Reduced early insulin secretion in the etiology of type 2 diabetes mellitus in Pima Indians

We report the results of cross-sectional, prospective, and longitudinal studies identifying etiologic metabolic factors in the susceptibility to type 2 diabetes mellitus of the Pima Indians of Arizona, whose prevalence and incidence rates of the disease are the highest in the world. Diabetic Pima Indians are metabolically prototypic, with obesity, insulin resistance, a reduced acute insulin response to glucose, and increased endogenous glucose production. Cross-sectional studies show that the acute insulin response is absent in diabetic subjects and lower in impaired than in normal glucose-tolerant subjects. Prospective studies using proportional hazards analyses indicate that insulin resistance and a relatively low acute insulin response predict diabetes independently of age, gender, and each other, with obesity increasing susceptibility by worsening one or both predictors. Longitudinal studies show that glucose tolerance deteriorates as the degree of obesity increases due to worsening insulin resistance and decreases in early insulin secretion. Furthermore, since the children of diabetic pregnancies are at much greater risk of developing diabetes at a young age than those of nondiabetic pregnancies, the diabetic uterine environment may induce insulin resistance and/or reduced insulin secretion: early evidence confirms that adult normal glucose-tolerant offspring show a substantially decreased acute insulin response--the clearest demonstration yet of an environmental condition increasing susceptibility to type 2 diabetes mellitus. However, the genetic determinants require elucidation: correlation of the acute insulin response with the age of parental diabetes onset in fathers as well as mothers indicates a mechanism independent of the diabetic uterine environment.     

PCLO variants are nominally associated with early-onset type 2 diabetes and insulin resistance in Pima Indians

OBJECTIVE—A prior genome-wide association (GWA) study in Pima Indians identified variants within PCLO that were associated with early-onset type 2 diabetes. PCLO encodes a presynaptic cytomatrix protein that functions as a Ca²⁺ sensor that may be involved in insulin secretion and/or insulin action. Therefore, PCLO was analyzed as a candidate gene for type 2 diabetes.RESEARCH DESIGN AND METHODS—Sequencing of PCLO identified four nonsynonymous variants and a 10–amino acid insertion. These variants, together with 100 additional variants identified by sequencing or chosen from databases, were genotyped for association analysis in the same 895 subjects analyzed in the prior GWA study (300 case subjects with diabetes onset at aged <25 years, 334 nondiabetic control subjects aged >45 years, and 261 discordant siblings of the case or control subjects for within-family analyses), as well as 415 nondiabetic Pima Indians who had been metabolically phenotyped for predictors of diabetes. Selected variants were further genotyped in a population-based sample of 3,501 Pima Indians.RESULTS—Four variants were modestly associated with early-onset type 2 diabetes in both general and within-family analyses (P = 0.004–0.04, recessive model), where the diabetes risk allele was also nominally associated with a lower insulin-mediated glucose disposal rate (P = 0.009–0.14, recessive model) in nondiabetic Pima Indians. However, their association with diabetes in the population-based sample was weaker (P = 0.02–0.20, recessive model).CONCLUSIONS—Variation within PCLO may have a modest effect on early-onset type 2 diabetes, possibly as a result of reduced insulin action, but has minimal, if any, impact on population-based risk for type 2 diabetes.The Pima Indians of Arizona have an extremely high prevalence of type 2 diabetes (1). Their diabetes is characterized by obesity, dysfunction of insulin secretion, insulin resistance (decreased insulin-mediated glucose disposal), and increased rate of endogenous glucose output (2). Studies have shown that type 2 diabetes, insulin action, acute insulin response to glucose, and obesity are highly heritable in this population (3–5). To identify genes that underlie the development of type 2 diabetes in Pima Indians, we recently completed a genome-wide association (GWA) study using the Affymetrix 100K SNP genotyping array (6). Two single nucleotide polymorphisms (SNPs), rs10487656 and rs10487657, that ranked among the top 1% for a general association with early-onset type 2 diabetes (defined as age of onset <25 years) mapped within an intron of the PCLO gene. PCLO is located on chromosome 7q21 and encodes for a presynaptic cytomatrix protein that functions as a Ca²⁺ sensor that could potentially have a role in insulin secretion and/or insulin action (7–10); therefore, PCLO was analyzed as a positional and physiological candidate gene for type 2 diabetes.     

