- Email: [email protected]
Adipokine genes and the insulin-resistance syndrome
International Congress Series 1253 (2003) 63 – 71
Adipokine genes and the insulin-resistance syndrome Alessandro Doria * Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, MA, USA Accepted 7 February 2003
Abstract Adipocytes secrete several molecules that are involved in the regulation of whole-body metabolism and other vital functions related to inflammation and immune responses. Genetic variability in the expression or activity of these cytokines, or adipokines, may be involved in the development of the insulin-resistance syndrome and type 2 diabetes. Two of these molecules—resistin and adiponectin—have been intensively studied during the past year. We and others have identified a single nucleotide polymorphism in the resistin gene that interacts with obesity in determining the risk of insulin-resistance and type 2 diabetes. A haplotype associated with several features of the insulinresistance syndrome has also been identified at the adiponectin locus. Whether these polymorphisms are themselves responsible for the association with insulin-resistance or are markers in linkage disequilibrium with as yet unidentified causal variants is unknown at this time. Further studies extending the polymorphism analysis to flanking regions and investigating variants for regulatory effects on gene expression are necessary to address this issue. Identification of diabetes-predisposing variants in adipokine genes will provide novel insights in the cellular mechanisms through which increased adiposity leads to insulin-resistance and type 2 diabetes, with possible implications on the development of new drugs to prevent these disorders. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Adipokine; Insulin; Resistin
A major breakthrough in insulin-resistance research has been the discovery that adipose tissue is an important endocrine organ regulating whole-body metabolism and other vital functions related to inflammation and immune responses. These actions are mediated by a
* Section on Genetics and Epidemiology, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, USA. Tel.: +1-617-732-2406; fax: +1-617-732-2667. E-mail address: [email protected] (A. Doria). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00136-5
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Fig. 1. The adipokine network in the regulation of metabolism and inflammation.
number of molecules that are secreted by adipocytes and act in an autocrine, paracrine, or endocrine fashion [1] (Fig. 1). Among those identified to date are leptin, adipsin, tumor necrosis factors a (TNF-a), resistin, and adiponectin [2 – 6]. Many of these cytokines, or ‘adipokines’, have been shown to modulate insulin action, FFA metabolism, and glucose uptake in target organs. While some controversy exists about their relative importance, these molecules are believed to play a critical role in adapting metabolic fluxes to the amount of stored energy [1]. With such profound effects on metabolism, variability in the adipokine genes has been proposed to be a determinant of insulin-resistance and other components of syndrome ‘X’, such as glucose intolerance, obesity, dyslipidemia, and high blood pressure. Specifically, it has been hypothesized that genetic variants affecting adipokines’ function, expression, or secretion may modulate the degree of insulin-resistance that is associated with any given body weight. Research aimed at identifying such variants is very active, fostered by the completion of the human genome sequence, improved understanding of genetic variation, and high-throughput technologies. Two adipokines having opposite effects on insulin action—resistin and adiponectin—have drawn particular interest during the past year.
1. Resistin Resistin is a 12.5-kDa, cysteine-rich protein that was identified by Steppan et al. [5] as being acutely upregulated during differentiation from preadipocytes to adipocytes. Resistin
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belongs to a family of resistin like molecules (RELMS) also known as FIZZ proteins [5,7] and is secreted by adipocytes, being detectable in serum [5]. While resistin’s function is still under study, there is strong evidence that this protein acts as a hormone inhibiting insulin action [5]. Administration of recombinant resistin to wild-type mice impairs glucose tolerance and insulin action, while an increase in insulin-sensitivity is observed when animals are treated with resistin-neutralizing antibodies [5]. Results are similar in 3T3-L1 adipocytes, suggesting that resistin may also have autocrine effects. While resistin’s role in human obesity is still unclear [8], circulating levels of this molecule are markedly increased in obese, insulin-resistant mice, and are decreased by treatment with insulin-sensitizing PPAR-g activators [5]. These findings point to increased resistin secretion as a possible mechanism through which obesity may lead to insulin-resistance, type 2 diabetes, and coronary artery disease. The resistin gene is placed on chromosome 19p13 at a short distance from the insulinreceptor. It is a small gene that spans less than 2 kb and includes four exons and three introns. Screening the entire gene for variants, we recently identified eight single nucleotide polymorphisms (SNPs) and a (GAT)n microsatellite [9]. Four SNPs are placed in the 5V flanking region, two in intron 2, and two in intron 3. Six SNPs are relatively frequent, with allele frequencies ranging from 0.09 to 0.43. All SNPs are in significant linkage disequilibrium, with only five haplotypes accounting for more than 80% of control chromosomes. To determine whether any of these variants are associated with an increased risk of insulin-resistance and type 2 diabetes, we typed each SNP in 312 cases with type 2 diabetes and 303 non-diabetic controls from the Joslin Clinic. While allele and genotype distribution were similar in the two groups at all polymorphic loci, we observed a significant interaction between one SNP (SNP6, IVS2 + 181G>A) and obesity in determining the risk of type 2 diabetes. In the total group, A/A homozygotes at this locus had a modest increase in the risk of diabetes as compared to carriers of other genotypes (odds ratio = 1.7, 95% C.I.: 0.8 –3.2). However, among obese subjects (%IBW above the median) the diabetes risk associated with A/A homozygosis was 4.8 (C.I. 1.1 –21.0), whereas it was not different from 1.0 (OR = 0.7, C.I. 0.2 –2.1) among lean individuals (%IBW below the median) (Fig. 2). The difference between the odds ratios was significant (v2 = 4.5, p = 0.034 with 1 df). Similar evidence of interaction was found when body weight was considered as a continuous variable in a logistic regression model ( p = 0.04 for the interaction between A/A homozygosis and % IBW). A possible explanation of these findings is that homozygosis for allele A of SNP 6 enhances resistin expression, but this does not affect insulin-sensitivity and the risk of diabetes unless resistin production is also boosted by excess adiposity. Studies of animal models have found increased serum levels of resistin in mice fed a high-fat diet or with genetic forms of obesity [5]. Whether the intronic SNP 6 can also enhance resistin expression or secretion is unknown at this time. Although introns have not been traditionally considered as having regulatory functions, there have been reports in the literature of intronic polymorphisms affecting gene expression, a notable example being SNP-43 in intron 3 of the calpain 10 gene [10]. Alternatively, this polymorphism may be a marker in linkage disequilibrium with other sequence differences affecting resistin expression that are placed outside the boundaries of the region that we screened.
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Fig. 2. Frequency of the SNP 6 A/A genotype in cases with type 2 diabetes (T2D) and non-diabetic controls (ND) according to whether their body weight is below (LEAN) or above (OBESE) the median (%IBW = 126.4).
Our results are consistent with the recent report by Wang et al. [11] who found a significant interaction between BMI and several SNPs at the resistin locus, including our SNP6, in determining the IVGTT insulin-sensitivity index Si in 119 non-diabetic individuals. On the other hand, in a non-diabetic population from Italy, we could not observe such interaction with obesity when fasting insulin was used as an index of insulin-sensitivity [9]. Instead, in this population there was a significant association between insulin-sensitivity and the ATG repeat in the 3VUTR [12]. If SNP 6 and the ATG repeats are mere markers, population differences in their linkage disequilibrium with as yet unknown causal variants may be responsible for these discrepancies. Three other reports have been published on the resistin gene, but none of these has investigated the interaction between SNP6 and obesity [13 – 15]. In one of these studies, SNP 3 in the 5V flanking region (g. 420C>G) was associated with an increased risk of obesity in a population from Quebec, but this finding could not be replicated among Scandinavian individuals [15].
2. Adiponectin Adiponectin, also known as APM1, Acrp30, or adipoQ, is an adipose tissue-specific protein of 247 amino acids sharing significant similarity with collagens VIII and X, and complement protein C1q (hence the name adipoQ) [6]. In contrast with resistin, which has low circulating levels, adiponectin is relatively abundant in serum representing 0.05% of circulating proteins. Adiponectin’s expression is reduced in the presence of obesity while it is increased by caloric restriction or treatment with insulin-sensitizing PPAR-g activators [16,17]. In animal models of obesity and diabetes, administration of adiponectin or its globular domain produces weight loss and improves insulin sensitivity and glucose tolerance [18 –20]. These effects result from actions on skeletal muscle to increase fatty
