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Perspectives in Diabetes The Peroxisome Proliferator–Activated Receptor-␥2 Pro12Ala Polymorphism Michael Stumvoll and Hans Ha¨ring

Peroxisome proliferator–activated receptor (PPAR)-␥ is a transcription factor with a key role in adipocyte differentiation. The Ala allele of the common Pro12Ala polymorphism in the isoform PPAR-␥2 is associated with reduced risk for type 2 diabetes. The effect on the individual is weak, but because of a prevalence of >75% of the high-risk Pro allele, the population-attributable risk is enormous. The in vivo effects of the polymorphism are secondary to alterations in adipose tissue, where PPAR-␥2 is predominantly expressed. Moderate reduction in transcriptional activity of PPAR-␥ as a result of the polymorphism modulates production and release of adipose-derived factors. Both decreased release of insulin-desensitizing free fatty acids, tumor necrosis factor-␣, and resistin and increased release of the insulin-sensitizing hormone adiponectin result in secondary improvement of insulin sensitivity of glucose uptake and suppression of glucose production. The population effect of this polymorphism may be modulated by environmental or genetic factors such as obesity, ethnicity, ratio of unsaturated to saturated fatty acids, and genetic background. Once diabetes has developed, the protective effect of the Ala allele may be lost, since increased vascular complications and more pronounced ␤-cell dysfunction have been reported. These observations, however, are currently unexplained. In conclusion, the Pro12Ala polymorphism in PPAR-␥2 represents the first genetic variant with a broad impact on the risk of common type 2 diabetes. The precise understanding of its mechanism may lead to novel diagnostic, preventive, and therapeutic approaches for improving the management of type 2 diabetes. Diabetes 51: 2341–2347, 2002

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eroxisome proliferator–activated receptor (PPAR)-␥ is a transcription factor that belongs to the same family of nuclear receptors as steroid and thyroid hormone receptors (1). It is activated by certain fatty acids, prostanoids, and thiazolidinediones, a novel class of insulin-sensitizing antidiabetic agents (2– 4). Upon activation, it heterodimerizes with the retinoid X receptor and binds to specific PPAR-responsive elements of DNA to promote transcription of numerous target genes (5). Although the isoform PPAR-␥1 is expressed in most tissues, PPAR-␥2 is specific for adipose tissue, where it plays a key role in regulating adipogenic differentiation (6). The PPAR-␥ gene is located on chromosome 3 (7), and the specific isoforms are a result of alternative mRNA splicing. A number of genetic variants in the PPAR-␥ gene have been identified. These include a very rare gain-of-function mutation (Pro115Gln) associated with obesity but not insulin resistance (8), two loss-of-function mutations (Val290Met and Pro467Leu) reported in three individuals with severe insulin resistance but normal body weight (9), the silent CAC478CAT mutation (10 –12), and the highly prevalent Pro12Ala polymorphism in PPAR-␥2 (Fig. 1). The latter is the result of a CCA-to-GCA missense mutation in codon 12 of exon B of the PPAR-␥ gene. This exon encodes the NH2-terminal residue that defines the adipocyte-specific PPAR-␥2 isoform. The Pro12Ala polymorphism in PPAR-␥2, which is the focus of this review, was first identified in 1997 (13), and the rare allele frequencies are ⬃12% in Caucasians, 10% in Native Americans, 8% in Samoans, 4% in Japanese, 3% in African-Americans, 2% in Nauruans, and 1% in Chinese (14,15). In Caucasians, the ethnic group with the highest frequency, this translates into a carrier prevalence of the polymorphism of almost 25%. ASSOCIATION WITH TYPE 2 DIABETES

From the University Hospital, Department of Endocrinology, Metabolism and Pathobiochemistry, Eberhard-Karls-Universita¨t, Tu¨bingen, Germany. Address correspondence and reprint requests to Dr. Michael Stumvoll, Medizinische Universita¨tsklinik, Otfried-Mu¨ller-Str. 10, D-72076, Tu¨bingen, Germany. E-mail: [email protected]. Received for publication 20 December 2001 and accepted in revised form 12 February 2002. FFA, free fatty acid; PPAR, peroxisome proliferator-activated receptor; TNF-␣, tumor necrosis factor-␣. DIABETES, VOL. 51, AUGUST 2002

The pathogenesis of type 2 diabetes is characterized by failure of ␤-cell function to compensate for decreased insulin sensitivity. The etiology is multifactorial, and twin studies clearly indicate a major role for involvement of genetic factors (16,17). Moreover, excess concordance rates in monozygotic versus dizygotic twins clearly suggest a contribution of genetic factors to both insulin resistance and ␤-cell dysfunction (18). However, due to enormous heterogeneity and probably polygenicity, very 2341

PPAR-␥2 Pro12Ala

FIG. 1. Organization of the PPAR-␥ gene.