Incidence of proteinuria in type 2 diabetes mellitus in the Pima Indians

Little is known of the natural history of nephropathy in type 2 (non-insulin-dependent) diabetes, yet type 2 diabetes is a major cause of end-stage renal disease in the United States. The incidence rate of heavy proteinuria was determined in Pima Indians participating in a longitudinal population study of diabetes and its complications. Heavy proteinuria was defined by a urine protein (g/liter) to urine creatinine (g/liter) ratio greater than or equal to 1.0 (greater than or equal to 113 mg protein/nmol creatinine), a level which corresponds to a urine protein excretion rate of about 1 g/day. The incidence rates of proteinuria in diabetic Pimas were 4, 12, 37, and 106 cases/1,000 person-years at risk in the periods 0 to 5, 5 to 10, 10 to 15, and 15 to 20 years after the diagnosis of diabetes. The cumulative incidence rates were 2%, 8%, 23%, and 50% at 5, 10, 15, and 20 years, respectively. The duration of diabetes, severity of diabetes as determined by the degree of hyperglycemia and type of treatment, and blood pressure were risk factors for proteinuria. The presence of heavy proteinuria was strongly associated with the development of renal insufficiency, defined by serum creatinine greater than or equal to 2.0 mg/dl (greater than or equal to 177 mumol/liter). The incidence of proteinuria in type 2 diabetes in Pima Indians was as high as that reported in type 1 diabetes in other populations and represents a frequent, serious complication of the disease.     

Effect of glycemia on mortality in Pima Indians with type 2 diabetes

The effect of plasma glucose concentration on overall and cause-specific mortality was examined in 1,745 Pima Indians (725 men, 1,020 women) > or = 15 years old with type 2 diabetes. During a median follow-up of 10.6 years (range 0.1-24.8), 533 subjects (275 men, 258 women) died; 113 of the deaths were attributable to cardiovascular disease, 96 to diabetes-related diseases (diabetic nephropathy for 92 of these), 249 to other natural causes, and 75 to external causes. After adjusting for age, sex, duration of diabetes, and BMI in a generalized additive proportional hazards model, higher baseline 2-h postload plasma glucose concentration predicted deaths from cardiovascular disease (P = 0.007) and diabetes-related diseases (P = 0.003), but not from other natural causes (P = 0.73). An increment of 5.6 mmol/l (100 mg/dl) in the 2-h plasma glucose concentration was associated with 1.2 times (95% CI 1.1-1.4) the death rate from cardiovascular disease, 1.3 times (95% CI 1.1-1.5) the death rate from diabetes-related diseases, and almost no change in the death rate from other natural causes (rate ratio = 1.0; 95% CI 0.94-1.1). In Pima Indians with type 2 diabetes, higher plasma glucose concentration predicts deaths from cardiovascular and diabetes-related diseases but has little or no effect on deaths from other natural or external causes.     

Association studies of insulin receptor substrate 1 gene (IRS1) variants in type 2 diabetes samples enriched for family history and early age of onset

The gene encoding insulin receptor substrate-1 (IRS1) represents a strong biological candidate for a contributory role in type 2 diabetes susceptibility. Indeed, functional studies have implicated the G971R variant, and a recent meta-analysis of 27 association studies suggested that carriage of 971R was associated with a 25% increase in disease risk. However, this association has not been evaluated in large samples. The present study genotyped the P512A and G971R IRS1 variants in 971 U.K. type 2 diabetic subjects ascertained for strong family history and/or early onset, as well as 1,257 control subjects matched by ethnicity. There was no evidence for association with type 2 diabetes for either variant. (For example, the odds ratio [OR] for carriage of 971R was 1.11 [95% CI 0.86-1.44, P = 0.44]) An updated meta-analysis (31 studies: 5,104 case and 7,418 control subjects) remained significant for the G971R association (P = 0.025), albeit with a diminished OR (1.15 [95% CI 1.02-1.31]). Additional studies of IRS1 variation will be required to obtain a robust estimate of the overall contribution of IRS1 variation to type 2 diabetes susceptibility, but the current study suggests that previous studies have overestimated the magnitude of this effect.     

