Metabolic effects of the Gly1057Asp polymorphism in IRS-2 and interactions with obesity

Insulin receptor substrate (IRS)-2 plays an important role in insulin signaling and its disruption results in diabetes in mice. In humans, the IRS-2 Gly1057Asp substitution was associated with lower risk of type 2 diabetes in lean individuals, but with a higher risk in obese individuals. To clarify the role of IRS-2 on the development of type 2 diabetes and obesity in Pima Indians, and particularly to investigate whether the effects of the Gly1057Asp polymorphism on metabolism are mediated by obesity, molecular scanning of the gene for mutations was performed and interaction of the polymorphism with obesity was tested. We identified the previously described Gly1057Asp mutation as well as a rare Asp819His mutation and four silent polymorphisms. The effect of the Gly1057Asp mutation on type 2 diabetes and obesity was tested in a large cohort of Pima Indians (n = 998). A subgroup of nondiabetic full-heritage Pima Indians (n = 233) had measurements of body composition, glucose tolerance, insulin action (M), endogenous glucose production (EGP; hyperinsulinemic clamp), acute insulin response (AIR, 25-g intravenous glucose tolerance test, n = 118 normal glucose-tolerant subjects), and percutaneous fat biopsy specimens from the periumbilical region (n = 160). A total of 132 nondiabetic subjects were included in longitudinal analyses. The frequency of the Asp1057 allele was 0.6. In cross-sectional analyses, subjects homozygous for the Asp1057 allele (Asp/Asp) had a higher prevalence of type 2 diabetes than heterozygote individuals and subjects homozygous for the Gly1057 allele (X/Gly, P = 0.04). There was no effect on BMI (P = 0.78) or gene-BMI interaction on the prevalence of type 2 diabetes (P = 0.57). In the nondiabetic subgroup, subjects with Asp/Asp had higher percent body fat (P = 0.01), BMI (P = 0.02), and waist circumference (P = 0.004), but there was no difference in metabolic characteristics (all P > 0.2). However, the relationship between percent body fat and fasting glucose, basal EGP, EGP during the clamp, AIR, and subcutaneous abdominal adipocyte size was significantly different in the Asp/Asp group (P for interaction = 0.02, 0.06, 0.0007, 0.08, and 0.006, respectively) compared with the X/Gly group, suggesting a more detrimental effect of Asp homozygosity on these traits with increasing percent body fat. In longitudinal analyses, among subjects in the upper tertile of change in percent body fat, those with Asp/Asp had a larger increase in fasting and postprandial glycemia and basal EGP and a larger decrease in M and AIR than subjects with X/Gly, independent of change in obesity (all P < 0.05). In conclusion, our findings suggest that the association of homozygosity for the Asp1057 allele in IRS-2 with type 2 diabetes in Pima Indians may be mediated by interaction of the polymorphism with obesity on several diabetes-related traits.     

The role of insulin receptor substrate-1 gene (IRS1) in type 2 diabetes in Pima Indians

The insulin receptor substrate-1 (IRS1) is a critical element in insulin-signaling pathways, and mutations in the IRS1 gene have been reported to have a role in determining susceptibility to traits related to type 2 diabetes. In gene expression studies of tissue biopsies from nondiabetic Pima Indians, IRS1 mRNA levels were reduced in adipocytes from obese subjects compared with lean subjects, and IRS1 mRNA levels were also reduced in skeletal muscle from insulin-resistant subjects compared with insulin-sensitive subjects (all P < 0.05). Based on these expression differences and the known physiologic role of IRS1, this gene was investigated as a candidate gene for susceptibility to type 2 diabetes in Pima Indians, a population with an extremely high incidence and prevalence of type 2 diabetes. Thirteen variants were identified, and among these variants, several were in complete linkage disequilibrium. Four genotypically unique variants were further genotyped in 937 DNA samples from full-heritage Pima Indians. Three of the variants were modestly associated with type 2 diabetes (P < 0.05), one of which was additionally associated with 2-h plasma insulin and glucose as well as insulin action at physiologic and maximally stimulating insulin concentrations (all P < 0.05). The association of variants in IRS1 with type 2 diabetes and type 2 diabetes-related phenotypes and the differential expression of IRS1 in adipocytes and skeletal muscle suggest a role of this gene in the pathogenesis of type 2 diabetes in Pima Indians.     

