A functional variant in the human betacellulin gene promoter is associated with type 2 diabetes.

Betacellulin (BTC) plays an important role in differentiation, growth, and antiapoptosis of pancreatic beta-cells. We characterized about 2.3 kb of the 5'-flanking region of human BTC gene and identified six polymorphisms (-2159A>G, -1449G>A, -1388C>T, -279C>A, -233G>C, and -226A>G). The G allele in the -226A>G polymorphism was more frequent in type 2 diabetic patients (n = 250) than in nondiabetic subjects (n = 254) (35.6% vs. 27.8%, P = 0.007), and the -2159G, -1449A, and -1388T alleles were in complete linkage disequilibrium with the -226G allele. The frequencies of the -279A and -233C alleles were low (7.0 and 2.0% in diabetic patients), and no significant differences were observed. In the diabetic group, insulin secretion ability, assessed by the serum C-peptide response to intravenous glucagon stimulation, was lower in patients with the -226G allele (G/G, 2.96 +/- 0.16 ng/ml; G/A, 3.65 +/- 0.18 ng/ml; A/A, 3.99 +/- 0.16 ng/ml at 5 min after stimulation; P = 0.008). Furthermore, in vitro functional analyses indicated that both the -226G and the -233C alleles caused an approximately 50% decrease in the promoter activity, but no effects of the -2159A>G, -1449G>A, -1388C>T, and -279C>A polymorphisms were observed. These results suggest that the -226A/G polymorphism of the BTC gene may contribute to the development of diabetes.

Extracellular superoxide dismutase gene polymorphism is associated with insulin resistance and the susceptibility to type 2 diabetes.

Oxidative stress has been implicated in pancreatic beta-cell damage, insulin resistance and vascular function in diabetic patients and the dysfunction of antioxidant enzymes may be associated with the pathogenesis of diabetes. Extracellular superoxide dismutase (EC-SOD) is found in the extracellular matrix of tissues and the major scavenger of superoxide radical. To investigate the role of genetic variability for the pathogenesis of type 2 diabetes, we scanned the protein coding exon and flanking introns of EC-SOD gene for mutation in Japanese type 2 diabetic patients. We identified two missense mutations, Ala40Thr (GCG-->ACG) and Arg213Gly (CGG-->GGG), and a silent mutation, Leu53Leu (CTG-->TTG). For one of these variants, the Ala40Thr polymorphism, the frequency of Thr allele and the number of subjects with Thr allele (Ala/Thr+Thr/Thr) were higher in type 2 diabetic patients (n=205) than those in non-diabetic subjects (n=220) (33.2% versus 24.1%, p=0.003 and 55.6% versus 42.7%, p=0.008, respectively). The patients with Thr allele also showed earlier age at diagnosis of diabetes (42.2+/-7.8 years versus 44.4+/-6.9 years, p=0.037) and higher prevalence of hypertension (53.5% versus 38.5%, p=0.032) than those without the allele. Insulin sensitivity, furthermore, was evaluated in 71 type 2 diabetic patients with short insulin tolerance test (SITT). The patients with Thr allele showed lower insulin sensitivity (Kitt value of SITT) than those without the allele (1.78+/-0.78%/min versus 2.33+/-1.02%/min, p=0.012), although no significant differences in other clinical and biochemical characteristics were observed between two groups. These results suggest that the genetic variant of EC-SOD gene is associated with insulin resistance and the susceptibility to type 2 diabetes.

Uncoupling protein 2 promoter polymorphism -866G/A affects its expression in beta-cells and modulates clinical profiles of Japanese type 2 diabetic patients.

Common uncoupling protein 2 (UCP2) promoter polymorphism -866G/A is reported to be associated with its expression in adipose tissue and the risk of obesity in Caucasians. On the other hand, several studies suggested that UCP2 expression in beta-cells is an important determinant of insulin secretion. In the Japanese population, morbid obesity is very rare, and insulin secretion capacity is relatively low as compared with Caucasians. Because UCP2 would link to insulin secretion and obesity, it might explain this ethnic difference. Here, we report that the UCP2 promoter with the A allele showed higher promoter activity in the INS-1 beta-cell line. The frequency of the A allele is higher in our Japanese study than that in Caucasians. Type 2 diabetic patients with the A allele need insulin therapy earlier and showed higher frequency of insulin treatment. Moreover glucose-induced early insulin secretion is significantly lower in patients with the A allele. However, there was no difference in allele frequency between obese and lean type 2 diabetic patients. In conclusion, UCP2 promoter polymorphism -866G/A does not affect obesity in Japanese type 2 diabetic patients but affects its transcription in beta-cells and modulates glucose-induced insulin secretion and eventually insulin requirement in Japanese type 2 diabetic patients. Higher A allele frequency in the Japanese population might partly explain the ethnic difference of insulin secretion capacity.

Functional variants in the glutathione peroxidase-1 (GPx-1) gene are associated with increased intima-media thickness of carotid arteries and risk of macrovascular diseases in japanese type 2 diabetic patients.

