Association of the Ser326Cys polymorphism in the OGG1 gene with type 2 DM

The association of the Ser326Cys polymorphism of the 8-oxoguanine glycosylase 1 (OGG1) gene with type 2 diabetes was examined using a Japanese population (n (M/W): 4585 (2085/2500); age: 62.6 ± 10.9 years). HbA1c levels and frequency of diabetic subjects were significantly higher in subjects with genotypes with Cys allele than in those without (p = 0.032 and 0.037, respectively). Multiple logistic regression analysis showed that genotypes with Cys allele were significantly associated with diabetes (OR: 1.32, p = 0.0289). In subjects whose glucose tolerance was classified by FPG and 2-h PG (n = 1.634), the association was more substantial (genotypes with Cys allele vs. without, OR: 1.70, p = 0.0059; genotypes Cys/Cys vs. Ser/Ser, OR: 2.19, p = 0.0008). In subjects with genotype Ser/Ser, the insulin secretion index, HOMA-β, increased in the subjects with glucose intolerance and decreased in the subjects with diabetes, while, in subjects with genotypes Ser/Cys + Cys/Cys, HOMA-β decreased as the glucose tolerance progressed (p for trend = 0.010).     

Association of P213S polymorphism of the L-selectin gene with type 2 diabetes and insulin resistance in Chinese population

Aims

L-selectin belongs to selectin family of adhesion molecule and participates in the generation and development of type 2 diabetes (T2D). In this study, we evaluated the relationship between the P213S polymorphism of L-selectin gene and T2D and insulin resistance in the Chinese population.

Methods

We genotyped P213S polymorphism in 801 patients with T2D and 834 healthy controls in the Chinese population using polymerase chain reaction–ligase detection reaction (PCR–LDR) technique. Plasma glucose, insulin, lipid, blood urea nitrogen, creatinine and uric acid levels were measured by biochemical technique.

Results

The frequency of 213PP genotype and P allele of the L-selectin gene in patients with T2D was significantly higher than that in controls (P = 0.007; P = 0.019, respectively). The relative risk of allele P suffered from T2D was 1.191 times higher than that of allele S. Moreover, the levels of FPG and HOMA-IR of PP and PS genotype carriers were significantly higher than those of SS genotype carriers in the T2D group (P < 0.05).

Conclusion

These findings indicated that the P213S polymorphism of L‐selectin gene may contribute to susceptibility to T2D and insulin resistance in the Chinese population, and P allele appears to be a risk factor for T2D.     

MicroRNA 144 impairs insulin signaling by inhibiting the expression of insulin receptor substrate 1 in type 2 diabetes mellitus

Background

Dysregulation of microRNA (miRNA) expression in various tissues and body fluids has been demonstrated to be associated with several diseases, including Type 2 Diabetes mellitus (T2D). Here, we compare miRNA expression profiles in different tissues (pancreas, liver, adipose and skeletal muscle) as well as in blood samples from T2D rat model and highlight the potential of circulating miRNAs as biomarkers of T2D. In parallel, we have examined the expression profiles of miRNAs in blood samples from Impaired Fasting Glucose (IFG) and T2D male patients.

Methodology/Principal Findings

Employing miRNA microarray and stem-loop real-time RT-PCR, we identify four novel miRNAs, miR-144, miR-146a, miR-150 and miR-182 in addition to four previously reported diabetes-related miRNAs, miR-192, miR-29a, miR-30d and miR-320a, as potential signature miRNAs that distinguished IFG and T2D. Of these microRNAs, miR-144 that promotes erythropoiesis has been found to be highly up-regulated. Increased circulating level of miR-144 has been found to correlate with down-regulation of its predicted target, insulin receptor substrate 1 (IRS1) at both mRNA and protein levels. We could also experimentally demonstrate that IRS1 is indeed the target of miR-144.

Conclusion

We demonstrate that peripheral blood microRNAs can be developed as unique biomarkers that are reflective and predictive of metabolic health and disorder. We have also identified signature miRNAs which could possibly explain the pathogenesis of T2D and the significance of miR-144 in insulin signaling.     

Evidence for early impairment of glucagon-like peptide 1-induced insulin secretion in human type 2 (non insulin-dependent) diabetes.