Identification of novel genes differentially expressed in omental fat of obese subjects and obese type 2 diabetic patients.

Obesity is associated with an increased risk for developing type 2 diabetes, insulin resistance, hypertension, dyslipidemia, cardiovascular disease, respiratory dysfunction, and certain forms of cancer. Insulin resistance in many type 2 diabetic patients is the result of increased visceral adiposity. To identify novel genes implicated in type 2 diabetes and/or obesity and to elucidate the molecular mechanisms underlying both diseases, we analyzed gene expression in omental fat from lean and obese nondiabetic subjects and obese type 2 diabetic patients using mRNA differential display and subtracted library techniques. After screening over 13,800 subtracted cDNA clones and 6,912 cDNA amplification products, we identified 2,078 cDNAs that showed potential differential expression in the omental fat of lean versus obese nondiabetic subjects versus obese type 2 diabetic patients. Data analysis showed that 70.7% of these clones corresponded to unknown genes (26.7% matched express sequence tags [ESTs]) and 29.3% corresponded to known genes. Reverse Northern and classic Northern analyses further confirmed that the expression of five of these cDNA clones was elevated in obese nondiabetic subjects and obese type 2 diabetic patients. Four candidate genes were further evaluated for tissue distribution, which showed expression primarily in adipose and skeletal muscle tissue, and chromosomal localization. We concluded that both mRNA differential display and subtracted cDNA libraries are powerful tools for identifying novel genes implicated in the pathogenesis of obesity and type 2 diabetes.     

Variations in peptide YY and Y2 receptor genes are associated with severe obesity in Pima Indian men

Peptide YY (PYY) and Y2 receptor (Y2R) may be important in the central regulation of body weight and food intake. To determine whether genetic variation in PYY and/or Y2R may contribute to morbid obesity in humans, these genes were sequenced in 83 extremely obese Pima Indians (BMI > or = 50 kg/m2). Sequencing of PYY identified three single nucleotide polymorphisms (SNPs) in the untranslated region. Sequencing of the Y2R coding region identified one missense (Ala172Thr) substitution and two silent substitutions. Eight additional SNPs in the 5' untranslated region of Y2R were identified from public databases. These SNPs were genotyped in 489 full-heritage adult Pimas (362 severely obese and 127 nondiabetic, nonobese subjects), who are not first-degree relatives, for association analysis. The PYY variants were not associated with obesity, whereas four variants from two haplotype blocks in Y2R were marginally associated (P = 0.054-0.067) with obesity. However, if the analysis was restricted to men (n = 167, 100 obese and 67 lean), the PYY variants and two SNPs in Y2R that were in complete linkage disequilibrium were significantly associated with severe obesity (P = 0.001 and P = 0.002, respectively). Our data suggest that the PYY-Y2R pathway may influence body weight through a sex-specific mechanism, but this finding requires confirmation in other populations.     

The L-α-lysophosphatidylinositol/GPR55 system and its potential role in human obesity