few genetic variants have been identified to account for a substantial proportion of common type 2 diabetes. First evidence for an association between the Pro12Ala polymorphisms in PPAR-␥2 and type 2 diabetes came from Japanese-Americans, in which a frequency of the rare Ala allele of 9.3% in subjects with normal glucose tolerance versus only 2.2% in patients with type 2 diabetes was observed (11). This type of association study, however, is frequently troubled by a lack of reproducibility. Nevertheless, in a study that used the powerful tool of transmission disequilibrium testing in 333 Scandinavian parent-offspring trios with abnormal glucose tolerance, only the Pro12Ala polymorphism remained significant among 16 variants with previously published evidence for association with type 2 diabetes (or related disorders) (19). This association of the Pro12Ala polymorphism with type 2 diabetes was recently replicated in two large population-based studies from Finland and Japan including well over 4,000 subjects (15,20). Although the initial publication reported a 75% risk reduction for diabetes conferred by the Ala allele (11), of five subsequent studies (12,21–24), only one (22) was able to reproduce a significant association. However, a metaanalysis including those five plus the aforementioned data set (19) demonstrated a significant risk reduction of 21% (19). The resulting population-attributable risk was estimated as ⬃25% (11). In other words, if the entire population carried the Ala allele, the prevalence for type 2 diabetes would be 25% lower. In contrast, mutations responsible for monogenic mature-onset diabetes of the young, though having a major effect on the individual’s glucose homeostasis, are so rare that they hardly affect a population’s risk of type 2 diabetes (25). This underlines the importance of alleles with weak individual effect but high population prevalence, such as the (so-called) wildtype Pro allele of PPAR-␥2 (75% prevalence in Caucasians), and clearly indicates the prominent role of PPAR-␥ among candidate genes for common type 2 diabetes. To make diagnostic, preventive, or therapeutic use of a polymorphism, it is necessary to understand how and in which metabolic, genetic, or environmental context the genotype influences the phenotype. A number of recent 2342

studies on metabolic pathways, diabetic and nondiabetic subphenotypes, and gene-environment and gene-gene interaction effects have provided some useful information regarding the potential mechanism by which the Pro12Ala polymorphism reduces the risk for type 2 diabetes. Generally, a genetic variant that affects the risk for type 2 diabetes must influence insulin sensitivity, insulin secretion, or susceptibility for obesity. EFFECT ON INSULIN SENSITIVITY

In the original article, significantly greater insulin sensitivity was reported in nondiabetic Ala carriers (11). However, Ala carriers also had a lower BMI, and after adjustment the difference in insulin sensitivity was no longer significant. It therefore remained unclear what the primary result of the polymorphism was. In one small study, the confounding influence of BMI (and other relevant variables) on insulin sensitivity was eliminated by pair-matching carriers of the polymorphism with wild-type control subjects (26). Glucose infusion rates during euglycemic-hyperinsulinemic clamp (the gold standard technique for measuring insulin sensitivity in humans) per se were not different; however, expressing them per unit insulin revealed a significantly greater insulin sensitivity index (26). In the largest study so far addressing this issue, a 7% greater insulin sensitivity was observed in 616 normal glucose-tolerant Swedish 70-year-old men carrying the Ala allele (27). In the same article, a 6% difference in the same direction using the minimal model estimate for insulin sensitivity was reported for 364 young Danes; however, it failed to reach statistical difference. A power calculation based on these data revealed a required sample size of ⬃2,000 to achieve significance. In a Chinese/Japanese sib-pair study, which by design controlled relatively well for genetic background and childhood environment, a greater estimated insulin sensitivity (homeostasis model analysis) was observed in carriers of the Ala allele (28). Interestingly, a more pronounced effect of the Pro12Ala polymorphism on insulin sensitivity was observed before the background of the common Gly972Arg polymorphism in insulin receptor substrate-1 (29). Taken together, these studies provide DIABETES, VOL. 51, AUGUST 2002

M. STUMVOLL AND H. HA¨RING

substantial evidence that the Pro12Ala polymorphism improves insulin sensitivity in humans. In subgroups with obesity, the difference in insulin sensitivity was more pronounced (22,30,31), suggesting an interaction of the polymorphism with factors originating from adipose tissue. In healthy, lean, and normal glucose tolerant subjects, greater insulin sensitivity of glucose disposal was tightly coupled to greater insulin sensitivity of lipolysis, as measured by isotope dilution (32). This finding was in principle confirmed in a larger cohort from the same laboratory by demonstrating significantly lower free fatty acid (FFA) concentrations during insulin stimulation (33). Based on these results, it appears possible that alterations in transcriptional activity of the Ala variant in adipocytes (where PPAR-␥2 is expressed) primarily enhance insulin’s action on suppression of lipolysis, resulting in a decreased release of FFAs. Secondarily, reduced availability of FFAs would then permit muscle to utilize more glucose and liver to suppress glucose production more efficiently upon insulin stimulation (34). The metabolic studies in humans were not designed to distinguish between insulin-sensitizing effects on peripheral glucose disposal and suppression of glucose production. Studies are therefore required that specifically address the question of whether this polymorphism affects glucose production and insulin clearance, two processes clearly influenced by FFA availability. EFFECT ON INSULIN SECRETION