To investigate a possible role of an enteroinsular axis involvement in the pathogenesis of type 2 diabetes, plasma glucagon-like peptide 1 (GLP-1) 7-36 amide response to nutrient ingestion was evaluated in type 2 diabetics affected by different degrees of beta-cell dysfunction. METHODS: 14 patients on oral hypoglycaemic treatment (group A: HbA1C = 8.1 +/- 1.8 %) and 11 age-matched diabetic patients on diet only (group B: HbA1C = 6.4 +/- 0.9) participated in the study. 10 healthy volunteers were studied as controls. In the postabsorptive state, a mixed meal (700 kCal) was administered to all subjects, and blood samples were regularly collected up to 180' for plasma glucose, insulin, glucagon, and GLP-1 determination. RESULTS: In the control group, the test meal induced a significant increase in plasma GLP-1 at 30' and 60' (p < 0.01); the peptide concentrations then returning toward basal levels. beta-cell function estimation by HOMA score confirmed a more advanced involvement in group A than in group B (p < 0.01). In contrast, the insulin resistance degree showed a similar result in the two groups (HOMA-R). In group A, first-phase postprandial insulin secretion (0 - 60') resulted, as expected, in being significantly reduced compared to healthy subjects (p < 0.001). In the same patients the mean fasting GLP-1 value was similar to controls, but the meal failed to increase plasma peptide levels, which even tended to decrease during the test (p < 0.01). In group B, food-mediated early insulin secretion was higher than in group A (p < 0.001), although significantly reduced when compared to controls (p < 0.01). Like group A, no GLP-1 response to food ingestion occurred in group B patients in spite of maintained basal peptide secretion. Whereas the test-meal did not significantly modify plasma glucagon levels in the control group, glucagon concentrations increased at 30' and 60' in both diabetic groups (p < 0.01). CONCLUSIONS: 1) The functional integrity of GLP-1 cells results as being seriously impaired even in the condition of mild diabetes; 2) the early peptide failure could contribute to the development of beta-cell deterioration which characterizes overt type 2 diabetes.     

Meta-analysis of the effect of KCNQ1 gene polymorphism on the risk of type 2 diabetes

The potassium voltage-gated channel, KQT-like subfamily member 1 (KCNQ1) is a member of 11 mammalian Kv channel families that plays a key role for the repolarization of the cardiac action potential as well as water and salt transport. Genome-wide association studies have identified KCNQ1 as a type 2 diabetes (T2D) susceptibility gene in populations of Asian descent. After that, a number of studies reported that the rs2237892, rs2237895, rs2237897, rs2283228, and rs231362 polymorphism in KCNQ1 has been implicated in T2D risk. However, studies on the association between these polymorphism and T2D remain conflicting. To derive a more precise estimation of the relationship, a meta-analysis of 114,140 patients and 167,322 controls from 30 published case–control studies was performed. Overall, significantly elevated T2D risk was associated with rs2237892, rs2237895, rs2237897, rs2283228, and rs231362 risk allele when all studies were pooled into the meta-analysis. In the subgroup analysis by ethnicity, sample size, and Hardy–Weinberg equilibrium status of controls, significantly increased risks were found for these polymorphisms. In conclusion, this meta-analysis suggests that rs2237892, rs2237895, rs2237897, rs2283228, and rs231362 polymorphisms in KCNQ1 are associated with elevated T2D risk.     

The absence of SIR2alpha protein has no effect on global gene silencing in mouse embryonic stem cells

The yeast sir2 gene plays a central role in mediating gene silencing and DNA repair in this organism. The mouse sir2alpha gene is closely related to its yeast homologue and encodes a nuclear protein expressed at particularly high levels in embryonic stem (ES) cells. We used homologous recombination to create ES cells null for sir2alpha and found that these cells did not have elevated levels of acetylated histones and did not ectopically express silent genes. Unlike yeast sir2 mutants, our sir2alpha null ES cells had normal sensitivity to insults such as ionizing radiation and heat shock, and they were able to silence invading retroviruses normally. These sir2alpha null cells were able to differentiate in culture normally. Our results failed to provide evidence that the mammalian SIR2alpha protein plays a role in gene silencing and suggest that the physiological substrate(s) for the SIR2alpha deacetylase may be nuclear proteins other than histones.     