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acid oxidation and on liver to increase sensitivity to the anti-gluconeogenesis effects of insulin [18 –20]. Thus, adiponectin is a hormone enhancing insulin sensitivity. Variants decreasing its expression or activity may predispose to insulin resistance and other components of the metabolic syndrome. In agreement with this hypothesis, a genome screen has detected linkage with insulin-resistance traits at 3q27 where the adiponectin gene is located [21]. We have recently identified a haplotype at the adiponectin locus that is associated with many correlates of the insulin-resistance syndrome [22]. This haplotype is defined by a T at SNP + 45T>G and a G at SNP + 276G>T. In a study of 413 non-diabetic subjects, homozygous carriers of the ‘TG’ haplotype had higher fasting glucose and insulin levels than heterozygous carriers of the ‘TG’ haplotype or non-carriers (X/X) ( p = 0.02 and p = 0.005, respectively) (Table 1). The association was even stronger for the insulin resistance index HOMAIR ( p = 0.003) (Table 1). TG/TG homozygotes also had significantly higher body weight ( p = 0.03), waist circumference ( p = 0.004), systolic ( p = 0.01) and diastolic ( p = 0.003) blood pressure, and the ratio of total to HDL cholesterol ( p = 0.01) (Table 1). Importantly, homozygosis for this haplotype was associated with lower serum levels of adiponectin ( p = 0.03), independent of gender, age, and body weight (Fig. 3). The association with increased body weight and insulin-resistance was confirmed in an independent study group of 614 Caucasian subjects. These results indicate that genetic variability at the adiponectin locus is a determinant of the insulin-resistance syndrome. Of particular importance is the observation that the same haplotype that is associated with insulin-resistance is also associated with lower circulating levels of adiponectin, independent of body weight or insulin levels. This finding indicates that hypo-adiponectinemia is a primary, genetically determined defect
Table 1 Clinical characteristics of non-diabetic subjects according to carrier status of adiponectin 45 – 276 haplotype TG Haplotype 45 – 276
N (%) M/F Age (years) IBW (%) Waist (cm) W – H ratio SBP (mmHg) DBP (mmHg) FBG (mg/dl) Insulin (AU/ml) HOMA Cholesterol Triglycerides HDL-Cholesterol Tot-Chol/HDL-Chol
TG/TG
TG/X
X/X
p
106 (27.0) 44/62 39 F 12 120 F 22 84.9 F 11 0.84 F 0.07 116 F 12 78 F 9 91 F 9 8.9 F 5.3 2.01 F 1.3 199 F 42 108 F 87 51 F 14 4.1 F 1.3
180 (45.8) 65/115 37 F 11 115 F 19 81.1 F 12 0.82 F 0.09 112 F 12 76 F 8 90 F 9 7.1 F 3.5 1.58 F 0.9 196 F 41 89 F 49 54 F 13 3.8 F 1.2
107 (27.2) 44/63 37 F 11 114 F 19 79.5 F 11 0.82 F 0.08 111 F 12 74 F 8 88 F 9 7.0 F 3.5 1.52 F 0.8 188 F 36 92 F 66 53 F 12 3.7 F 1.1
0.38 0.32 0.03 0.004 0.04 0.01 0.003 0.02 0.005y 0.003y 0.11 0.06y 0.13 0.01
X denotes any haplotype other than TG. y Significance was tested on log-transformed values.
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Fig. 3. MeanFS.E. plasma adiponectin levels in 32 homozygotes for the ‘TG’ haplotype (TG/TG) and 32 non-TG carriers (X/X). Each genotype group included an equal number of men and women. The ‘TG/TG’ genotype was found to be a significant determinant of plasma adiponectin levels ( p=0.03, after adjusting for gender, %IBW, and age). Adiponectin levels were measured by Dr. Philipp Scherer at Albert Einstein College of Medicine, NY.
contributing to the etiology of obesity and insulin-resistance. This notion is in agreement with the recent report of decreased adiponectin levels early in the course of obesity in longitudinal studies of obesity-prone monkeys [23]. It must be noticed that neither polymorphism affects known regulatory regions, one being a synonymous substitution and the other being located in an intron. Thus, these variants are probably markers of a haplotype(s) containing a causal polymorphism affecting plasma adiponectin levels. At this regard, there are at least two known polymorphisms ( + 712A>G and + 2019del/insA) that are in almost complete linkage disequilibrium with the ‘TG’ haplotype. Of particular interest is the location of SNP + 2019 in the 3VUTR of the cDNA—a region that plays a pivotal role in the control of gene expression by binding proteins that regulate mRNA processing, translation, or degradation [24]. While this SNP is not placed in known cis-acting domains such as the adenylate/uridylate-rich elements (AREs), it may disrupt other regulatory elements that are postulated to exist in this region [24]. Further studies examining the relation between mRNA stability and + 2019 genotype are needed to tell whether this variant is indeed the culprit at this locus. Association between polymorphisms at the adiponectin locus and insulin-resistance or type 2 diabetes has been reported by other studies during the past year. In Japanese, Hara et al. [25] found an association between type 2 diabetes and SNP + 45 and + 276, while Stumvoll et al. [26] detected an association between SNP + 45 and indices of insulin sensitivity in Germans. Of notice, in both studies the association at position + 45 was with allele ‘G’ [25,26], whereas in our study the association with insulin-resistance involves
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allele ‘T’ (as part of the ‘TG’ haplotype). An as yet different pattern has been recently reported in French Caucasians, in whom the association with adiponectin levels and type 2 diabetes (but not insulin-resistance) concerns two polymorphisms in the 5Vflanking region [27]. Similar to resistin, these discrepancies may reflect differences among populations in the linkage disequilibrium structure, resulting in association of the disease haplotype with different SNP alleles in different populations. Calpain 10 is an example of such situation [10]. Alternatively, there may be several diabetes-predisposing variants at this locus, the effect of which is conditional to environmental or genetic factors characteristic of individual populations.