Currently, strong evidence for a direct effect of the Pro12Ala polymorphism on insulin secretion is lacking. However, in Japanese subjects with manifest type 2 diabetes, a lower ␤-cell function index (homeostasis model analysis) was reported in carriers of the Ala allele (15). Interestingly, lipid infusion designed to elevate plasma FFA concentrations fourfold resulted in a decrease in insulin secretion during hyperglycemic clamp in carriers of the Ala allele, but an increase in control subjects with two Pro alleles (35). These findings might provide a partial explanation for the above observation that ␤-cell function deteriorates more in Ala carriers once diabetes has developed (15). Conceivably, superimposition of secondary mechanisms—such as chronic exposure to elevated FFAs and/or hyperglycemia, which are characteristic for overt type 2 diabetes—alters (or even reverses) the effect of the genetic variant. Expression of PPAR-␥, though not specifically of the isoform PPAR-␥, in ␤-cells has been demonstrated (36). However, whether any of the above effects on ␤-cell function were direct or secondary remains open and requires further and more detailed studies in, for example, heterozygous PPAR-␥ knockout animals or humans homozygous for the Ala allele. EFFECT ON OBESITY

Because PPAR-␥ plays a key role in adipocyte differentiation and body fat mass is a strong determinant of insulin sensitivity and type 2 diabetes, the influence of the Pro12Ala polymorphism on susceptibility for obesity has been of major interest. In Pima Indians, suggestive linkage (logarithm of odds ⫽ 2.0) with percentage body fat was reported for the 3p24.2-p22 locus, which is close to the region harboring the PPAR-␥ gene (3p25-p24.2) (37). DIABETES, VOL. 51, AUGUST 2002

Cross-sectional studies with small to moderate sample sizes yielded inconsistent results by demonstrating either no difference (21,23,28,38,39) or a modestly greater BMI in carriers of the Ala allele (10,40,41). Two sufficiently large studies (⬎1,000 nondiabetic subjects) found a lower BMI in Finns (11) and no difference in Japanese (15). On the other hand, longitudinal studies in selected populations with relatively small sample sizes consistently suggested greater weight gain in association with the Ala allele (20,42,43). Thus, although cross-sectional evidence convincingly argues against susceptibility of obesity conferred by the Ala allele, the issue remains somewhat unclear. In particular, the interpretation of the longitudinal analysis is complicated by the fact that greater insulin sensitivity per se predicts future weight gain (44). Weight gain may be related to the greater insulin sensitivity particular of lipolysis (33), which would result in overproportionate retention of FFAs in stored triglycerides during physiological insulin stimulation (e.g., postprandially). In this scenario, obesity would be a consequence of increased insulin sensitivity and not directly of the Ala allele. It may be of note in this context that under a high-fat diet, heterozygous PPAR-␥– deficient mice (an animal model for reduced PPAR-␥ activity) were protected from the development of insulin resistance caused by adipocyte hypertrophy, compared with wild-type littermates (45). Clearly, any effect of this polymorphism on measures of obesity would be extremely subtle, and its demonstration would be crucially dependent on subject selection and interaction with ethnic background and other genetic or environmental factors. For example, an interaction effect of the Pro12Ala polymorphism with the Trp64Arg polymorphism in the ␤3-adrenergic receptor (which by itself probably does not influence body weight [46]) has been reported (47). Moreover, a greater BMI was observed in Ala carriers when the dietary polyunsaturated fat–to– saturated fat ratio was low, suggesting gene-nutrient interaction via the PPAR-␥ locus (48). CELLULAR MECHANISM

PPAR-␥ is a master transcriptional regulator involved in the expression of probably hundreds of genes (1). Two studies have directly examined the transcriptional activity of the Ala variant of PPAR-␥ in comparison to the Pro variant in experimental cell models (11,49). Reduced binding of the Ala variant to the PPAR-␥–responsive DNA elements was observed in transient transfection assays (11,49). Moreover, reduced transcription of specific genes (lipoprotein lipase and acyl-CoA oxidase) was reported for cells overexpressing the Ala variant compared with cells overexpressing the wild-type protein (11). These studies clearly indicate reduced transcriptional activity of PPAR-␥ as a result of the Pro-to-Ala exchange. Because PPAR-␥2 is exclusively expressed in fat cells, any metabolic effects of the polymorphism, including those on glucose homeostasis, are likely to be secondary to alterations in adipose tissue. There is evidence from humans that the Pro12Ala polymorphism promotes the suppression of FFA release by insulin (32,33). It is unclear, however, which genes are specifically affected by the transcriptional changes con2343

PPAR-␥2 Pro12Ala

FIG. 2. Effect of the Pro12Ala polymorphism and other factors on PPAR-␥ activity in adipocytes and release of mediators relevant for glucose homeostasis.