Alterations of insulin signaling in type 2 diabetes: A review of the current evidence from humans

A generally accepted view posits that insulin resistant condition in type 2 diabetes is caused by defects at one or several levels of the insulin-signaling cascade in skeletal muscles, adipose tissue and liver, that quantitatively constitute the bulk of the insulin-responsive tissues. Hence, the gradual uncovering of the biochemical events defining the intracellular signaling of insulin has been quickly followed by clinical studies on humans attempting to define the molecular defect(s) responsible for the establishment of the insulin resistant state. While the existence of molecular defects within the insulin signal transduction machinery is undisputed, contrasting data exist on what is the principal molecular alteration leading to insulin resistance. Such discrepancies in the literature may depend on: 1) different subject characteristics, 2) methodological differences 3) small cohorts of subjects, and – not least – 4) intrinsic limitations in studying every detail of the insulin signaling cascade. Here, we review the studies on humans exploring the defects of the insulin signaling cascade generated by insulin resistance and type 2 diabetes, focusing on muscle and adipose tissue – which account for most of the glucose disposal capacity of the body – with focus on the unresolved discrepancies present in the literature.     

Troglitazone has no effect on K(ATP) channel opener induced-relaxations in rat aorta and in human saphenous veins from patients with type 2 diabetes

Troglitazone, a thiazolidinedione derivative, is an oral antidiabetic agent that enhances insulin sensitivity in insulin-resistant states. K(ATP) channels, on the other hand, have important roles protecting cardiovascular system in ischemic and/or hypoxic states. They are also important in the control of vascular tone, and therefore of blood pressure. We tested whether troglitazone can directly affect vascular K(ATP) channel opener-induced relaxations in vitro. 1, 10 or 100 microM troglitazone incubations for 30 min did not alter cromakalim (a K(ATP) channel opener)--induced relaxations in endothelium-denuded aortas from rat, saphenous veins from type 2 diabetic and nondiabetic patients. In addition, we compared the sensitivity to cromakalim in diabetic saphenous veins with that of nondiabetic veins. The concentration-response curve for cromakalim was shifted to the right in diabetic vein. pD2 values for cromakalim were 6.85+/-0.08 vs. 6.61+/-0.04 (p<0.05) in nondiabetic (n:10) and diabetic (n:7) veins respectively. % maximum response of cromakalim was also significantly decreased by 24+/-3% in diabetic veins. However, responsiveness of veins to phenylephrine or sodium nitroprusside were similar in both groups. The results obtained may be clinically useful 1. suggesting that in ischaemic and/or hypoxic insults troglitazone may not worsen vascular dilatation, through K(ATP) channel, in diabetic patients who are more prone to these conditions than healthy people, 2. providing an evidence that diabetes causes an impaired dilatation of human saphenous vein through K(ATP) channels. This may partly be related with diabetes-induced vascular complications, such as vasospasm and even hypertension. Accordingly, since saphenous veins are used as conduit vessels in coronary by-pass graft surgery, the results also suggest that the defective dilatation through K(ATP) channels may play a role on the performance of saphenous vein grafts in type 2 diabetes.     

Dependency of the metabolic effect of sc-injected human regular insulin on intra-abdominal fat in patients with type 2 diabetes.

Ten patients with type 2 diabetes were enrolled in an isoglycemic glucose clamp study to determine the impact of intra-abdominal fat, subcutaneous abdominal fat and total abdominal fat on the metabolic effect of a single bolus (0.2 IU/kg) of sc-injected human regular insulin. The maximum metabolic effect associated highly and negatively with intra-abdominal fat (r = - 0.72, p < 0.02) and with the homeostasis model assessment insulin resistance score (HOMA, r = - 0.71, p < 0.03). Likewise, the total metabolic effect of sc-injected insulin correlated strongly and negatively with intra-abdominal fat (r = - 0.77, p < 0.01), HOMA (r = - 0.74, p < 0.02) and HbA (1c) (r = - 0.70, p < 0.03). Stepwise multiple regression analyses showed that the highest metabolic effect was only significantly predicted by intra-abdominal fat, indicating a high negative correlation with the maximum effect (beta = - 0.72) whereas time to maximum metabolic effect showed a strong (beta = 0.72) and positive correlation with HOMA. In combination with the HOMA, it is intra-abdominal fat, and not subcutaneous abdominal fat, which explains 50 - 75 % of the variability of the effect of sc human regular insulin in patients with type 2 diabetes.     

