased mitochondrial metabolism.

Figure 1 Glucose-induced insulin secretion, sulfonylureas, and the HNF-1a mutation. Abbreviations: GLUT-2, glucose transporter in hepatocyte and pancreatic y6-cell; ATP, adenosine triphosphate; K-ATP, potassium-ATP channel. Source: Adapted from Ref. 63.

of insulin action, namely, adipose tissue, skeletal muscle, and liver. Currently, two structurally diverse PPARy agonists are used in clinical practice: pioglitazone (Actos®) and rosiglitazone (Avandia®). Troglitazone was withdrawn from the market in March 2000 because of its association with idiosyncratic hepatotoxicity, an effect that had been postulated to be influenced by mutations of the glutathione-S-transferase genotype (72). In a study by Watanabe et al. (72) genotype analysis was performed on 110 patients who had been prescribed troglitazone, evaluating 68 polymorphic sites in 51 candidate genes relating to drug metabolism, apoptosis, production and elimination of reactive oxygen species, and also the signal transduction pathways of PPARy2 and insulin. A strong correlation with transaminase elevations was observed only in patients with the combined glutathione-S-transferase GSTT1-GSTM1 null genotype.

The interaction between Thiazolidinediones and the PPAR represents another model of pharmacogenomics. Thiazolidinediones has been shown to decrease plasma glucose concentrations in patients with type 2 DM (73-75), but clinical studies have shown that 10-25% of the patients treated with thiazolidinediones do not achieve a 15% reduction in fasting plasma glucose or do not convert from impaired glucose tolerance to normal glucose tolerance (76). The molecular reasons for the differential responses have not been determined, although it has been postulated that that differences in the PPARy genotype may modify the response to thiazolidinedione treatment. Different genetic variants of the PPARy gene have been shown to affect drug action in vitro (77). The most common variant in the PPARy gene, the Pro12Ala variant, occurs at a frequency of 12% to 15% (78-83), whereas other mutations are very rare (84-86). In a clinical study, the Pro12Ala and the Pro12Pro variants in the PPARy gene, however, were not associated with a favorable response to pioglitazone in patients with type 2 DM (87). Although these preliminary results would suggest that these variants in the PPARy gene do not determine the response to pioglitazone, it is unclear whether other variants in the gene, or indeed variants in downstream pathways from this receptor, are important determinants of efficacy or adverse effects, such as fluid retention. Interestingly, in an animal model it has been shown that the CD36 fatty acid transporter gene is an important determinant of the insulin-sensitizing actions and subsequent metabolic effects of pioglitazone (88). The fatty acid transporter CD36 is one of a number of molecules that mediate the uptake of (free fatty acids) FFA by adipocytes and muscle cells and is a well-known target for PPAR ligands (89,90). Clearly this is an area for future research.

HYPERLIPIDEMIA

Hyperlipidemia, the elevation of lipid concentrations in plasma, is the manifestation of a disorder in the synthesis and degradation of plasma lipoproteins. The major concern in patients with hyperlipidemia is their increased risk of cardiovascular disease. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-determining enzyme for cholesterol synthesis. They reduce cholesterol by stimulating an increase in low-density-lipoprotein (LDL) receptors on hepatocyte membranes, thereby increasing the clearance of LDL from the circulation. Their main effect is to reduce LDL-cholesterol (LDL-C), but they may also reduce triglycerides to a modest extent and increase HDL-cholesterol (HDL-C). They are generally considered to be the most effective of all lipid-lowering drugs currently available. Large-scale clinical trials in the primary and secondary prevention of coronary heart disease (CHD) have unequivocally demonstrated the efficacy of statins in reducing the risk of cardiovascular events (91-96).

HMG-CoA reductase inhibitors are a generally well-tolerated class of drug, although in a minority of patients severe adverse effects, such as myopathy or rhabdomyo-lysis, may occur. The incidence of these potentially life-threatening side effects increases with increasing dose or with the coadminstration of drugs that affect the kinetics or dynamics of statins. The withdrawal of cerivastatin as a result of deaths from rhabdomyo-lysis illustrates the clinical importance of such interactions. On the other hand, not all patients respond to statin therapy with a reduction in CHD risk. Large clinical trials with statins have demonstrated varying reductions in cardiovascular events associated with similar changes in LDL-C, suggesting that at least some of the benefit of statin therapy may be derived from nonlipid mechanisms of disease attenuation, such as the modification of the inflammatory response, endothelial function, plaque stability, and thrombus formation (97).