Genetic susceptibility to nephropathy in Pima Indians with type 2 diabetes mellitus

Summary: In Pima Indians, the incidence of end-stage renal disease, nearly all of which is attributable to type 2 diabetes mellitus, is more than 20 times that in the general United States population. Studies in the Pimas indicate that factors other than diabetes per se enhance susceptibility to the development of diabetic nephropathy. Aggregation of renal disease in families, a relationship between parental blood pressure and diabetic nephropathy in the offspring, and an association between higher prediabetic blood pressure and the occurrence of renal disease after the onset of diabetes all point to individual differences in susceptibility. Although clustering of environmental exposures may be responsible for these findings, they may also represent genetic transmission of susceptibility to renal disease. Recently, linkage analyses were performed in 98 diabetic sib-pairs, both affected by diabetic nephropathy. Two adjacent markers on chromosome 7 met the criteria for suggestive linkage with two-point analysis. Positioned between these markers is the gene coding for aldose reductase. Polymorphisms of this locus are associated with diabetic microvascular complications in other populations. Linkage studies provide evidence that familial aggregation of diabetic renal disease reflects, in part, genetic transmission of susceptibility that appears to be independent of the transmission of diabetes.     

胃旁路术对2型糖尿病大鼠脂肪组织胰岛素受体β及胰岛素受体底物-1表达的影响

目的：观察胃旁路术对2型糖尿病大鼠（GK大鼠）脂肪组织胰岛素受体β（IR-β）及胰岛素受体底物-1（IRS-1）表达的影响,探讨其改善胰岛素抵抗的机制。方法30只8周龄雄性GK大鼠（糖尿病模型）采用数字表法分为手术组（行胃旁路手术）、假手术组（与手术组大鼠相同部位切断后原位端端吻合）和饮食配对组（与手术组大鼠同种和同等质量的饮食）,每组10只,另10只8周龄雄性SD大鼠作为空白对照组（自由进食及饮水）。检测术前与术后4周各组大鼠空腹血糖（FPG）和空腹胰岛素（FINS）水平,计算术前及术后4周胰岛素抵抗指数（HOMA-IR）,应用蛋白印迹（Western blot）技术检测各组术后4周脂肪组织IR-β和IRS-1的表达。结果手术组术后4周FPG及 HOMA-IR 较术前明显降低（分别为5.13±0.22比11.73±0.37,2.16±0.18比5.10±0.29;均P<0.05）,并能达到空白对照组术后水平（P>0.05）;而假手术组和饮食配对组较术前无显著变化（均P＞0.05）;术后4周手术组IR-β及IRS-1表达量均明显高于其他3组（均P<0.05）。结论胃旁路术能上调2型糖尿病大鼠胰岛素信号转导通路中IR-β及IRS-1的表达,改善脂肪组织胰岛素抵抗,提高胰岛素的敏感性。     

Variants in ARHGEF11, a candidate gene for the linkage to type 2 diabetes on chromosome 1q, are nominally associated with insulin resistance and type 2 diabetes in Pima Indians

A prior genome-wide linkage scan in Pima Indians indicated a young-onset (aged <45 years) type 2 diabetes susceptibility locus on chromosome 1q21-q23. ARHGEF11, which encodes the Rho guanine nucleotide exchange factor 11, was analyzed as a positional candidate gene for this linkage because this protein may stimulate Rho-dependent signals, such as the insulin signaling cascade. The ARHGEF11 gene, and two adjacent genes NTRK1 and INSRR, were sequenced in 24 Pima Indians who were not first-degree relatives. Sequencing of the coding regions, 5' and 3' untranslated regions and putative promoter regions of these genes, identified 28 variants in ARHGEF11, 11 variants in NTRK1, and 8 variants in INSSR. These 47 variants, as well as 84 additional public database variants within/between these genes, were genotyped for association analysis in the same group of Pima Indians who had participated in the linkage study (n = 1,228). An R1467H in ARHGEF11, and several additional noncoding variants that were in high linkage disequilibrium with this variant, were nominally associated with young-onset type 2 diabetes (P = 0.01; odds ratio 3.39) after adjusting for sex, family membership, and Pima heritage. The risk allele H had a frequency of 0.10. In a subgroup of 262 nondiabetic, full-heritage Pima Indians who had undergone detailed metabolic testing, the risk allele H also was associated with a lower mean insulin-mediated glucose disposal rate and a lower mean nonoxidative glucose storage rate after adjusting for age, sex, nuclear family membership, and percentage of body fat (P < or = 0.01). These findings suggest that variation within ARHGEF11 nominally increases risk of type 2 diabetes, possibly as a result of increased insulin resistance.     