Association of the -112A>C polymorphism of the uncoupling protein 1 gene with insulin resistance in Japanese individuals with type 2 diabetes

The -112A>C polymorphism (rs10011540) of the gene for uncoupling protein 1 (UCP1) has been associated with type 2 diabetes mellitus in Japanese individuals. The aim of the present study was to investigate the effects of this polymorphism, as well as the well-known -3826A>G polymorphism (rs1800592), on clinical characteristics of type 2 diabetes. We determined the genotypes of the two polymorphisms in 93 Japanese patients with type 2 diabetes. Intramyocellular lipid content and hepatic lipid content (HLC) were measured by magnetic resonance spectroscopy. No significant differences in age, sex, BMI, or HbA1c level were detected between type 2 diabetic patients with the -112C allele and those without it. However, homeostasis model assessment for insulin resistance (p=0.0089) and HLC (p=0.012) was significantly greater in patients with the -112C allele. We did not detect an association of the -3826A>G polymorphism (rs1800592) of UCP1 gene with any measured parameters. These results suggest that insulin resistance caused by the -112C allele influences the susceptibility to type 2 diabetes.     

A novel dominant-negative mutation of the hepatocyte nuclear factor-1alpha gene in Japanese early-onset type 2 diabetes.

We investigated the presence and the function of hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations in 26 Japanese subjects with type 2 diabetes. The subjects were between 20 and 39 years of age on diagnosis and had diabetic first-degree relatives. Two different frameshift mutations were found in 2 subjects (8 %). One novel mutation, T539fsdelC (deletion of C in codon 539 for Thr), is predicted to generate a protein of normal 539 residues at the N-terminus followed by an abnormal 119 amino acid protein. The mutation, P291fsinsC (insertion of C in codon 291 for Pro) should lead to production of a truncated protein of 315 amino acids. Transfection reporter assay using MIN6 and HepG2 cells revealed both mutations to have null function in the transactivation of reporter gene expression. When transfected with wild-type gene, these mutations behaved as dominant-negative regulators in both cells. An equimolar amount of T539fsdelC reduced wild-type activity by approximately 80% in MIN6 cells, while the same concentration of P291fsinsc reduced it by 30%. The sequences responsible for the transactivation activity of HNF-1alpha are confined largely to amino acids 547-628, so that the T539fsdelC mutation, which affects this entire region, replacing amino acids 540-631 with an abnormal 119 amino acid protein, may acquire a potent dominant-negative function.     

Association of the C242T polymorphism in the NADPH oxidase p22 phox gene with carotid atherosclerosis in Slovenian patients with type 2 diabetes

Oxidative stress plays an important role in the pathogenesis of diabetes and its complications. Genetic variations of enzymes producing reactive oxygen species could change their activity, thus contributing to the susceptibility to oxidative stress. The aim of this study was to examine the role of the NADPH oxidase C242T polymorphism in the development of carotid atherosclerosis in patients with type 2 diabetes. 286 diabetic patients and 150 healthy controls were enrolled in the study. Carotid atherosclerosis was quantified ultrasonographically as carotid intima-media thickness, plaque score (0–6) and plaque type (1–5). Diabetic patients were divided into low and high risk groups based on ultrasound phenotypes of carotid atherosclerosis. Genotypes were determined by real-time PCR. Levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) were measured by enzyme-linked immunosorbent assay (ELISA). Diabetic patients demonstrated a statistically significant difference compared to healthy controls in the following parameters: age, BMI, waist circumference, smoking prevalence, glucose, triglyceride and 8-OHdG serum levels. Control subjects had significantly higher levels of HDL, LDL and total cholesterol than diabetics (p?<?0.001). The NADPH C242T polymorphism was not related with clinical characteristics, lipid parameters and 8-OHdG serum levels. We found no significant difference in the NADPH genotype distribution between diabetics and controls (p?=?0.19) nor between low and high risk subgroups of diabetics (mean CIMT: p?=?0.67; plaque score: p?=?0.49, plaque type: p?=?0.56). In the present study the NADPH C242T polymorphism was not associated with the degree of oxidative stress and carotid atherosclerosis. Further studies will show if it can be used as a genetic marker for carotid atherosclerosis in diabetic patients.     