Atherosclerosis in type 2 diabetic patients has been linked to increased oxidative stress. Glutathione peroxidase-1 (GPx-1) plays an important role in the antioxidant defense of the vascular wall. To assess the association between variants in the GPx-1 gene and atherosclerosis, we screened the gene in 184 Japanese type 2 diabetic patients and identified four polymorphisms (-602A/G, +2C/T, Ala(5)/Ala(6), and Pro198Leu). Among these polymorphisms, -602G, +2T, Ala(6), and 198Leu were in strong linkage disequilibrium with each other. The patients were divided into two groups on the basis of the codon 198 polymorphism, Pro/Pro (n = 151) and Pro/Leu (n = 33), to analyze clinical characteristics. The mean intima-media thickness (IMT) of common carotid arteries (P = 0.0028) and the prevalence of cardiovascular disease (P = 0.035) and peripheral vascular disease (P = 0.027) were significantly higher in the Pro/Leu group than in the Pro/Pro group. In vitro functional analyses indicated that the combination of polymorphisms (Ala(6)/198Leu) of the GPx-1 gene had a 40% decrease in enzyme activity, and the combination of polymorphisms (-602G/+2T) had a 25% decrease in transcriptional activity. These results suggest that functional variants in the GPx-1 gene are associated with increased IMT of carotid arteries and risk of cardiovascular and peripheral vascular diseases in type 2 diabetic patients.

Human IAPP-induced pancreatic ß cell toxicity and its regulation by autophagy.

Pancreatic islets in patients with type 2 diabetes mellitus (T2DM) are characterized by loss of ß cells and formation of amyloid deposits derived from islet amyloid polypeptide (IAPP). Here we demonstrated that treatment of INS-1 cells with human IAPP (hIAPP) enhances cell death, inhibits cytoproliferation, and increases autophagosome formation. Furthermore, inhibition of autophagy increased the vulnerability of ß cells to the cytotoxic effects of hIAPP. Based on these in vitro findings, we examined the pathogenic role of hIAPP and its relation to autophagy in hIAPP-knockin mice. In animals fed a standard diet, hIAPP had no toxic effects on ß cell function; however, hIAPP-knockin mice did not exhibit a high-fat-diet-induced compensatory increase in ß cell mass, which was due to limited ß cell proliferation and enhanced ß cell apoptosis. Importantly, expression of hIAPP in mice with a ß cell-specific autophagy defect resulted in substantial deterioration of glucose tolerance and dispersed cytoplasmic expression of p62-associated toxic oligomers, which were otherwise sequestrated within p62-positive inclusions. Together, our results indicate that increased insulin resistance in combination with reduced autophagy may enhance the toxic potential of hIAPP and enhance ß cell dysfunction and progression of T2DM.

Protective role of human insulin against the cytotoxicity associated with human mutant S20G islet amyloid polypeptide.

AIMS/INTRODUCTION: Islet amyloid polypeptide (IAPP) is a main component of islet amyloid in type 2 diabetes and cosecreted from ß-cell with insulin. Clinical evidence from the patients with S20G mutation of the IAPP gene, as well as experimental evidence that insulin could inhibit amyloid formation of IAPP, suggests that a gradual reduction of insulin could be related to the cytotoxicity associated with S20G-IAPP through long-term deterioration of ß-cells in type 2 diabetes. Our objective was to show an effect of human insulin on S20G-IAPP associated cytotoxicity. MATERIALS AND METHODS: We analyzed the cytotoxicity associated with S20G-IAPP by controlling human insulin expression using adenovirus vectors with micro ribonucleic acid specifically against human insulin in endocrine AtT-20ins cells, which express human insulin permanently. Additionally, we carried out a follow-up study of circulating IAPP and insulin in type 2 diabetic patients. RESULTS: S20G-IAPP expression was associated with a decrease in viability and an increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-positive cells in AtT-20ins cells. Furthermore, downregulation of human insulin enhanced the cytotoxicity associated with S20G-IAPP, and induced the cytotoxicity associated with wild-type (WT)-IAPP. Reduction of ubiquitin carboxy-terminal hydrolase L1 activity enhanced cytotoxicity under the downregulation of human insulin expression in both S20G- and WT-IAPP transduced cells. A 5-year follow up of type 2 diabetic patients showed a disproportionate increase of serum fasting IAPP-to-insulin ratio from baseline. CONCLUSIONS: Human insulin plays a protective role against the cytotoxicity associated with S20G-IAPP, as well as WT-IAPP. The findings could suggest long-term deterioration of insulin secretion associates with IAPP linked cytotoxicity in type 2 diabetes.

Expression of wild-type and mutant S20G hIAPP in physiologic knock-in mouse models fails to induce islet amyloid formation, but induces mild glucose intolerance.