3. Summary and conclusions A number of molecules secreted by the adipose tissues are involved in the regulation of insulin action and other metabolic functions. Evidence is emerging that genetic variability in two of these adipokines—resistin and adiponectin—is a significant determinant of insulin-resistance and type 2 diabetes. Sequence differences associated with these traits have been identified in both genes, but whether these are causal variants or mere markers is unknown at this time. Solving this problem will require extending the analysis to 5Vand 3Vflanking regions to determine how far the haplotype(s) associated with insulin-resistance extend from these genes, and investigating all haplotype-specific variants for regulatory effects on gene expression. Ultimately, identification of diabetes-predisposing variants will provide critical insights in these molecules’ function and the cellular mechanisms by which increased adiposity leads to insulin-resistance and type 2 diabetes. Knowledge of these mechanisms might suggest additional strategies for preventing type 2 diabetes and its burden of morbidity and mortality.
Acknowledgements The research described in this article was supported by NIH Grant R01 DK55523 and a Research Award from the American Diabetes Association.
References [1] A.R. Saltiel, You are what you secrete, Nat. Med. 7 (2001) 887 – 888. [2] Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold, J.M. Friedman, Positional cloning of the mouse obese gene and its human homologue, Nature 372 (1994) 425 – 432. [3] B.S. Rosen, K.S. Cook, J. Yaglom, D.L. Groves, J.E. Volanakis, D. Damm, T. White, B.M. Spiegelman, Adipsin and complement factor D activity: an immune-related defect in obesity, Science 244 (1989) 1483 – 1487. [4] G.S. Hotamisligil, N.S. Shargill, B.M. Spiegelman, Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance, Science 259 (1993) 87 – 91. [5] C.M. Steppan, S.T. Bailey, S. Bhat, E.J. Brown, R.R. Banerjee, C.M. Wright, H.R. Patel, R.S. Ahima, M.A. Lazar, The hormone resistin links obesity to diabetes, Nature 409 (2001) 307 – 312.
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[6] P.E. Scherer, S. Williams, M. Fogliano, G. Baldini, H.F. Lodish, A novel serum protein similar to C1q, produced exclusively in adipocytes, J. Biol. Chem. 270 (1995) 26746 – 26749. [7] I.N. Holcomb, R.C. Kabakoff, B. Chan, et al., FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family, EMBO J. 19 (2000) 4046 – 4055. [8] D.B. Savage, C.P. Sewter, E.S. Klenk, D.G. Segal, A. Vidal-Puig, R.V. Considine, S. O’Rahilly, Resistin/ Fizz3 expression in relation to obesity and peroxisome proliferator-activated receptor-gamma action in humans, Diabetes 50 (2001) 2199 – 2202. [9] X. Ma, J.H. Warram, V. Trischitta, A. Doria, Genetic variants at the resistin locus and risk of type 2 diabetes in Caucasians, J. Clin. Endocrinol. Metab. 87 (2002) 4407 – 4410. [10] Y. Horikawa, N. Oda, N.J. Cox, et al., Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus, Nat. Genet. 26 (2000) 163 – 175. [11] H. Wang, W.S. Chu, C. Hemphill, S.C. Elbein, Human resistin gene: molecular scanning and evaluation of association with insulin sensitivity and type 2 diabetes in Caucasians, J. Clin. Endocrinol. Metab. 87 (2002) 2520 – 2524. [12] A. Pizzuti, A. Argiolas, R. Di Paola, et al., An ATG repeat in the 3V-untranslated region of the human resistin gene is associated with a decreased risk of insulin resistance, J. Clin. Endocrinol. Metab. 87 (2002) 4403 – 4406. [13] F. Sentinelli, S. Romeo, M. Arca, E. Filippi, F. Leonetti, M. Banchieri, U. Di Mario, M.G. Baroni, Human resistin gene, obesity, and type 2 diabetes: mutation analysis and population study, Diabetes 51 (2002) 860 – 862. [14] H. Osawa, H. Onuma, A. Murakami, M. Ochi, T. Nishimiya, K. Kato, I. Shimizu, Y. Fujii, J. Ohashi, H. Makino, Systematic search for single nucleotide polymorphisms in the resistin gene: the absence of evidence for the association of three identified single nucleotide polymorphisms with Japanese type 2 diabetes, Diabetes 51 (2002) 863 – 866. [15] J.C. Engert, M.C. Vohl, S.M. Williams, et al., 5VFlanking variants of resistin are associated with obesity, Diabetes 51 (2002) 1629 – 1634. [16] Y. Arita, S. Kihara, N. Ouchi, M. Takahashi, et al., Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity, Biochem. Biophys. Res. Commun. 257 (1999) 79 – 83. [17] W.S. Yang, W.J. Lee, T. Funahashi, S. Tanaka, Y. Matsuzawa, C.L. Chao, C.L. Chen, T.Y. Tai, L.M. Chuang, Weight reduction increases plasma levels of an adipose-derived anti-inflammatory protein, adiponectin, J. Clin. Endocrinol. Metab. 