ferred by the polymorphism. Conceivably, the proportion of large versus small adipocytes is involved. Activators of PPAR-␥ have been shown to promote differentiation of preadipocytes to small adipocytes (50), and in small adipocytes lipolysis is more insulin sensitive than in large adipocytes (51). In addition, alterations in fat distribution (more subcutaneous and less visceral [52]) as a result of the polymorphism could mediate effects on lipolysis. In humans, PPAR-␥ expression in visceral adipose tissue relative to subcutaneous adipose tissue is increased in obese subjects (53). Because visceral adipose tissue is metabolically more harmful (54), the Ala allele would be expected to have an even greater impact in obese subjects, as in fact was shown to be the case (30). Interestingly, heterozygous PPAR-␥– deficient transgenic mice had smaller adipocytes and greater insulin sensitivity than wild-type mice (45). These mice were also characterized by greater insulin sensitivity of both glucose disposal and suppression of glucose production (45,55). In these studies, insulin suppression of FFA release was not determined but would represent a prime candidate for mediating the insulin effects on glucose homeostasis. In addition to fatty acids, adipose tissues release a number of peptide hormones that influence insulin sensitivity. These include the cytokine tumor necrosis factor-␣ (TNF-␣) (56), resistin (57), and adiponectin (apM-1) (58). Although the insulin desensitizing evidence for TNF-␣ and resistin is weak for humans, adiponectin concentrations were clearly shown to be positively correlated with insulin sensitivity, even after adjusting for body fat (59), to decrease with deteriorating glucose tolerance (59), and to increase after weight reduction (60). Moreover, intravenous administration of recombinant adiponectin to rodent models of insulin resistance restored normal insulin sensitivity (61). Because all three peptides are under transcriptional control of PPAR-␥ (57,62– 64), any of them (and possibly other peptides yet to be identified) could mediate the Pro12Ala effect on insulin sensitivity (Fig. 2). In humans, however, it has not been studied yet whether the 2344

Pro12Ala polymorphism influences systemic or local adipocytokine concentrations. It has been difficult to explain why, paradoxically, both reduced transcriptional activity (due to heterozygous knockout or the Pro12Ala polymorphism) and pharmacological activation (by thiazolidinediones) of PPAR-␥ results in improved insulin sensitivity (65). Recent studies in heterozygous PPAR-␥– deficient mice, however, have contributed to unraveling this problem. Alterations in white adipose tissue triglycerides, hepatic triglycerides, hepatic energy consumption, and muscle energy metabolism were markedly different between the two levels of PPAR-␥ activation (63), indicating that entirely different metabolic pathways must be mediating the insulin-sensitizing effect of supraphysiological stimulation (thiazolidinediones) and moderate reduction (heterozygous knockout or Pro12Ala) of PPAR-␥ activity. Comparison with other naturally occurring variants in PPAR-␥ may be useful for elucidating the molecular mechanisms. The Val290Met and Pro467Leu mutations drastically reduced transcriptional activity of PPAR-␥ in a dominant-negative fashion in vitro and resulted in a severely insulin resistant though lean phenotype in vivo (9). Structure-function simulations suggested that these mutations affect the orientation of helix 12 of PPAR-␥, which is important for the interaction with ligands and coactivators (9). On the other hand, these mutations are less likely to affect ligand-independent transcriptional activity, a process strongly controlled by the NH2 terminus (66), the domain that harbors codon 12. Thus, the net result of an amino acid exchange that decreases transcriptional activity of PPAR-␥ critically depends on the localization within the PPAR-␥ molecule. In support of this concept, antidiabetic properties of PPAR-␥ antagonists (67) or PPAR-␥ modulators (68 –70) have been demonstrated. Finally, it is necessary to point out that the CCA-to-GCA missense mutation in PPAR-␥ (which causes the Pro12Ala exchange) need not necessarily be the functionally relevant mutation, but rather in linkage disequilibrium with it. DIABETES, VOL. 51, AUGUST 2002

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For example, the functional mutation could reside in the promoter region of the PPAR-␥ gene and result in reduced expression of PPAR-␥ protein. The Pro12Ala polymorphism would then merely represent a genetic marker for the relevant mutation. However, the demonstrated reduction in transcriptional activity argues against such a scenario (11,49). In addition, a systematic screening of 70 diabetic individuals failed to reveal additional missense mutations elsewhere in the PPAR-␥ gene or its promoter (19). All in all, a strong case exists for Pro12Ala itself being the etiologic exchange. SUMMARY AND CONCLUSIONS

The transcription factor PPAR-␥ is a master regulator of the relationships between nutrients, susceptibility to obesity, control of peptides released from adipocytes, and insulin sensitivity. The alanine allele of the common Pro12Ala polymorphisms in the isoform PPAR-␥2 is associated with a 25% reduced risk for type 2 diabetes. It thus represents the first genetic variant with a broad impact on the risk of common type 2 diabetes. The effect of this polymorphism is probably mediated by increased insulin sensitivity, which may be secondary to more efficient suppression of FFA release from fat tissue, where the isoform PPAR-␥2 is exclusively expressed. Modulation of expression and release of adipocytokines that influence insulin sensitivity are likely also to be involved, but this remains to be demonstrated in humans. The effects on muscle, liver, and possibly other tissues ultimately influencing glucose homeostasis are secondary. The underlying molecular mechanism of this polymorphism is a moderate reduction of the ligand-independent activity of PPAR-␥. Some findings for the Pro12Ala variant differed depending on superimposition of environmental factors. This clearly suggests that so-called “gene-environment interaction” may well hinge on genetic variations, such as those occurring in the transcription factor PPAR-␥. A number of issues remain to be confirmed and further explored, including the increased vascular complications and more pronounced ␤-cell dysfunction associated with the Ala allele once diabetes has developed. Moreover, the likely interaction with independent modulators such as obesity, ethnicity, ratio of unsaturated to saturated fatty acids, and other common genetic polymorphisms requires further studies. The understanding of how specific modulation of PPAR-␥ influences metabolism in humans may accelerate the development of novel pharmacological agents useful for preventing or treating type 2 diabetes and related disorders. ACKNOWLEDGMENTS