GPR55 is a putative cannabinoid receptor, and l-α-lysophosphatidylinositol (LPI) is its only known endogenous ligand. We investigated 1) whether GPR55 is expressed in fat and liver; 2) the correlation of both GPR55 and LPI with several metabolic parameters; and 3) the actions of LPI on human adipocytes. We analyzed CB1, CB2, and GPR55 gene expression and circulating LPI levels in two independent cohorts of obese and lean subjects, with both normal or impaired glucose tolerance and type 2 diabetes. Ex vivo experiments were used to measure intracellular calcium and lipid accumulation. GPR55 levels were augmented in the adipose tissue of obese subjects and further so in obese patients with type 2 diabetes when compared with nonobese subjects. Visceral adipose tissue GPR55 correlated positively with weight, BMI, and percent fat mass, particularly in women. Hepatic GPR55 gene expression was similar in obese and type 2 diabetic subjects. Circulating LPI levels were increased in obese patients and correlated with fat percentage and BMI in women. LPI increased the expression of lipogenic genes in visceral adipose tissue explants and intracellular calcium in differentiated visceral adipocytes. These findings indicate that the LPI/GPR55 system is positively associated with obesity in humans.     

A functional Tyr1306Cys variant in LARG is associated with increased insulin action in vivo

Diminished insulin sensitivity is a characteristic feature of type 2 diabetes. Inhibition of insulin action, resulting in reduced skeletal muscle glucose uptake, is mediated in part through stimulation of RhoA activity. One regulator of RhoA activity is leukemia-associated Rho guanine nucleotide exchange factor (LARG). The LARG gene maps to a region on chromosome 11q23-24 that shows genetic linkage to BMI and type 2 diabetes in Pima Indians. Because of its role in RhoA activation, the LARG gene was analyzed as a positional candidate gene for this linkage. Sequencing of the LARG gene and genotyping of variants identified several polymorphisms that were associated with in vivo rates of insulin-mediated glucose uptake, at both physiological and maximally stimulating insulin concentrations, among 322 nondiabetic Pima Indians who had undergone a hyperinsulinemic-euglycemic clamp. The strongest association with rate of glucose uptake was found with a Tyr1306Cys polymorphism (P < 0.0001, adjusted for age, sex, percent body fat, and nuclear family membership). In transient transfection studies in NIH3T3 cells, the LARG(Cys1306) protein had reduced activity compared with LARG(Tyr1306) protein (P < 0.05). We propose that the Tyr1306Cys substitution in LARG, through its differential activation of RhoA, increases insulin sensitivity in nondiabetic Pima Indians.     

Melanocortin 4 receptor gene variation is associated with severe obesity in Pima Indians

Heterozygous coding mutations in the melanocortin 4 receptor gene (MC4R) are the most common genetic causes of severe human obesity identified to date. To determine whether MC4R has a role in causing severe obesity in Pima Indians, we sequenced the coding region of this gene in 426 full-heritage, non-first-degree related, adult Pima Indians (300 severely obese and 126 nondiabetic nonobese control subjects). Three coding variations were detected as heterozygotes only in severely obese subjects. One variation, detected in three obese subjects, was a novel single-base insertion (A) at nucleotide 100, and it predicted a frameshift and premature STOP at codon 37. The second variant, detected in 10 obese subjects, predicted a previously identified arginine-to-glutamine substitution at codon 165, and the third variant, detected in one obese subject, predicted a novel glycine-to-serine substitution at codon 231. Three polymorphisms were also identified in the 5' untranslated region, but these variants were detected in both obese and lean subjects and had similar allele frequencies. We conclude that variations in MC4R may account for a small portion of obesity in Pima Indians, but they do not explain the overall high prevalence of obesity in this Native American population.     

The 3'-untranslated region polymorphism of the gene for skeletal muscle-specific glycogen-targeting subunit of protein phosphatase 1 in the type 2 diabetic Japanese population.