Statins Reduce Risk in Specific Populations

Allelic variants in several candidate genes have been identified as markers for CHD, and their relationship with the response to statin therapy (based on metabolic, angiographic, and clinical outcomes) has been evaluated through retrospective analyses in many of the large statin trials. Although treatment with statin therapy results in similar improvements in lipoprotein profiles in all patients (among both carriers and noncarriers of the variant allele), it appears that statins preferentially benefit individuals in terms of the number of cardiovascular events and mortality who carry the high-risk variant genotypes for these risk factors, as compared with the individuals who have the wild-type genotype (98). Several polymorphic candidate genes have been identified as predictors of disease severity; these are summarized in Table 4.

Apolipoprotein E

Apolipoprotein E (ApoE), a constituent of very low-density lipoprotein (VLDL), is derived from the liver and serves in the transport and redistribution of lipids among various tissues throughout the body (99). Carriers of the e4 allele have a 40% increased risk of developing CHD when compared with carriers of the e2 or s3 alleles (100). During the follow-up period of a substudy of the Scandinavian Simvastatin Survival Study (4S), placebo-treated carriers of the e4 allele were almost twice as likely to die as noncarriers (15.7% and 9.0%), corresponding to a mortality risk ratio of 1.8 (101). Treatment with simvastatin resulted in a 61% reduction in the number of deaths in carriers of the e4 allele (from 15.7% to 6.0%), whereas in non-e4 carriers, simvastatin was associated with a 43% decrease in mortality (from 9.0% to 5.1%).

Table 4 Genetic Markers of Statin Response

Marker

Nature

High-risk variant allele

References

Apolipoprotein E

Constituent of liver-

e4 (compared with e2

(100)

(ApoE)

derived very low-density lipoprotein Transport and redistribution of lipids

and e3 alleles)

ß-Fibrinogen

Deposited in atherosclerotic plaques

-455G/A (wild type-455G/G)

(105)

Cholesterol ester

Mediates exchange of

Taql polymorphism B1

(110)

transfer protein

lipids between

allele (compared with

(CETP)

lipoproteins

B2 allele)

Hepatic lipase (HL)

Lipolytic enzyme involved in the metabolism of triglycerides, LDL, and HDL

C allele (compared with T allele)

(114,115)

Lipoprotein lipase

Involved in the

Asp(9)Asn LPL

(116)

(LPL)

metabolism of triycerides in

mutation

lipoproteins

PIA2 polymorphism

Platelet

Induces neointimal

(122)

glycoprotein III

proliferation

Stromelysin-1

Associated with connective tissue remodeling in atherogenesis and plaque rupture

6A (compared with 5A allele)

(123)

Abbreviations: VLDL, very low-density lipoproteins; HDL, high-density lipoproteins; LDL, low-density lipoproteins.

Abbreviations: VLDL, very low-density lipoproteins; HDL, high-density lipoproteins; LDL, low-density lipoproteins.

fi-Fibrinogen

Polymorphisms in the B-fibrinogen gene, particularly the -455G/A single-nucleotide polymorphism (SNP), have been associated with differences in the plasma levels of fibrinogen and the severity of arterial disease. It has thus been postulated that patients with the -455A allele have an increased rate of progression of CHD when compared with the wild type (-455GG) because their fibrinogen levels may increase more when the acute-phase response is triggered (102-104). The -455A allele was identified in 257 (4% homozygous, 34% heterozygous) out of 697 men enrolled in a study from the Regression Growth Evaluation Statin Study (REGRESS) (105). All patients had similar baseline lipid values and disease history, but the -455A homozygotes had significantly higher baseline fibrinogen levels (but less angiographic evidence of CHD) when compared with the other genotypes. However, after a two-year period of follow-up, placebo-treated patients with the -455AA genotype experienced a significantly greater progression of CHD as assessed by coronary angiographic parameters when compared with the -455GA and -455GG genotypes. The authors hypothesize that the -455A allele may promote a stronger acute-phase response in fibrinogen and that the resulting higher fibrinogen levels may form the pathogenetic basis for the stronger progression of coronary atherosclerosis. Despite similar reductions in LDL-C in the pravastatin-treated groups, only carriers of the A allele demonstrated angiographic regression of the disease. Although carriers of the high-risk allele, particularly the homozygotes, were associated with higher plasma fibrinogen levels and more rapid progression of the atherosclerotic lesions, pravastatin therapy seemed to offset this deleterious effect. The fact that the more rapid progression in the -455AA genotype was not apparent in the pravastatin group may be explained by a much larger positive effect of pravastatin treatment than the deleterious influence of the fibrinogen -455G/A polymorphism on the development of the disease.