Dr. Stumvoll is currently supported by a Heisenberg Grant of the Deutsche Forschungsgemeinschaft. REFERENCES 1. Desvergne B, Wahli W: Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20:649 – 688, 1999 2. Olefsky JM: Treatment of insulin resistance with peroxisome proliferatoractivated receptor gamma agonists. J Clin Invest 106:467– 472, 2000 3. Schoonjans K, Auwerx J: Thiazolidinediones: an update. Lancet 355:1008 – 1010, 2000 4. Saltiel AR, Olefsky JM: Thiazolidinediones in the treatment of insulin resistance and type II diabetes (Review). Diabetes 45:1661–1669, 1996 DIABETES, VOL. 51, AUGUST 2002

5. Spiegelman BM: PPAR-gamma: adipogenic regulator and thiazolidinedione receptor (Review). Diabetes 47:507–514, 1998 6. Auwerx J: PPARgamma, the ultimate thrifty gene. Diabetologia 42:1033– 1049, 1999 7. Beamer BA, Negri C, Yen CJ, Gavrilova O, Rumberger JM, Durcan MJ, Yarnall DP, Hawkins AL, Griffin CA, Burns DK, Roth J, Reitman M, Shuldiner AR: Chromosomal localization and partial genomic structure of the human peroxisome proliferator activated receptor-gamma (hPPAR gamma) gene. Biochem Biophys Res Commun 233:756 –759, 1997 8. Ristow M, Muller Wieland D, Pfeiffer A, Krone W, Kahn CR: Obesity associated with a mutation in a genetic regulator of adipocyte differentiation. N Engl J Med 339:953–959, 1998 9. Barroso I, Gurnell M, Crowley VE, Agostini M, Schwabe JW, Soos MA, Maslen GL, Williams TD, Lewis H, Schafer AJ, Chatterjee VK, O’Rahilly S: Dominant negative mutations in human PPARgamma associated with severe insulin resistance, diabetes mellitus and hypertension. Nature 402:880 – 883, 1999 10. Valve R, Sivenius K, Miettinen R, Pihlajamaki J, Rissanen A, Deeb SS, Auwerx J, Uusitupa M, Laakso M: Two polymorphisms in the peroxisome proliferator-activated receptor-gamma gene are associated with severe overweight among obese women. J Clin Endocrinol Metab 84:3708 –3712, 1999 11. Deeb SS, Fajas L, Nemoto M, Pihlajamaki J, Mykkanen L, Kuusisto J, Laakso M, Fujimoto W, Auwerx J: A Pro12Ala substitution in PPARg2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nat Genet 20:284 –287, 1998 12. Meirhaeghe A, Fajas L, Helbecque N, Cottel D, Lebel P, Dallongeville J, Deeb S, Auwerx J, Amouyel P: A genetic polymorphism of the peroxisome proliferator-activated receptor gamma gene influences plasma leptin levels in obese humans. Hum Mol Genet 7:435– 440, 1998 13. Yen CJ, Beamer BA, Negri C, Silver K, Brown KA, Yarnall DP, Burns DK, Roth J, Shuldiner AR: Molecular scanning of the human peroxisome proliferator activated receptor gamma (hPPAR gamma) gene in diabetic Caucasians: identification of a Pro12Ala PPAR gamma 2 missense mutation. Biochem Biophys Res Commun 241:270 –274, 1997 14. Vigouroux C, Fajas L, Khallouf E, Meier M, Gyapay G, Lascols O, Auwerx J, Weissenbach J, Capeau J, Magre J: Human peroxisome proliferatoractivated receptor-gamma2: genetic mapping, identification of a variant in the coding sequence, and exclusion as the gene responsible for lipoatrophic diabetes. Diabetes 47:490 – 492, 1998 15. Mori H, Ikegami H, Kawaguchi Y, Seino S, Yokoi N, Takeda J, Inoue I, Seino Y, Yasuda K, Hanafusa T, Yamagata K, Awata T, Kadowaki T, Hara K, Yamada N, Gotoda T, Iwasaki N, Iwamoto Y, Sanke T, Nanjo K, Oka Y, Matsutani A, Maeda E, Kasuga M: The Pro12 3 Ala substitution in PPAR-␥ is associated with resistance to development of diabetes in the general population: possible involvement in impairment of insulin secretion in individuals with type 2 diabetes. Diabetes 50:891– 894, 2001 16. Medici F, Hawa M, Pyke DA, Leslie RD: Concordance rate for type II diabetes mellitus in monozygotic twins: actuarial analysis. Diabetologia 42:146 –150, 1999 17. Poulsen P, Kyvik KO, Vaag A, Beck Nielsen H: Heritability of type II (non-insulin-dependent) diabetes mellitus and abnormal glucose tolerance– a population-based twin study. Diabetologia 42:139 –145, 1999 18. Lehtovirta M, Kaprio J, Forsblom C, Eriksson J, Tuomilehto J, Groop L: Insulin sensitivity and insulin secretion in monozygotic and dizygotic twins. Diabetologia 43:285–293, 2000 19. Altshuler D, Hirschhorn JN, Klannemark M, Lindgren CM, Vohl M-C, Nemesh J, Lane CD, Schaffner SF, Bolk A, Brewer C, Tuomi T, Gaudet D, Hudson TJ, Daly M, Groop L, Lander ES: The common PPARg Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes. Nat Gen 26:76 – 80, 2000 20. Douglas JA, Erdos MR, Watanabe RM, Braun A, Johnston CL, Oeth P, Mohlke KL, Valle TT, Ehnholm C, Buchanan TA, Bergman RN, Collins FS, Boehnke M, Tuomilehto J: The peroxisome proliferator–activated receptor-␥2 Pro12A1a variant: association with type 2 diabetes and trait differences. Diabetes 50:886 – 890, 2001 21. Clement K, Hercberg S, Passinge B, Galan P, Varroud-Vial M, Shuldiner AR, Beamer BA, Charpentier G, Guy-Grand B, Froguel P, Vaisse C: The Pro115Gln and Pro12Ala PPAR gamma gene mutations in obesity and type 2 diabetes. Int J Obes Relat Metab Disord 24:391–393, 2000 22. Hara K, Okada T, Tobe K, Yasuda K, Mori Y, Kadowaki H, Hagura R, Akanuma Y, Kimura S, Ito C, Kadowaki T: The Pro12Ala polymorphism in PPAR gamma2 may confer resistance to type 2 diabetes. Biochem Biophys Res Commun 271:212–216, 2000 23. Mancini FP, Vaccaro O, Sabatino L, Tufano A, Rivellese AA, Riccardi G, Colantuoni V: Pro12Ala substitution in the peroxisome proliferator-acti2345