Adiponectin receptor 1 gene (ADIPOR1) as a candidate for type 2 diabetes and insulin resistance

Considerable data support adiponectin as an important adipose-derived insulin sensitizer that enhances fatty acid oxidation and alters hepatic gluconeogenesis. Adiponectin acts by way of two receptors, ADIPOR1 and ADIPOR2. ADIPOR1 is widely expressed in tissues, including muscle, liver, and pancreas, and binds the globular form of adiponectin with high affinity. To test the hypothesis that sequence variations in or near the ADIPOR1 gene contribute to the risk of developing type 2 diabetes and the metabolic syndrome, we screened the eight exons (including the untranslated exon 1) of the ADIPOR1 gene with flanking intronic sequences and the 5' and 3' flanking sequences. We identified 22 single nucleotide polymorphisms (SNPs) in Caucasian and African-American subjects, of which a single nonsynonymous SNP (N44K) in exon 2 was present only in African-American subjects. We typed 14 sequence variants that had minor allele frequencies >5%. No SNP was associated with type 2 diabetes in Caucasians or African Americans, and no SNP was a determinant of insulin sensitivity or insulin secretion among nondiabetic members of high-risk Caucasian families. However, the two alleles of a SNP in the 3' untranslated region were expressed unequally, and ADIPOR1 mRNA levels were significantly lower among transformed lymphocytes from diabetic African-American individuals than among control cell lines. This altered gene expression might suggest a role for ADIPOR1 in the metabolic syndrome.     

Normal coding sequence of insulin gene in Pima Indians and Nauruans, two groups with highest prevalence of type II diabetes

The nucleotide sequence of the insulin gene was determined in American Pima Indians and Micronesian Nauruans, two populations in whom the prevalence of non-insulin-dependent (type II) diabetes mellitus is the highest in the world. The insulin gene was amplified by the polymerase chain reaction to generate single-stranded DNA suitable for direct sequencing. The nucleotide sequences of the coding and adjacent regions of the insulin gene in six Pima Indians and two Nauruans with type II diabetes were identical to previously published insulin gene sequences of nondiabetic subjects.     

Predominant effect of kidney disease on mortality in Pima Indians with or without type 2 diabetes

BACKGROUND: We examined the effect of kidney disease (KD) on mortality in nondiabetic and diabetic Pima Indians aged > or = 45 years old. METHODS: Deaths and person-years of follow-up were stratified in a time-dependent fashion into categories of (1) no proteinuria and normal serum creatinine (SCr); (2) proteinuria and normal SCr; (3) high SCr [SCr > or = 133 micromol/L (1.5 mg/dL) in men, > or = 124 micromol/L (1.4 mg/dL) in women] but not on renal replacement therapy (RRT); or (4) RRT. RESULTS: Among 1993 subjects, 55.8% had type 2 diabetes at baseline. Overall death rates increased with declining kidney function in both the nondiabetic and diabetic subjects (P < 0.0001). Death rates were similar in nondiabetic and diabetic subjects with comparable levels of kidney function, although the number of deaths among nondiabetic subjects with advanced KD was small. Infections and malignancy were the leading causes of death in nondiabetic subjects with KD. Among diabetic subjects, overall mortality increased with diabetes duration (P = 0.0001) and was highest in those on RRT (P < 0.0001). High SCr was associated with higher death rates from cardiovascular disease (CVD), diabetic nephropathy (DN), infections, and malignancy. CONCLUSION: Death rates increased comparably with worsening kidney function in both nondiabetic and diabetic subjects and were similar in nondiabetic and diabetic subjects without KD. KD was associated with excess mortality from DN, CVD, infections, and malignancy in diabetic subjects, and from infections in those without diabetes.     

Molecular analysis of KCNJ10 on 1q as a candidate gene for Type 2 diabetes in Pima Indians