86 (2001) 3815 – 3819. [18] J. Fruebis, T.S. Tsao, S. Javorschi, D. Ebbets-Reed, M.R. Erickson, F.T. Yen, B.E. Bihain, H.F. Lodish, Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 2005 – 2010. [19] A.H. Berg, T.P. Combs, X. Du, M. Brownlee, P.E. Scherer, The adipocyte-secreted protein Acrp30 enhances hepatic insulin action, Nat. Med. 7 (2001) 947 – 953. [20] T. Yamauchi, J. Kamon, H. Waki, Y. Terauchi, et al., The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity, Nat. Med. 7 (2001) 941 – 946. [21] A.H. Kissebah, G.E. Sonnenberg, J. Myklebust, M. Goldstein, et al., Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 14478 – 14483. [22] C. Menzaghi, T. Ercolino, R. Di Paola, A.H. Berg, J.H. Warram, P.E. Scherer, V. Trischitta, A. Doria, A haplotype at the adiponectin locus is associated with obesity and other features of the insulin resistance syndrome, Diabetes 51 (2002) 2306 – 2312. [23] K. Hotta, T. Funahashi, N.L. Bodkin, H.K. Ortmeyer, Y. Arita, B.C. Hansen, Y. Matsuzawa, Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys, Diabetes 50 (2001) 1126 – 1133. [24] B. Conne, A. Stutz, J.D. Vassalli, The 3Vuntranslated region of messenger RNA. A molecular ‘hotspot’ for pathology? Nat. Med. 6 (2000) 637 – 641. [25] K. Hara, P. Boutin, Y. Mori, et al., Genetic variation in the gene encoding adiponectin is associated with an increased risk of type 2 diabetes in the Japanese population, Diabetes 51 (2002) 536 – 540. [26] M. Stumvoll, O. Tschritter, A. Fritsche, H. Staiger, W. Renn, M. Weisser, F. Machicao, H. Haring, Asso-
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ciation of the T-g polymorphism in adiponectin (exon 2) with obesity and insulin sensitivity: interaction with family history of type 2 diabetes, Diabetes 51 (2002) 37 – 41. [27] F. Vasseur, N. Helbecque, C. Dina, et al., Single-nucleotide polymorphism haplotypes in the both proximal promoter and exon 3 of the APM1 gene modulate adipocyte-secreted adiponectin hormone levels and contribute to the genetic risk for type 2 diabetes in French Caucasians, Hum. Mol. Genet. 11 (2002) 2607 – 2614.
Adipokine genes and the insulin-resistance syndrome Alessandro Doria * Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, MA, USA Accepted 7 February 2003
Abstract Adipocytes secrete several molecules that are involved in the regulation of whole-body metabolism and other vital functions related to inflammation and immune responses. Genetic variability in the expression or activity of these cytokines, or adipokines, may be involved in the development of the insulin-resistance syndrome and type 2 diabetes. Two of these molecules—resistin and adiponectin—have been intensively studied during the past year. We and others have identified a single nucleotide polymorphism in the resistin gene that interacts with obesity in determining the risk of insulin-resistance and type 2 diabetes. A haplotype associated with several features of the insulinresistance syndrome has also been identified at the adiponectin locus. Whether these polymorphisms are themselves responsible for the association with insulin-resistance or are markers in linkage disequilibrium with as yet unidentified causal variants is unknown at this time. Further studies extending the polymorphism analysis to flanking regions and investigating variants for regulatory effects on gene expression are necessary to address this issue. Identification of diabetes-predisposing variants in adipokine genes will provide novel insights in the cellular mechanisms through which increased adiposity leads to insulin-resistance and type 2 diabetes, with possible implications on the development of new drugs to prevent these disorders. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Adipokine; Insulin; Resistin
A major breakthrough in insulin-resistance research has been the discovery that adipose tissue is an important endocrine organ regulating whole-body metabolism and other vital functions related to inflammation and immune responses. These actions are mediated by a
* Section on Genetics and Epidemiology, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, USA. Tel.: +1-617-732-2406; fax: +1-617-732-2667. E-mail address: [email protected] (A. Doria). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00136-5
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Fig. 1. The adipokine network in the regulation of metabolism and inflammation.