PPAR-␥2 Pro12Ala

vated receptor-␥2 is not associated with type 2 diabetes. Diabetes 48:1466 – 1468, 1999 24. Ringel J, Engeli S, Distler A, Sharma AM: Pro12Ala missense mutation of the peroxisome proliferator activated receptor gamma and diabetes mellitus. Biochem Biophys Res Commun 254:450 – 453, 1999 25. Bell GI, Polonsky KS: Diabetes mellitus and genetically programmed defects in beta-cell function. Nature 414:788 –791, 2001 26. Jacob S, Stumvoll M, Becker R, Koch M, Nielsen M, Lo¨ blein K, Maerker E, Volk A, Renn W, Balletshofer B, Machicao F, Rett K, Ha¨ ring HU: The PPARgamma2 polymorphism Pro12Ala is associated with better insulin sensitivity in the offspring of type 2 diabetic patients. Horm Metab Res 32:413– 416, 2000 27. Ek J, Andersen G, Urhammer SA, Hansen L, Carstensen B, Borch-Johnson K, Drivsholm T, Berglund L, Hansen T, Lithell H, Pedersen O: Studies of the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor-g2 (PPAR-g2) gene in relation to insulin sensitivity among glucose tolerant Caucasians. Diabetologia 44:1170 –1176, 2001 28. Chuang L-M, Hsueh W, Chen Y-DI, Ho LT, Sheu WHH, Pei D, Nakatsuka CH, Cox D, Pratley RE, Lei HH, Tai TY: Sibling-based association study of the PPARg 2 Pro12Ala polymorphism and metabolic variables in Chinese and Japanese hypertension families: a SAPPHIRr study. J Mol Med 79:656 – 664. 2002 29. Stumvoll M, Stefan N, Fritsche A, Madaus A, Tschritter O, Koch M, Machicao F, Ha¨ ring H: Interaction effect between common polymorphisms in PPARg2 (Pro12Ala) and IRS-1 (Gly972Arg) on insulin sensitivity. J Mol Med 80:33–38, 2002 30. Koch M, Rett K, Maerker E, Volk A, Haist K, Deninger M, Renn W, Ha¨ ring HU: The PPARgamma2 amino acid polymorphism Pro 12 Ala is prevalent in offspring of type II diabetic patients and is associated to increased insulin sensitivity in a subgroup of obese subjects. Diabetologia 42:758 – 762, 1999 31. Globerman H, Zauberman Y, Makarov T, Beamer BA, Yen CJ, Shuldiner AR, Harel C, Karnieli E: Analysis of the peroxisome proliferator activated receptor gamma (PPARgamma) gene in HAIRAN syndrome with obesity. Clin Endocrinol (Oxf) 52:479 – 485, 2000 32. Stumvoll M, Wahl HG, Lo¨ blein K, Becker R, Machicao F, Jacob S, Ha¨ ring H: The Pro12Ala polymorphism in the peroxisome proliferator–activated receptor-␥2 gene is associated with increased antilipolytic insulin sensitivity. Diabetes 50:876 – 881, 2001 33. Stumvoll M, Ha¨ ring H: Reduced lipolysis as possible cause for greater weight gain in subjects with the Pro12Ala polymorphism in PPAR␥2. Diabetologia 45:152–153, 2001 34. Boden G: Role of fatty acids in the pathogenesis of insulin resistance and NIDDM (Review). Diabetes 46:3–10, 1997 35. Stefan N, Fritsche A, Ha¨ ring H, Stumvoll M: Effect of experimental elevation of free fatty acids on insulin secretion and insulin sensitivity in healthy carriers of the Pro12Ala polymorphism of peroxisome-proliferator–activated receptor-␥2 gene. Diabetes 50:1143–1148, 2001 36. Dubois M, Pattou F, Kerr-Conte J, Gmyr V, Vanderwalle B, Desreumaux P, Auwerx J, Schoonjans K, Lefebvre J: Expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) in normal human pancreatic islet cells. Diabetologia 43:1165–1169, 2000 37. Norman RA, Thompson DB, Foroud T, Garvey WT, Bennett PH, Bogardus C, Ravussin E: Genomewide search for genes influencing percent body fat in Pima Indians: suggestive linkage at chromosome 11q21– q22: Pima Diabetes Gene Group. Am J Hum Genet 60:166 –173, 1997 38. Swarbrick MM, Chapman CM, McQuillan BM, Hung J, Thompson PL, Beilby JP: A Pro12Ala polymorphism in the human peroxisome proliferator-activated receptor-gamma 2 is associated with combined hyperlipidaemia in obesity. Eur J Endocrinol 144:277–282, 2001 39. Oh EY, Min KM, Chung JH, Min YK, Lee MS, Kim KW, Lee MK: Significance of Pro12Ala mutation in peroxisome proliferator-activated receptor-gamma2 in Korean diabetic and obese subjects. J Clin Endocrinol Metab 85:1801–1804, 2000 40. Cole SA, Mitchell BD, Hsueh WC, Pineda P, Beamer BA, Shuldiner AR, Comuzzie AG, Blangero J, Hixson JE: The Pro12Ala variant of peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2) is associated with measures of obesity in Mexican Americans. Int J Obes Relat Metab Disord 24:522–524, 2000 41. Beamer BA, Yen CJ, Andersen RE, Muller D, Elahi D, Cheskin LJ, Andres R, Roth J, Shuldiner AR: Association of the Pro12Ala variant in the peroxisome proliferator–activated receptor-␥2 gene with obesity in two Caucasian populations. Diabetes 47:1806 –1808, 1998 42. Lindi V, Sivenius K, Niskanen L, Laakso M, Uusitupa MI: Effect of the Pro12Ala polymorphism of the PPAR-␥2 gene on long-term weight change in Finnish non-diabetic subjects. Diabetologia 44:925–926, 2001 2346