A newly identified 3'-untranslated region (UTR) polymorphism of the gene for skeletal muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PPP1R3) was associated with insulin resistance and type 2 diabetes in Pima Indians (Xia J, Scherers W, Cohen PTW, Majer M, Xi T, Norman RA, Knowler WC, Bogardus C, Prochazka M: A common variant in PP1R3 associated with insulin resistance and type 2 diabetes. Diabetes 47:1519-1524, 1998). Thus, we investigated the frequency of polymorphism of the adenine- and thymine-rich element (ARE-1 and its variant ARE-2) in 426 Japanese type 2 diabetic and 380 nondiabetic subjects using a polymerase chain reaction (PCR)-restriction enzyme fragment length polymorphism (RFLP) method. The allele frequency of the ARE-2 variant in diabetic subjects was higher than that in nondiabetic subjects (0.34 vs. 0.29; P < 0.05), even though its frequency in Japanese subjects was lower (P < 0.001) than the reported value in Pima Indians (0.56). An aspartate polymorphism at codon 905 was 100% coupled to the ARE-2 allele, and its allele frequency was higher also in diabetic subjects. Although a serine substitution at codon 883 was partially linked with the ARE-2 allele, there was no difference between diabetic and nondiabetic subjects. These results indicate that the frequency of polymorphism of the PPP1R3 gene (ARE-2 and Asp905) is different between two ethnic groups and is increased in Japanese people with type 2 diabetes, suggesting that these variants may be a possible marker for searching for diabetogenic genes.     

Low plasma adiponectin concentrations do not predict weight gain in humans

Low concentrations of plasma adiponectin, the most abundant adipose-specific protein, are observed in obese individuals and predict the development of type 2 diabetes. Administration of adiponectin to rodents prevented diet-induced weight gain, suggesting a potential etiologic role of hypoadiponectinemia in the development of obesity. Our aim was to prospectively examine whether low plasma adiponectin concentrations predict future weight gain in Pima Indians, explaining the predictive effect of adiponectin on the development of type 2 diabetes. We measured plasma adiponectin concentrations in 219 nondiabetic Pima Indians (112 M/107 F, age 31 +/- 9 years, body weight 96 +/- 20 kg [mean +/- SD]) in whom body weight and height were measured and BMI calculated at baseline and follow-up. Cross-sectionally, plasma adiponectin concentrations were negatively associated with body weight (r = -0.28, P = 0.0001). Prospectively, plasma adiponectin concentrations at baseline were not associated with change in weight or BMI before or after adjustment for time of follow-up or after additional adjustment for age at follow-up and sex (all P > 0.3). Our data suggest that low plasma adiponectin concentrations do not play an etiologic role in development of obesity in Pima Indians. Therefore, the predictive effect of low plasma adiponectin concentrations on the development of type 2 diabetes seems to be mediated by factors other than increased adiposity.     

High white blood cell count is associated with a worsening of insulin sensitivity and predicts the development of type 2 diabetes.

Chronic low-grade inflammation may be involved in the pathogenesis of insulin resistance and type 2 diabetes. We examined whether a high white blood cell count (WBC), a marker of inflammation, predicts a worsening of insulin action, insulin secretory function, and the development of type 2 diabetes in Pima Indians. We measured WBC in 352 nondiabetic Pima Indians (215 men and 137 women, aged 27 +/- 6 years [means +/- SD], body fat 32 +/- 8%, WBC 8,107 +/- 2,022 cells/mm(3)) who were characterized for body composition (by hydrodensitometry or dual-energy X-ray absorptiometry), glucose tolerance (by 75-g oral glucose tolerance test), insulin action (M; by hyperinsulinemic clamp), and acute insulin secretory response (AIR; by 25-g intravenous glucose challenge). Among 272 subjects who were normal glucose tolerant (NGT) at baseline, 54 developed diabetes over an average follow-up of 5.5 +/- 4.4 years. Among those who remained nondiabetic, 81 subjects had follow-up measurements of M and AIR. Cross-sectionally, WBC was related to percent body fat (r = 0.32, P < 0.0001) and M (r = -0.24, P < 0.0001), but not to AIR (r = 0.06, P = 0.4). In a multivariate analysis, when adjusted for age and sex, both percent body fat (P < 0.0001) and M (P = 0.03) were independently associated with WBC. A high WBC value predicted diabetes (relative hazard 90th vs. 10th percentiles [95%CI] of 2.7 [1.3-5.4], P = 0.007) when adjusted for age and sex. The predictive effect of WBC persisted after additional adjustment for established predictors of diabetes, i.e., percent body fat, M, and AIR (relative hazard 2.6 [1.1-6.2], P = 0.03). After adjustment for follow-up duration, a high WBC at baseline was associated with a subsequent worsening of M (P = 0.003), but not a worsening of AIR. A high WBC predicts a worsening of insulin action and the development of type 2 diabetes in Pima Indians. These findings are consistent with the hypothesis that a chronic activation of the immune system may play a role in the pathogenesis of type 2 diabetes.     