UNLABELLED: Aims/Introduction: Human islet polypeptide S20G mutation (hIAPP(S20G)) is associated with earlier onset type 2 diabetes and increased amyloidogenicity and cytotoxicity in vitro vs wild-type hIAPP (hIAPP(WT)), suggesting that amyloidogenesis may be pathogenic for type 2 diabetes. We compared the contributions of hIAPP(S20G) and hIAPP(WT) toward intra islet amyloid formation and development of type 2 diabetes in a unique physiologic knock-in mouse model. MATERIALS AND METHODS: We replaced the mouse IAPP gene (M allele) with hIAPP(WT) (W allele) and hIAPP(S20G) (G allele) via homologous recombination and backbred transgenic mice against C57Bl/6 strain 5 generations to minimize genetic variation. Mice (3 month old) were maintained on control (CD) or high fat diet (HFD) for 15 months and studied at 3 month intervals by oral glucose tolerance testing (OGTT) and pancreas histology to assess glucose homeostastis, amyloidogeneisis, islet mass, ß cell replication, and apoptosis. RESULTS: IAPP blood levels were indistinguishable in all mice. WW and GW mice maintained on both diets lacked intraislet amyloid at all ages. On both diets relative to MM controls WW and GW mice exhibit glucose intolerance (P < 0.008) with no differences in insulin secretion. However, GW mice secreted significantly more insulin (P < 0.03 that WW mice on both diets throughout the study. By 12 months on the high fat diet all mice increased their ß cell mass about 3-fold and were indistinguishable. CONCLUSIONS: Physiologic expression of hIAPP(WT) and hIAPP(S20G) in C57Bl/6 mice produces mild glucose intolerance with inappropriately normal insulin secretion that is independent of intraislet amyloid formation. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00166.x, 2011).

Progressive deterioration of insulin secretion in Japanese type 2 diabetic patients in comparison with those who carry the S20G mutation of the islet amyloid polypeptide gene: A long-term follow-up study.

UNLABELLED: Aims/Introduction: In order to clarify the enhanced ß-cell dysfunction in type 2 diabetic patients carrying the S20G mutation of the islet amyloid polypeptide gene (S20G-patients), we first estimated the decline of insulin secretion in Japanese type 2 diabetic patients without the S20G mutation (non-S20G-T2D-patients) by long-term observation, and then compared it with that of the S20G-patients. MATERIALS AND METHODS: We followed 70 non-S20G-T2D-patients (body mass index <30 kg/m(2)) for more than 10 years and six S20G-patients for more than 5 years. We measured fasting C-peptide (F-CP) every 1-2 years and carried out a glucagon test at least once during the follow-up period. F-CP and a 5-min value of C-peptide after glucagon injection (5'-CP) were used as the indices of insulin secretion. We excluded patients who had renal dysfunction and/or anti-insulin antibodies in the insulin-treated patients. The individual annual declines were calculated from the slopes of the regression lines between C-peptide levels and duration (years after diagnosis). RESULTS: The mean individual annual declines of both F-CP and 5'-CP were significantly greater in the S20G-patients than the non-S20G-T2D-patients (F-CP; 0.047 ± 0.026 vs 0.011 ± 0.037 nmol/L/year, P = 0.025, 5'-CP; 0.139 ± 0.055 vs 0.022 ± 0.012 nmol/L/year, P = 0.008). CONCLUSIONS: We established the annual decline of insulin secretion in the Japanese type 2 diabetic patients by the long-term observation. The results show that the decline of insulin secretion is more rapid in the S20G-patients than the non-S20G-T2D-patients. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00102.x, 2011).

Autophagy protects against human islet amyloid polypeptide-associated apoptosis.

UNLABELLED: Aims/Introduction: Islets in type 2 diabetes are characterized by deposition of islet amyloid polypeptide (IAPP) as well as ß-cell dysfunction. The unique amyloidogenic character of human (h)IAPP is associated with cytotoxicity. Autophagy is a ubiquitous system of cellular recycling that contributes to cell survival. Thus, we examined whether autophagy could ameliorate hIAPP-associated cytotoxicity. MATERIALS AND METHODS: First, we used a COS-1 cell model, lacking endogenous IAPP that might affect cytotoxicity related to exogenous hIAPP. Next, we used the mouse ß-cell line, MIN-6 cells. Both cells were transfected with hIAPP or rat (r)IAPP expression constructs, or transfected with bicistronic vectors expressing green fluorescent protein (GFP) and either hIAPP or rIAPP for flow cytometry analysis. Cell viability and apoptosis markers were studied in relation to chemical or genetic modulation of autophagy. RESULTS: The viability of cells expressing hIAPP was significantly decreased as compared with those expressing rIAPP and the cleavage of pro-caspase-3 was elevated in hIAPP-transfected cells. The formation of autophagosomes and the conversion of microtubule-associated protein light chain 3B I to II were elevated in hIAPP-expressing cells. The viability of hIAPP-expressing cells was increased after treatment with rapamycin, an inducer of autophagy, and decreased after treatment with 3-methyladenine, an inhibitor of autophagy. In MIN-6 cells, annexin positive cells were increased by 3-methyladenine and decreased by rapamycin using flow cytometry. Knocking down of the autophagy protein 5 gene decreased hIAPP-transfected cell viability. CONCLUSIONS: Autophagy is co-localized with hIAPP expression and it plays a protective role in hIAPP-associated apoptosis. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00065.x, 2010).