Cholesteryl Ester Transfer Protein

Cholesteryl ester transfer protein (CETP) has a central role in the metabolism of HDL-C, mediating the exchange of lipids between lipoproteins. The CETP-enzyme has a central role in reverse cholesterol transport (RCT), whereby cholesterol from peripheral tissues is transported back to the liver where it is preferentially excreted into bile (106). This results in the net transfer of cholesteryl ester from HDL to other lipoproteins and the subsequent uptake of cholesterol by hepatocytes (107). This is illustrated in Figure 2. The presence of a polymorphism in the CETP gene (which is also called TaqlB) is associated with elevated concentrations of CETP, which in turn leads to reduced concentrations of HDL-C, a strong and independent risk factor for the development of CHD (107,109). Individuals with the TaqlB polymorphism in the CETP gene may therefore be at higher risk for the development of CHD (110). In the study of 807 men by Kuivenhoven et al. (107), the term B1 was used to denote the presence of Taql, and B2 was used to denote absence of Taql. The respective frequencies of the B1B1, B1B2, and B2B2 genotypes were 35%, 49%, and 16%, respectively. The Bl allele was associated with lower HDL-C concentrations and higher CETP concentrations in all patients. Baseline LDL-C concentrations were similar in all genotypes, but on following a 2-year period of follow-up, placebo-treated patients with the B1B1 genotype showed the most pronounced angiographic progression of atherosclerosis, when compared with the B1B2 and B2B2 genotypes. Although patients with each genotype achieved similar reductions in LDL-C from pravastatin therapy, both the B1 homozygotes and heterozygotes experienced less (angiographically-determined) progression of coronary disease when compared with individuals with the respective genotype who received placebo. Conversely, no significant differences in disease progression were observed between the

Figure 2 The proteins involved in HDL-mediated reverse cholesterol transport. Abbreviations: VLDL, very low-density lipoproteins; HDL, high-density lipoproteins; LDL, low-density lipopro-teins; ATP, adenosine triphosphate.

placebo and pravastatin-treated B2B2 homozygotes. However, there was only a small decrease in plaque regression in the pravastatin-treated group (the decrease in mean luminal diameter was 0.05 + 0.16 mm for the B1B1 genotype, 0.07 + 0.20 mm for the B1B2 genotype, and 0.09 + 0.16 mm for the B2B2 genotype), and whether this leads to a long-lasting clinical benefit is unclear.

Hepatic Lipase

Hepatic lipase (HL) is a plasma lipolytic enzyme that plays an important role in the metabolism of triglycerides, LDL, and HDL. Increased HL activity has been associated with reduced HDL levels and smaller HDL particles, and also an increased number of small, dense LDL particles (111,112). The presence of a C-T substitution at position -514 in the promoter region of the HL gene accounts for approximately one-quarter of the variance in HL activity in men and women (113,114). The presence of the C allele has been associated with higher HL activity, more atherogenic LDL particles, and lower levels of anti-atherogenic HDL lipoproteins (115). Results from one study suggest that this promoter region gene polymorphism is responsible for the differential lipoprotein and angiographic response to lipid-lowering therapy (114). In the 49 men included in the analysis, 25 had the CC genotype, 20 had the CT, and 4 had the TT genotype at position -514 of the HL gene promoter. At baseline, men with the CC genotype had greater HL activity, lower HDL-C, and lower LDL buoyancy, when compared with those with the TT genotype. Lipid-lowering therapy (lovastatin plus colestipol or niacin plus colestipol) was associated with an 18% decrease in HL activity and a 12% increase in LDL buoyancy in patients with the CC genotype, whereas HL activity and LDL buoyancy did not significantly change in patients with the TT genotype. Furthermore, lipid-lowering therapy was also associated with a 2.1% reduction in coronary stenosis among men with the CC genotype, whereas progression of stenosis was observed in men with the TT genotype.