Identification of a major gene responsible for type 1 diabetes in the Komeda diabetes-prone rat.

Type 1 diabetes mellitus is an autoimmune disease involving both environmental and genetic factors. Genetic analyses in humans and rodents have shown that the major histocompatibility complex (MHC) is a major genetic factor and that several other genes may be involved in the development of the disease. We performed genetic analysis of type 1 diabetes in a newly established animal model, the Komeda diabetes-prone (KDP) rat, and found that most of the genetic predisposition to diabetes is accounted for by two major susceptibility genes, MHC and Iddm/kdp1. In addition, we identified a nonsense mutation in the Casitas B-lineage lymphoma b (Cblb) gene by positional cloning of Iddm/kdp1. In this paper, I review our positional cloning analysis of Iddm/kdp1 and propose a two-gene model of the development of type 1 diabetes in which two major susceptibility genes, Cblb and MHC, determine autoimmune reaction and tissue specificity to pancreatic beta-cells, respectively.     

Altered signaling and cell cycle regulation in embryonal stem cells with a disruption of the gene for phosphoinositide 3-kinase regulatory subunit p85alpha

The p85alpha regulatory subunit of class I(A) phosphoinositide 3-kinases (PI3K) is derived from the Pik3r1 gene, which also yields alternatively spliced variants p50alpha and p55alpha. It has been proposed that excess monomeric p85 competes with functional PI3K p85-p110 heterodimers. We examined embryonic stem (ES) cells with heterozygous and homozygous disruptions in the Pik3r gene and found that wild type ES cells express virtually no monomeric p85alpha. Although, IGF-1-stimulated PI3K activity associated with insulin receptor substrates was unaltered in all cell lines, p85alpha-null ES cells showed diminished protein kinase B activation despite increased PI3K activity associated with the p85beta subunit. Furthermore, p85alpha-null cells demonstrated growth retardation, increased frequency of apoptosis, and altered cell cycle regulation with a G(0)/G(1) cell cycle arrest and up-regulation of p27(KIP), whereas signaling through CREB and MAPK was enhanced. These phenotypes were reversed by re-expression of p85alpha via adenoviral gene transfer. Surprisingly, all ES cell lines could be differentiated into adipocytes. In these differentiated ES cells, however, compensatory p85beta signaling was lost in p85alpha-null cells while increased signaling by CREB and MAPK was still observed. Thus, loss of p85alpha in ES cells induced alterations in IGF-1 signaling and regulation of apoptosis and cell cycle but no defects in differentiation. However, differentiated ES cells partially lost their ability for compensatory signaling at the level of PI3K, which may explain some of the defects observed in mice with homozygous deletion of the Pik3r1 gene.     

Association of humoral immunity to human Hsp60 with the IL-6 gene polymorphism C-174G in patients with type 2 diabetes and controls.

The relationship between humoral immunity to hsp60 and type 2 diabetes along with other relevant metabolic, inflammatory and immunogenetic variables was studied in 76 non-diabetic and 74 diabetic persons aged 55-74 years selected from the population-based KORA Survey 2000. Antibodies to human hsp60 were measured in serum samples by ELISA. Hsp60 antibodies were detected in all but two individuals in a considerable range of titres (22-1,856 AU/ml). There was no significant association to age and sex, or to key clinical or metabolic parameters (BMI, WHR, HbA1c, total cholesterol, LDL cholesterol, HDL cholesterol, systolic and diastolic blood pressure, albumin, uric acid) or immunological parameters (CRP, IL-6, sIL-6R, TNFalpha, sTNFalpha R60, sTNFalpha R80). Analysis of antibody-positive individuals revealed an association between hsp60 antibodies and diabetes at borderline significance (p = 0.047), which was lost when the two antibody-negative individuals were included. Genetic analyses indicated that this association was significant in carriers of the C allele of the IL-6 promoter region polymorphism at nucleotide -174 (p = 0.02), but not in GG genotype carriers. We conclude that humoral immunity to human hsp60 may be enhanced in those diabetic patients carrying the -174C allele of the IL-6 gene. This finding may contribute to an understanding of the relationship between the -174C allele and increased risk of atherosclerosis.     