Genetic variation in the human winged helix/forkhead transcription factor gene FOXC2 in Pima Indians

FOXC2 is a winged helix gene that has been shown to counteract obesity, hypertriglyceridemia, and diet-induced insulin resistance in rodents. Therefore, FOXC2 was analyzed as a candidate gene for susceptibility to type 2 diabetes in Pima Indians. Four variants were identified by sequencing the coding region, as well as 638 bp of the 5' region and 300 bp of the 3' region of the gene. Two single nucleotide polymorphisms (SNPs) were found in the putative promoter region, a C-512T transition and a G-350T. In addition, two SNPs were found in the 3' region, a C1548T and a C1702T. The G-350T and the C1702T variants were in complete linkage disequilibrium, and the C1548T variant was relatively rare; therefore, only the C-512T and G-350T variants were additionally genotyped in 937 full-blooded Pima Indians. Neither of these polymorphisms were associated with type 2 diabetes; however, the C-512T variant was associated with BMI (P = 0.03) and percentage of body fat (P = 0.02) in male and female Pima subjects, as well as with basal glucose turnover and fasting plasma triglycerides in women. Our data indicate that variation in FOXC2 may have a minor role in body weight control and seems to be involved in the regulation of basal glucose turnover and plasma triglyceride levels in women, but this gene does not significantly contribute to the etiology of type 2 diabetes in Pima Indians.     

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.     

Common Polymorphisms in the Adiponectin Gene ACDC Are Not Associated With Diabetes in Pima Indians

Adiponectin is an abundant adipose tissue-derived protein with important metabolic effects. Plasma adiponectin levels are decreased in obese individuals, and low adiponectin levels predict insulin resistance and type 2 diabetes. Two variants in the adiponectin gene ACDC have been previously associated with plasma adiponectin levels, obesity, insulin resistance, and type 2 diabetes. To determine the role of genetic variation in ACDC in susceptibility to obesity and type 2 diabetes in Pima Indians, we screened the promoter, exons, and exon-intron boundaries of the gene to identify allelic variants. We identified 17 informative polymorphisms that comprised four common (minor allele frequency >15%) linkage disequilibrium clusters consisting of 1-4 variants each. We genotyped one representative polymorphism from each cluster in 1,338 individuals and assessed genotypic association with type 2 diabetes, BMI, serum lipid levels, serum adiponectin levels, and measures of insulin sensitivity and secretion. None of the ACDC variants were associated with type 2 diabetes, BMI, or measures of insulin sensitivity or secretion. One variant, single nucleotide polymorphism (SNP)-12823, was associated with serum adiponectin levels (P = 0.002), but this association explained only 2% of the variance of serum adiponectin levels. Our findings suggest that these common ACDC polymorphisms do not play a major role in susceptibility to obesity or type 2 diabetes in this population.     