number of molecules that are secreted by adipocytes and act in an autocrine, paracrine, or endocrine fashion [1] (Fig. 1). Among those identified to date are leptin, adipsin, tumor necrosis factors a (TNF-a), resistin, and adiponectin [2 – 6]. Many of these cytokines, or ‘adipokines’, have been shown to modulate insulin action, FFA metabolism, and glucose uptake in target organs. While some controversy exists about their relative importance, these molecules are believed to play a critical role in adapting metabolic fluxes to the amount of stored energy [1]. With such profound effects on metabolism, variability in the adipokine genes has been proposed to be a determinant of insulin-resistance and other components of syndrome ‘X’, such as glucose intolerance, obesity, dyslipidemia, and high blood pressure. Specifically, it has been hypothesized that genetic variants affecting adipokines’ function, expression, or secretion may modulate the degree of insulin-resistance that is associated with any given body weight. Research aimed at identifying such variants is very active, fostered by the completion of the human genome sequence, improved understanding of genetic variation, and high-throughput technologies. Two adipokines having opposite effects on insulin action—resistin and adiponectin—have drawn particular interest during the past year.
1. Resistin Resistin is a 12.5-kDa, cysteine-rich protein that was identified by Steppan et al. [5] as being acutely upregulated during differentiation from preadipocytes to adipocytes. Resistin
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belongs to a family of resistin like molecules (RELMS) also known as FIZZ proteins [5,7] and is secreted by adipocytes, being detectable in serum [5]. While resistin’s function is still under study, there is strong evidence that this protein acts as a hormone inhibiting insulin action [5]. Administration of recombinant resistin to wild-type mice impairs glucose tolerance and insulin action, while an increase in insulin-sensitivity is observed when animals are treated with resistin-neutralizing antibodies [5]. Results are similar in 3T3-L1 adipocytes, suggesting that resistin may also have autocrine effects. While resistin’s role in human obesity is still unclear [8], circulating levels of this molecule are markedly increased in obese, insulin-resistant mice, and are decreased by treatment with insulin-sensitizing PPAR-g activators [5]. These findings point to increased resistin secretion as a possible mechanism through which obesity may lead to insulin-resistance, type 2 diabetes, and coronary artery disease. The resistin gene is placed on chromosome 19p13 at a short distance from the insulinreceptor. It is a small gene that spans less than 2 kb and includes four exons and three introns. Screening the entire gene for variants, we recently identified eight single nucleotide polymorphisms (SNPs) and a (GAT)n microsatellite [9]. Four SNPs are placed in the 5V flanking region, two in intron 2, and two in intron 3. Six SNPs are relatively frequent, with allele frequencies ranging from 0.09 to 0.43. All SNPs are in significant linkage disequilibrium, with only five haplotypes accounting for more than 80% of control chromosomes. To determine whether any of these variants are associated with an increased risk of insulin-resistance and type 2 diabetes, we typed each SNP in 312 cases with type 2 diabetes and 303 non-diabetic controls from the Joslin Clinic. While allele and genotype distribution were similar in the two groups at all polymorphic loci, we observed a significant interaction between one SNP (SNP6, IVS2 + 181G>A) and obesity in determining the risk of type 2 diabetes. In the total group, A/A homozygotes at this locus had a modest increase in the risk of diabetes as compared to carriers of other genotypes (odds ratio = 1.7, 95% C.I.: 0.8 –3.2). However, among obese subjects (%IBW above the median) the diabetes risk associated with A/A homozygosis was 4.8 (C.I. 1.1 –21.0), whereas it was not different from 1.0 (OR = 0.7, C.I. 0.2 –2.1) among lean individuals (%IBW below the median) (Fig. 2). The difference between the odds ratios was significant (v2 = 4.5, p = 0.034 with 1 df). Similar evidence of interaction was found when body weight was considered as a continuous variable in a logistic regression model ( p = 0.04 for the interaction between A/A homozygosis and % IBW). A possible explanation of these findings is that homozygosis for allele A of SNP 6 enhances resistin expression, but this does not affect insulin-sensitivity and the risk of diabetes unless resistin production is also boosted by excess adiposity. Studies of animal models have found increased serum levels of resistin in mice fed a high-fat diet or with genetic forms of obesity [5]. Whether the intronic SNP 6 can also enhance resistin expression or secretion is unknown at this time. Although introns have not been traditionally considered as having regulatory functions, there have been reports in the literature of intronic polymorphisms affecting gene expression, a notable example being SNP-43 in intron 3 of the calpain 10 gene [10]. Alternatively, this polymorphism may be a marker in linkage disequilibrium with other sequence differences affecting resistin expression that are placed outside the boundaries of the region that we screened.