Lipoprotein Lipase

Lipoprotein lipase (LPL) is an enzyme that is involved in the metabolism of triglycerides in lipoproteins, such as chylomicrons and VLDL (111). Common mutations in the LPL gene that lead to deficient LPL activity have been associated with hypertriglyceridemia and low HDL levels, which results in affected individuals being at increased risk for premature CHD (116). In one study, the nature and frequency of the aspartic acid to aspara-gine substitution at position 9 in exon 2 of the LPL gene (Asp9Asn) has been examined (117). The authors hypothesized that the presence of the Asp9Asn LPL polymorphism in a given patient would increase susceptibility to atherosclerosis and therefore be associated with greater progression of the CHD. Indeed, the Asp9Asn mutation was identified in 4.8% of the population (38 men), and carriers of the polymorphism were more likely to have a positive family history of CHD and a lower HDL-C level at baseline, when compared with patients without the mutation. After a two-year period of follow-up, placebo-treated carriers of the Asp9Asn LPL polymorphism showed greater progression of angiographically significant CHD when compared with the noncarriers. Although the lipid-lowering effect of pravastatin was attenuated in patients carrying the Asp9Asn substitution, the deleterious effects of this polymorphism on the progression of atherosclerosis could apparently be reversed by pravastatin.

Platelet Glycoprotein III

Studies have identified an association between the polymorphism of the gene encoding platelet glycoprotein Ilia (PlA2 polymorphism) and acute coronary syndromes, subacute stent thrombosis, restenosis development following coronary stent implantation, and increased platelet aggregability (118-122). Statin therapy has been shown to reduce the increased rate of restenosis associated with the high risk PIA2 allele and significantly improved clinical outcomes in a consecutive series of patients undergoing coronary stent implantation (122). In a study of 650 patients (78% of whom were homozygous for the PIA1 allele, and 22% of the patients carried the PIA2 allele) by Walter et al. (122), restenosis rates at 6 months were significantly reduced by statin therapy. Although the extent of cholesterol lowering by statin therapy was identical in both carriers of the PIA2 allele and patients homozygous for PIA1 and there were no significant differences in baseline parameters, statin-treated carriers of the PIA2 allele had a 22.3% lower rate of restenosis than P/A2 carriers without statin therapy (28.6% vs. 50.9%, respectively; p = 0.01). Furthermore, statin-treated patients homozygous for the P/A1 allele had a slight reduction in the restenosis rate when compared with the P/41 homozygotes who did not receive statin therapy. In the patients who received placebo, the P/A2 allele was associated with an increased rate of restenosis when compared with the P/A1 allele (50.9% vs. 34%; p = 0.01), although there were no differences in the rate of restenosis observed between statin-treated carriers of either allele. The significantly lower restenosis rate observed in statin-treated carriers of the P/42 allele was associated with a significant improvement in the six-month event-free survival (49.3% in P/42 carriers without statin treatment vs. 28.2% in statin-treated P/A2 carriers; p < 0.01), whereas statin therapy had a minimal effect on six-month event-free survival among those homozygous for P/A1.

Siromelys/n-1

Stromelysin-1, a matrix metalloproteinase, is involved with the connective tissue remodeling processes associated with atherogenesis and atherosclerotic plaque rupture (123). The 6A allele of the 5A6A polymorphism in the stromelysin-1 gene promoter region has been linked to a rapidly progressive form of coronary stenosis due to atherosclerosis (124). In a sub-study of the REGRESS, de Maat et al. hypothesized that the presence of the stromelysin-1 6A allele may be associated with an increased risk of clinical events or requirement for repeat angioplasty due to clinical restenosis (123,125). Greater than 75% of the 494 men evaluated in this analysis carried the variant allele (6A) (50% heterozygous and 26% homozygous), and after two years of follow-up, no significant differences in angio-graphic measures of coronary artery obstruction were found among the three genotypes that were treated with pravastatin or between placebo and pravastatin-treated patients. However, nearly twice as many clinical events [myocardial infarction (MI), CHD death, symptom-driven percutaneous transluminal coronary angioplasty or coronary artery bypass graft surgery, stroke and transient ischemic attack, and death] were observed in the placebo-treated patients with the 5A6A and 6A6A genotypes (26% in both groups) when compared with those homozygous for the 5A allele (12%) (p < 0.05). It must be stated, however, that in this study the baseline risk in the 5A5A group was already lower than for the 5A6A and 6A6A groups. Compared with the placebo, treatment with pravastatin was associated with a 48% increase in frequency of clinical events among patients with the 5A5A genotype, whereas patients with the 5A6A and 6A6A genotypes experienced a 71% and a 54% reduction in clinical events, respectively. Pravastatin was also associated with an increase in the frequency of symptom-driven repeat angioplasty among patients with the 5A5A genotype (from 11% to 28%), whereas patients with the 5A6A and 6A6A genotypes experienced reductions in the frequency of repeat angioplasty (from 37.5% to 0%, and 40% to 15%, respectively p < 0.002) compared with the placebo.