Heterogeneity in gene loci associated with type 2 diabetes on human chromosome 20q13.1

Human chromosome 20q12-q13.1 has been linked to type 2 diabetes mellitus (T2DM) in multiple studies. We screened a 5.795-Mb region for diabetes-related susceptibility genes in a Caucasian cohort of 310 controls and 300 cases with T2DM and end-stage renal disease (ESRD), testing 390 SNPs for association with T2DM-ESRD. The most significant SNPs were found in the perigenic regions: HNF4A (hepatocyte nuclear factor 4alpha), SLC12A5 (potassium-chloride cotransporter member 5), CDH22 (cadherin-like 22), ELMO2 (engulfment and cell motility 2), SLC13A3 (sodium-dependent dicarboxylate transporter member 3), and PREX1 (phosphatidylinositol 3,4,5-triphosphate-dependent RAC exchanger 1). Haplotype analysis found six haplotype blocks globally associated with disease (p<0.05). We replicated the PREX1 SNP association in an independent case-control T2DM population and inferred replication of CDH22, ELMO2, SLC13A3, SLC12A5, and PREX1 using in silico perigenic analysis of two T2DM Genome-Wide Association Study data sets. We found substantial heterogeneity between study results.     

Association of single-nucleotide polymorphisms in the suppressor of cytokine signaling 2 (SOCS2) gene with type 2 diabetes in the Japanese

Several previous linkage scans in type 2 diabetes (T2D) families indicated a putative susceptibility locus on chromosome 12q15-q22, while the underlying gene for T2D has not yet been identified. We performed a region-wide association analysis on 12q15-q22, using a dense set of >500 single-nucleotide polymorphisms (SNPs), in 1492 unrelated Japanese individuals enrolled in this study. We identified an association between T2D and a haplotype block spanning 13.6 kb of genomic DNA that includes the entire SOCS2 gene. Evolutionary-based haplotype analysis of haplotype-tagging SNPs followed by a "sliding window" haplotypic analysis indicated SNPs that mapped to the 5' region of the SOCS2gene to be associated with T2D with high statistical significance. The SOCS2 gene was expressed ubiquitously in human and murine tissues, including pancreatic beta-cell lines. Adenovirus-mediated expression of the SOCS2 gene in MIN6 cells or isolated rat islets significantly suppressed glucose-stimulated insulin secretion. Our data indicate that SOCS2 may play a role in susceptibility to T2D in the Japanese.     

Expression of the neurofibromatosis type 2 gene in human tissues.

The neurofibromatosis Type 2 tumor suppressor gene is implicated in the hereditary tumor syndrome NF2, hallmarked by bilateral vestibular schwannomas, meningiomas, and ocular non-neoplastic features. The gene product has characteristics of a membrane cytoskeleton-linking protein but the mechanism of tumor suppression by the NF2 protein remains to be elucidated. The NF2 gene is widely expressed in mouse and rat tissues. In humans, most of the expression data have accumulated through Northern blot analysis, RT-PCR and, more recently, Western blot analysis, providing information on whole tissues and organs rather than on specific cell types. We report here an extensive survey of NF2 gene expression in human tissues using a combination of mRNA in situ hybridization (mRNA ISH) and immunohistochemistry (IH) with a panel of monoclonal antibodies (MAbs) supplemented by tissue immunoprecipitation experiments with affinity-purified polyclonal antibodies. Expression was observed in many different cell types, most of which appear functionally normal in individuals affected by NF2. Surprisingly, expression could not be consistently documented in Schwann cells and arachnoidal cells by IH or by mRNA ISH in formalin-fixed tissue. However, consistent immunostaining of Schwann cells was seen in frozen sections. (J Histochem Cytochem 47:1471-1479, 1999)