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Fig. 2. Frequency of the SNP 6 A/A genotype in cases with type 2 diabetes (T2D) and non-diabetic controls (ND) according to whether their body weight is below (LEAN) or above (OBESE) the median (%IBW = 126.4).
Our results are consistent with the recent report by Wang et al. [11] who found a significant interaction between BMI and several SNPs at the resistin locus, including our SNP6, in determining the IVGTT insulin-sensitivity index Si in 119 non-diabetic individuals. On the other hand, in a non-diabetic population from Italy, we could not observe such interaction with obesity when fasting insulin was used as an index of insulin-sensitivity [9]. Instead, in this population there was a significant association between insulin-sensitivity and the ATG repeat in the 3VUTR [12]. If SNP 6 and the ATG repeats are mere markers, population differences in their linkage disequilibrium with as yet unknown causal variants may be responsible for these discrepancies. Three other reports have been published on the resistin gene, but none of these has investigated the interaction between SNP6 and obesity [13 – 15]. In one of these studies, SNP 3 in the 5V flanking region (g. 420C>G) was associated with an increased risk of obesity in a population from Quebec, but this finding could not be replicated among Scandinavian individuals [15].
2. Adiponectin Adiponectin, also known as APM1, Acrp30, or adipoQ, is an adipose tissue-specific protein of 247 amino acids sharing significant similarity with collagens VIII and X, and complement protein C1q (hence the name adipoQ) [6]. In contrast with resistin, which has low circulating levels, adiponectin is relatively abundant in serum representing 0.05% of circulating proteins. Adiponectin’s expression is reduced in the presence of obesity while it is increased by caloric restriction or treatment with insulin-sensitizing PPAR-g activators [16,17]. In animal models of obesity and diabetes, administration of adiponectin or its globular domain produces weight loss and improves insulin sensitivity and glucose tolerance [18 –20]. These effects result from actions on skeletal muscle to increase fatty
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acid oxidation and on liver to increase sensitivity to the anti-gluconeogenesis effects of insulin [18 –20]. Thus, adiponectin is a hormone enhancing insulin sensitivity. Variants decreasing its expression or activity may predispose to insulin resistance and other components of the metabolic syndrome. In agreement with this hypothesis, a genome screen has detected linkage with insulin-resistance traits at 3q27 where the adiponectin gene is located [21]. We have recently identified a haplotype at the adiponectin locus that is associated with many correlates of the insulin-resistance syndrome [22]. This haplotype is defined by a T at SNP + 45T>G and a G at SNP + 276G>T. In a study of 413 non-diabetic subjects, homozygous carriers of the ‘TG’ haplotype had higher fasting glucose and insulin levels than heterozygous carriers of the ‘TG’ haplotype or non-carriers (X/X) ( p = 0.02 and p = 0.005, respectively) (Table 1). The association was even stronger for the insulin resistance index HOMAIR ( p = 0.003) (Table 1). TG/TG homozygotes also had significantly higher body weight ( p = 0.03), waist circumference ( p = 0.004), systolic ( p = 0.01) and diastolic ( p = 0.003) blood pressure, and the ratio of total to HDL cholesterol ( p = 0.01) (Table 1). Importantly, homozygosis for this haplotype was associated with lower serum levels of adiponectin ( p = 0.03), independent of gender, age, and body weight (Fig. 3). The association with increased body weight and insulin-resistance was confirmed in an independent study group of 614 Caucasian subjects. These results indicate that genetic variability at the adiponectin locus is a determinant of the insulin-resistance syndrome. Of particular importance is the observation that the same haplotype that is associated with insulin-resistance is also associated with lower circulating levels of adiponectin, independent of body weight or insulin levels. This finding indicates that hypo-adiponectinemia is a primary, genetically determined defect
Table 1 Clinical characteristics of non-diabetic subjects according to carrier status of adiponectin 45 – 276 haplotype TG Haplotype 45 – 276
N (%) M/F Age (years) IBW (%) Waist (cm) W – H ratio SBP (mmHg) DBP (mmHg) FBG (mg/dl) Insulin (AU/ml) HOMA Cholesterol Triglycerides HDL-Cholesterol Tot-Chol/HDL-Chol
TG/TG
TG/X
X/X
p
106 (27.0) 44/62 39 F 12 120 F 22 84.9 F 11 0.84 F 0.07 116 F 12 78 F 9 91 F 9 8.9 F 5.3 2.01 F 1.3 199 F 42 108 F 87 51 F 14 4.1 F 1.3
180 (45.8) 65/115 37 F 11 115 F 19 81.1 F 12 0.82 F 0.09 112 F 12 76 F 8 90 F 9 7.1 F 3.5 1.58 F 0.9 196 F 41 89 F 49 54 F 13 3.8 F 1.2
107 (27.2) 44/63 37 F 11 114 F 19 79.5 F 11 0.82 F 0.08 111 F 12 74 F 8 88 F 9 7.0 F 3.5 1.52 F 0.8 188 F 36 92 F 66 53 F 12 3.7 F 1.1
0.38 0.32 0.03 0.004 0.04 0.01 0.003 0.02 0.005y 0.003y 0.11 0.06y 0.13 0.01
X denotes any haplotype other than TG. y Significance was tested on log-transformed values.