Expression of Mfn2, the Charcot-Marie-Tooth neuropathy type 2A gene, in human skeletal muscle: effects of type 2 diabetes, obesity, weight loss, and the regulatory role of tumor necrosis factor alpha and interleukin-6

The primary gene mutated in Charcot-Marie-Tooth type 2A is mitofusin-2 (Mfn2). Mfn2 encodes a mitochondrial protein that participates in the maintenance of the mitochondrial network and that regulates mitochondrial metabolism and intracellular signaling. The potential for regulation of human Mfn2 gene expression in vivo is largely unknown. Based on the presence of mitochondrial dysfunction in insulin-resistant conditions, we have examined whether Mfn2 expression is dysregulated in skeletal muscle from obese or nonobese type 2 diabetic subjects, whether muscle Mfn2 expression is regulated by body weight loss, and the potential regulatory role of tumor necrosis factor (TNF)alpha or interleukin-6. We show that mRNA concentration of Mfn2 is decreased in skeletal muscle from both male and female obese subjects. Muscle Mfn2 expression was also reduced in lean or in obese type 2 diabetic patients. There was a strong negative correlation between the Mfn2 expression and the BMI in nondiabetic and type 2 diabetic subjects. A positive correlation between the Mfn2 expression and the insulin sensitivity was also detected in nondiabetic and type 2 diabetic subjects. To determine the effect of weight loss on Mfn2 mRNA expression, six morbidly obese subjects were subjected to weight loss by bilio-pancreatic diversion. Mean expression of muscle Mfn2 mRNA increased threefold after reduction in body weight, and a positive correlation between muscle Mfn2 expression and insulin sensitivity was again detected. In vitro experiments revealed an inhibitory effect of TNFalpha or interleukin-6 on Mfn2 expression in cultured cells. We conclude that body weight loss upregulates the expression of Mfn2 mRNA in skeletal muscle of obese humans, type 2 diabetes downregulates the expression of Mfn2 mRNA in skeletal muscle, Mfn2 expression in skeletal muscle is directly proportional to insulin sensitivity and is inversely proportional to the BMI, TNFalpha and interleukin-6 downregulate Mfn2 expression and may participate in the dysregulation of Mfn2 expression in obesity or type 2 diabetes, and the in vivo modulation of Mfn2 mRNA levels is an additional level of regulation for the control of muscle metabolism and could provide a molecular mechanism for alterations in mitochondrial function in obesity or type 2 diabetes.     

Overnight lowering of free fatty acids with Acipimox improves insulin resistance and glucose tolerance in obese diabetic and nondiabetic subjects.

Obesity is commonly associated with elevated plasma free fatty acid (FFA) levels, as well as with insulin resistance and hyperinsulinemia, two important cardiovascular risk factors. What causes insulin resistance and hyperinsulinemia in obesity remains uncertain. Here, we have tested the hypothesis that FFAs are the link between obesity and insulin resistance/hyperinsulinemia and that, therefore, lowering of chronically elevated plasma FFA levels would improve insulin resistance/hyperinsulinemia and glucose tolerance in obese nondiabetic and diabetic subjects. Acipimox (250 mg), a long-acting antilipolytic drug, or placebo was given overnight (at 7:00 P.M., 1:00 A.M., 7:00 A.M.) to 9 lean control subjects, 13 obese nondiabetic subjects, 10 obese subjects with impaired glucose tolerance, and 11 patients with type 2 diabetes. Euglycemic-hyperinsulinemic clamps and oral glucose tolerance tests (75 g) were performed on separate mornings after overnight Acipimox or placebo treatment. In the three obese study groups, Acipimox lowered fasting levels of plasma FFAs (by 60-70%) and plasma insulin (by approximately 50%). Insulin-stimulated glucose uptake during euglycemic-hyperinsulinemic clamping was more than twofold higher after Acipimox than after placebo. Areas under the glucose and insulin curves during oral glucose tolerance testing were both approximately 30% lower after Acipimox administration than after placebo. We conclude that lowering of elevated plasma FFA levels can reduce insulin resistance/hyperinsulinemia and improve oral glucose tolerance in lean and obese nondiabetic subjects and in obese patients with type 2 diabetes.     