43. Nicklas BJ, van Rossum EF, Berman DM, Ryan AS, Dennis KE, Shuldiner AR: Genetic variation in the peroxisome proliferator–activated receptor-␥2 gene (Pro12Ala) affects metabolic responses to weight loss and subsequent weight regain. Diabetes 50:2172–2176, 2001 44. Swinburn BA, Nyomba BL, Saad MF, Zurlo F, Raz I, Knowler WC, Lillioja S, Bogardus C, Ravussin E: Insulin resistance associated with lower rates of weight gain in Pima Indians. J Clin Invest 88:168 –173, 1991 45. Kubota N, Terauchi Y, Miki H, Tamemoto H, Yamauchi T, Komeda K, Satoh S, Nakano R, Ishii C, Sugiyama T, Eto K, Tsubamoto Y, Okuno A, Murakami K, Sekihara H, Hasegawa G, Naito M, Toyoshima Y, Tanaka S, Shiota K, Kitamura T, Fujita T, Ezaki O, Aizawa S, Nagai R, Tobe K, Kimura S, Kadowaki T: PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol Cell 4:597– 609, 1999 46. Allison DB, Heo M, Faith MS, Pietrobelli A: Meta-analysis of the association of the Trp64Arg polymorphism in the beta3 adrenergic receptor with body mass index. Int J Obes Relat Metab Disord 22:559 –566, 1998 47. Hsueh WC, Cole SA, Shuldiner AR, Beamer BA, Blangero J, Hixson JE, MacCluer JW, Mitchell BD: Interactions between variants in the ␤3adrenergic receptor and peroxisome proliferator–activated receptor-␥2 genes and obesity. Diabetes Care 24:672– 677, 2001 48. Luan J, Browne PO, Harding AH, Halsall DJ, O’Rahilly S, Chatterjee VK, Wareham NJ: Evidence for gene-nutrient interaction at the PPAR␥ locus. Diabetes 50:686 – 689, 2001 49. Masugi J, Tamori Y, Mori H, Koike T, Kasuga M: Inhibitory effect of a proline-to-alanine substitution at codon 12 of peroxisome proliferatoractivated receptor-gamma 2 on thiazolidinedione-induced adipogenesis. Biochem Biophys Res Commun 268:178 –182, 2000 50. Okuno A, Tamemoto H, Tobe K, Ueki K, Mori Y, Iwamoto K, Umesono K, Akanuma Y, Fujiwara T, Horikoshi H, Yazaki Y, Kadowaki T: Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats. J Clin Invest 101:1354 –1361, 1998 51. Abbott WG, Foley JE: Comparison of body composition, adipocyte size, and glucose and insulin concentrations in Pima Indian and Caucasian children. Metabolism 36:576 –579, 1987 52. Adams M, Montague CT, Prins JB, Holder JC, Smith SA, Sanders L, Digby JE, Sewter CP, Lazar MA, Chatterjee VK, O’Rahilly S: Activators of peroxisome proliferator-activated receptor gamma have depot-specific effects on human preadipocyte differentiation. J Clin Invest 100:3149 – 3153, 1997 53. Lefebvre AM, Laville M, Vega N, Riou JP, van Gaal L, Auwerx J, Vidal H: Depot-specific differences in adipose tissue gene expression in lean and obese subjects. Diabetes 47:98 –103, 1998 54. Montague CT, O’Rahilly S: The perils of portliness: causes and consequences of visceral adiposity. Diabetes 49:883– 888, 2000 55. Miles PD, Barak Y, He W, Evans RM, Olefsky JM: Improved insulinsensitivity in mice heterozygous for PPAR-gamma deficiency. J Clin Invest 105:287–292, 2000 56. Kroder G, Bossenmaier B, Kellerer M, Capp E, Stoyanov B, Muhlhofer A, Berti L, Horikoshi H, Ullrich A, Haring H: Tumor necrosis factor-alpha- and hyperglycemia-induced insulin resistance: evidence for different mechanisms and different effects on insulin signaling. J Clin Invest 97:1471–1477, 1996 57. Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA: The hormone resistin links obesity to diabetes. Nature 409:307–312, 2001 58. Hu E, Liang P, Spiegelman BM: AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271:10697–10703, 1996 59. Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, Tataranni PA: Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 86:1930 –1935, 2001 60. Yang WS, Lee WJ, Funahashi T, Tanaka S, Matsuzawa Y, Chao CL, Chen CL, Tai TY, Chuang LM: Weight reduction increases plasma levels of an adipose-derived anti-inflammatory protein, adiponectin. J Clin Endocrinol Metab 86:3815–3819, 2001 61. Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T: The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:941–946, 2001 62. Hofmann C, Lorenz K, Braithwaite SS, Colca JR, Palazuk BJ, Hotamisligil GS, Spiegelman BM: Altered gene expression for tumor necrosis factor-a and its receptor during drug and dietary modulation of insulin resistance. Endocrinology 134:264 –270, 1994 DIABETES, VOL. 51, AUGUST 2002