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Fig. 3. MeanFS.E. plasma adiponectin levels in 32 homozygotes for the ‘TG’ haplotype (TG/TG) and 32 non-TG carriers (X/X). Each genotype group included an equal number of men and women. The ‘TG/TG’ genotype was found to be a significant determinant of plasma adiponectin levels ( p=0.03, after adjusting for gender, %IBW, and age). Adiponectin levels were measured by Dr. Philipp Scherer at Albert Einstein College of Medicine, NY.
contributing to the etiology of obesity and insulin-resistance. This notion is in agreement with the recent report of decreased adiponectin levels early in the course of obesity in longitudinal studies of obesity-prone monkeys [23]. It must be noticed that neither polymorphism affects known regulatory regions, one being a synonymous substitution and the other being located in an intron. Thus, these variants are probably markers of a haplotype(s) containing a causal polymorphism affecting plasma adiponectin levels. At this regard, there are at least two known polymorphisms ( + 712A>G and + 2019del/insA) that are in almost complete linkage disequilibrium with the ‘TG’ haplotype. Of particular interest is the location of SNP + 2019 in the 3VUTR of the cDNA—a region that plays a pivotal role in the control of gene expression by binding proteins that regulate mRNA processing, translation, or degradation [24]. While this SNP is not placed in known cis-acting domains such as the adenylate/uridylate-rich elements (AREs), it may disrupt other regulatory elements that are postulated to exist in this region [24]. Further studies examining the relation between mRNA stability and + 2019 genotype are needed to tell whether this variant is indeed the culprit at this locus. Association between polymorphisms at the adiponectin locus and insulin-resistance or type 2 diabetes has been reported by other studies during the past year. In Japanese, Hara et al. [25] found an association between type 2 diabetes and SNP + 45 and + 276, while Stumvoll et al. [26] detected an association between SNP + 45 and indices of insulin sensitivity in Germans. Of notice, in both studies the association at position + 45 was with allele ‘G’ [25,26], whereas in our study the association with insulin-resistance involves
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allele ‘T’ (as part of the ‘TG’ haplotype). An as yet different pattern has been recently reported in French Caucasians, in whom the association with adiponectin levels and type 2 diabetes (but not insulin-resistance) concerns two polymorphisms in the 5Vflanking region [27]. Similar to resistin, these discrepancies may reflect differences among populations in the linkage disequilibrium structure, resulting in association of the disease haplotype with different SNP alleles in different populations. Calpain 10 is an example of such situation [10]. Alternatively, there may be several diabetes-predisposing variants at this locus, the effect of which is conditional to environmental or genetic factors characteristic of individual populations.
3. Summary and conclusions A number of molecules secreted by the adipose tissues are involved in the regulation of insulin action and other metabolic functions. Evidence is emerging that genetic variability in two of these adipokines—resistin and adiponectin—is a significant determinant of insulin-resistance and type 2 diabetes. Sequence differences associated with these traits have been identified in both genes, but whether these are causal variants or mere markers is unknown at this time. Solving this problem will require extending the analysis to 5Vand 3Vflanking regions to determine how far the haplotype(s) associated with insulin-resistance extend from these genes, and investigating all haplotype-specific variants for regulatory effects on gene expression. Ultimately, identification of diabetes-predisposing variants will provide critical insights in these molecules’ function and the cellular mechanisms by which increased adiposity leads to insulin-resistance and type 2 diabetes. Knowledge of these mechanisms might suggest additional strategies for preventing type 2 diabetes and its burden of morbidity and mortality.
Acknowledgements The research described in this article was supported by NIH Grant R01 DK55523 and a Research Award from the American Diabetes Association.
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