Influence of obesity and type 2 diabetes on gluconeogenesis and glucose output in humans: a quantitative study

The contribution of gluconeogenesis (GNG) to endogenous glucose output (EGO) in type 2 diabetes is controversial. Little information is available on the separate influence of obesity on GNG. We measured percent GNG (by the 2H2O technique) and EGO (by 6,6-[2H]glucose) in 37 type 2 diabetic subjects (9 lean and 28 obese, mean fasting plasma glucose [FPG] 8.3 +/- 0.3 mmol/l) and 18 control subjects (6 lean and 12 obese) after a 15-h fast. Percent GNG averaged 47 +/- 5% in lean control subjects and was significantly increased in association with both obesity (P < 0.01) and diabetes (P = 0.004). By multivariate analysis, percent GNG was independently associated with BMI (partial r = 0.27, P < 0.05, with a predicted increase of 0.9% per BMI unit) and FPG (partial r = 0.44, P = 0.0009, with a predicted increase of 2.7% per mmol/l of FPG). In contrast, EGO was increased in both lean and obese diabetic subjects (15.6 +/- 0.5 micromol x min(-1) x kg(-1) of fat-free mass, n = 37, P = 0.002) but not in obese nondiabetic control subjects (13.1 0.7, NS) as compared with lean control subjects (12.4 +/- 1.4). Consequently, gluconeogenic flux (percent GNG x EGO) was increased in obesity (P = 0.01) and markedly elevated in diabetic subjects (P = 0.0004), whereas glycogenolytic flux was reduced only in association with obesity (P = 0.05). Fasting plasma glucagon levels were significantly increased in diabetic subjects (P < 0.05) and positively related to EGO, whereas plasma insulin was higher in obese control subjects than lean control subjects (P = 0.05) and unrelated to measured glucose fluxes. We conclude that the percent contribution of GNG to glucose release after a 15-h fast is independently and quantitatively related to the degree of overweight and the severity of fasting hyperglycemia. In obese individuals, reduced glycogenolysis ensures a normal rate of glucose output. In diabetic individuals, hyperglucagonemia contributes to inappropriately elevated rates of glucose output from both GNG and glycogenolysis.     

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.     

Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes

Type 2 diabetes is characterized by impaired insulin secretion. Some but not all studies suggest that a decrease in beta-cell mass contributes to this. We examined pancreatic tissue from 124 autopsies: 91 obese cases (BMI >27 kg/m(2); 41 with type 2 diabetes, 15 with impaired fasting glucose [IFG], and 35 nondiabetic subjects) and 33 lean cases (BMI <25 kg/m(2); 16 type 2 diabetic and 17 nondiabetic subjects). We measured relative beta-cell volume, frequency of beta-cell apoptosis and replication, and new islet formation from exocrine ducts (neogenesis). Relative beta-cell volume was increased in obese versus lean nondiabetic cases (P = 0.05) through the mechanism of increased neogenesis (P < 0.05). Obese humans with IFG and type 2 diabetes had a 40% (P < 0.05) and 63% (P < 0.01) deficit and lean cases of type 2 diabetes had a 41% deficit (P < 0.05) in relative beta-cell volume compared with nondiabetic obese and lean cases, respectively. The frequency of beta-cell replication was very low in all cases and no different among groups. Neogenesis, while increased with obesity, was comparable in obese type 2 diabetic, IFG, or nondiabetic subjects and in lean type 2 diabetic or nondiabetic subjects. However, the frequency of beta-cell apoptosis was increased 10-fold in lean and 3-fold in obese cases of type 2 diabetes compared with their respective nondiabetic control group (P < 0.05). We conclude that beta-cell mass is decreased in type 2 diabetes and that the mechanism underlying this is increased beta-cell apoptosis. Since the major defect leading to a decrease in beta-cell mass in type 2 diabetes is increased apoptosis, while new islet formation and beta-cell replication are normal, therapeutic approaches designed to arrest apoptosis could be a significant new development in the management of type 2 diabetes, because this approach might actually reverse the disease to a degree rather than just palliate glycemia.     

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.