M. STUMVOLL AND H. HA¨ RING

63. Yamauchi T, Kamon J, Waki H, Murakami K, Motojima K, Komeda K, Ide T, Kubota N, Terauchi Y, Tobe K, Miki H, Tsuchida A, Akanuma Y, Nagai R, Kimura S, Kadowaki T: The mechanisms by which both heterozygous peroxisome proliferator-activated receptor gamma (PPARgamma) deficiency and PPARgamma agonist improve insulin resistance. J Biol Chem 276:41245– 41254, 2001 64. Maeda N, Takahashi M, Funahashi T, Kihara S, Nishizawa H, Kishida K, Nagaretani H, Matsuda M, Komuro R, Ouchi N, Kuriyama H, Hotta K, Nakamura T, Shimomura I, Matsuzawa Y: PPAR␥ ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes 50:2094 –2099, 2001 65. Kahn CR, Chen L, Cohen SE: Unraveling the mechanism of action of thiazolidinediones. J Clin Invest 106:1305–1307, 2000 66. Werman A, Hollenberg A, Solanes G, Bjorbaek C, Vidal-Puig A, Flier JS: Ligand-independent activation domain in the N terminus of peroxisome proliferator-activated receptor g (PPARg). J Biol Chem 272: 20230 –20235. 2002 67. Mukherjee R, Hoener PA, Jow L, Bilakovics J, Klausing K, Mais DE,

DIABETES, VOL. 51, AUGUST 2002

Faulkner A, Croston GE, Paterniti JR Jr: A selective peroxisome proliferator-activated receptor-gamma (PPARgamma) modulator blocks adipocyte differentiation but stimulates glucose uptake in 3T3–L1 adipocytes. Mol Endocrinol 14:1425–1433, 2000 68. Oberfield JL, Collins JL, Holmes CP, Goreham DM, Cooper JP, Cobb JE, Lenhard JM, Hull-Ryde EA, Mohr CP, Blanchard SG, Parks DJ, Moore LB, Lehmann JM, Plunket K, Miller AB, Milburn MV, Kliewer SA, Willson TM: A peroxisome proliferator-activated receptor gamma ligand inhibits adipocyte differentiation. Proc Natl Acad Sci U S A 96:6102– 6106, 1999 69. Elbrecht A, Chen Y, Adams A, Berger J, Griffin P, Klatt T, Zhang B, Menke J, Zhou G, Smith RG, Moller DE: L-764406 is a partial agonist of human peroxisome proliferator-activated receptor gamma: the role of Cys313 in ligand binding. J Biol Chem 274:7913–7922, 1999 70. Reginato MJ, Bailey ST, Krakow SL, Minami C, Ishii S, Tanaka H, Lazar MA: A potent antidiabetic thiazolidinedione with unique peroxisome proliferator-activated receptor gamma-activating properties. J Biol Chem 273: 32679 –32684, 1998

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