Thus, it appears that patients with the 5A5A genotype receive no additional benefit from pravastatin, indicating that the response to pharmacological intervention showed a genotype-specific effect, solely benefiting carriers of the 6A allele. The results from the Lopid Coronary Angiography Trial (LOCAT), using gemfibrozil, corroborate the findings from REGRESS and suggest that both statins and fibrates offer a protective effect on disease progression solely in carriers of the 6A allele, whereas the degree of protection in patients with the 5A5A genotype is marginal (126,127).

Statins and the Genetics of Metabolism and Disposition

Pravastatin is not significantly metabolized by the CYP450 system; the CYP3A family metabolizes lovastatin, simvastatin, atorvastatin, and cerivastatin, whereas CYP2C9

metabolizes fluvastatin (128). Fluvastatin is a synthetic HMG-CoA reductase inhibitor and is a racemic mixture of (—)-3S, 5R-fluvastatin and (+ )-3R, 5S-fluvastatin. The latter has a 30-fold higher therapeutic activity (129). Fluvastatin is eliminated from the human body almost entirely by hepatic biotransformation, primarily being metabolized to 5-hydroxy-, 6-hydroxy-, and N-deisopropyl-fluvastatin by CYP2C9. In vitro data show that about 50-80% of fluvastatin metabolic clearance is due to CYP2C9 (130). Kirchheiner et al. (130) studied the impact of the two frequent CYP2C9 amino acid polymorphisms on enantio-specific fluvastatin pharmacokinetics and pharmacodynamics in healthy volunteers (130). The pharmacokinetics of both enantiomers of fluvastatin depended on the CYP2C9 genotype, with a threefold group mean difference in the active enantiomer and even greater differences in the inactive enantiomer. The CYP2C9*2 variant did not have any significant influence on fluvastatin kinetics. However, differences in plasma concentrations were not reflected in cholesterol-lowering after 14 days of fluvastatin intake in healthy volunteers. The latter result is unsurprising for a number of reasons. First, the baseline cholesterol levels differed between the genotype groups because the study groups were stratified for the CYP2C9 genotype but not for the baseline cholesterol concentration. Second, fluvastatin steady-state kinetics were not measured after 15 days of treatment, and kinetic differences between genotype groups might have been smaller at steady-state than after a single dose. Third, the sample size of this study was determined for pharmacokinetics analyses, and a larger sample would be necessary to assess the pharmacodynamic impact. As discussed previously, the pharmacodynamic effect of statins is influenced by multiple variables including other pharmacogenomic predictors of HMG-CoA reductase inhibitor efficacy, such as polymorphisms in the LDL receptor, CETP, ApoE, and the ^-fibrinogen genes.

Drug transporters are increasingly recognized to be important in drug disposition and response. Polymorphisms in the MDR1 gene, encoding P-glycoprotein (P-gp), affect the pharmacokinetics of many commonly used drugs, including statins, many of which are substrates for P-gp (131,132). P-gp is a member of the large ATP-binding cassette (ABCB1) family of proteins. It is found in the small intestine on the brush border of enterocytes and may thus influence the oral bioavailability of statin therapy (128,133). However, the extent of this has not yet been quantified. It is also evident that many substrates of P-gp are also CYP3A4 substrates, and this is well demonstrated in the case of statins. The overlap between CYP3A4 and P-gp substrates may have resulted in part from the coordinated regulation and tissue expression of CYP3A4 and MDR1 in organs, such as the liver and intestine. Interestingly, both genes are located on the same chromosome in close proximity, 7q22.1 and 7q21.1 for CYP3A4 and MDR1, respectively (134). Future studies of the relevant statin should therefore take into account both the metabolizing and transport capabilities of individuals, as failure to correct this confounding factor may lead to contradictory data from different studies.

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