The KCNJ10 gene is located within a region on chromosome 1q linked to type 2 diabetes in the Pima Indians and six other populations. We therefore investigated this gene as a potential type 2 diabetes candidate gene in Pima Indians. KCNJ10 consists of two exons, spans approximately 33 kb, and we identified eight single-nucleotide polymorphisms (SNPs), including one (SNP2) in the coding region leading to a Glu359Lys substitution. Association studies were carried out in a case-control group composed of 149 affected and 150 unaffected Pimas, and the linkage analysis was performed in a linkage set of 1,338 Pimas. SNP1 in the promoter and SNP2 in the intron, which were in a complete linkage disequilibrium, and SNP5 in the 3' untranslated region showed association with diabetes in the case-control group (P = 0.02 and P = 0.01, respectively). When genotyped in the linkage set, only the KCNJ10-SNP1 variant showed a modest association with type 2 diabetes (P = 0.01). KCNJ10-SNP1 is in a strong linkage disquilibrium with SNP14 of the adjacent KCNJ9 locus, which we previously found to be associated with type 2 diabetes. After adjustment for KCNJ10-SNP1, the original linkage score at this locus was marginally reduced from 3.1 to 2.9. We conclude that these variants in KCNJ10 are unlikely to be the cause of linkage of type 2 diabetes with 1q in Pima Indians.     

Polymorphisms in the oxygen-regulated protein 150 gene (ORP150) are associated with insulin resistance in Pima Indians

The ORP150 gene that encodes the human oxygen-regulated protein (150 kDa) maps to chromosome 11q23, a region previously reported to be linked to type 2 diabetes and obesity in Pima Indians. This gene was also found to be differentially expressed in global gene expression studies comparing muscle mRNA from insulin-resistant versus insulin-sensitive subjects. Therefore, ORP150 was analyzed as a candidate gene for susceptibility to diabetes. Twelve variants were identified, and three unique representative polymorphisms were genotyped in 1,338 Pima Indians. None of these polymorphisms were associated with diabetes, but two polymorphisms were significantly associated with measures of insulin resistance. These data indicate that ORP150 has a role in insulin action but does not have a major role in determining susceptibility to type 2 diabetes in Pima Indians.     

Variants in hepatocyte nuclear factor 4alpha are modestly associated with type 2 diabetes in Pima Indians

Single nucleotide polymorphisms (SNPs) within the hepatocyte nuclear factor 4alpha (HNF4alpha) gene are associated with type 2 diabetes in Finns and Ashkenazi Jews. Previous studies in both populations have reported linkage to type 2 diabetes near the HNF4alpha locus on chromosome 20q12-13. To investigate whether HNF4alpha is a diabetes susceptibility gene in Pima Indians, a population with the highest reported prevalence of type 2 diabetes but with no evidence for linkage of the disease on chromosome 20q, 19 SNPs across the promoter and coding region of HNF4alpha were genotyped for association analysis. In a group of 1,037 Pima Indians (573 diabetic and 464 nondiabetic subjects), three SNPs in HNF4alpha (rs3212183 and rs2071197 located in introns 3 and 1, respectively, and rs6031558, an extremely rare SNP located in the P2 promoter region) were modestly associated with type 2 diabetes (rs3212183 odds ratio [OR] 1.34 [95% CI 1.07-1.67], P = 0.009; rs2071197 1.34 [1.07-1.66], P = 0.008; and rs6031558 3.18 [1.03-9.84], P = 0.04, adjusted for age, sex, birth year, heritage, and family membership). We conclude that variants in HNF4alpha do not appear to be major determinants for type 2 diabetes in Pima Indians; however, HNF4alpha may have a minor role in type 2 diabetes susceptibility within this Native American population.     

Human insulin receptor substrate-2: gene organization and promoter characterization.

Insulin receptor substrate-2 (IRS-2) belongs to a family of cytoplasmic adaptor proteins, which link insulin, IGF-1, and cytokine receptor tyrosine kinases to signaling pathways regulating metabolism, growth, and differentiation (1-3). IRS-2-deficient mice display all characteristics of type 2 diabetes, suggesting that dysfunction of the IRS-2 gene may contribute to the pathogenesis of human type 2 diabetes (4). Based on its progesterone inducibility, we have recently cloned and sequenced a full-length human IRS-2 cDNA containing an open reading frame (ORF) of 4,014 bp and 5'- and 3'-untranslated regions (UTRs) of 516 and 2,466 bp (5). Although the IRS-2 gene has previously been thought to lack introns within the coding region (6,7), the amino acid sequence predicted from our cDNA sequence differed at its very COOH-terminal end from an IRS-2 protein sequence derived from genomic IRS-2 sequences. Therefore, we carefully analyzed the genomic structure of the IRS-2 gene and found that the IRS-2 gene contains an intron that disrupts the ORF. Characterization of promoter and 5'-flanking regions of IRS-2 by sequencing, reporter gene assays, and chromatin structure analysis suggests that elements conferring progesterone inducibility are not located immediately upstream of the gene promoter.