Mutations in the small heterodimer partner gene increase morbidity risk in Japanese type 2 diabetes patients.

Mutations in the small heterodimer partner gene (NR0B2; alias SHP) are associated with high birth weight and mild obesity in Japanese children. SHP mutations may also be associated with later obesity and insulin resistance syndrome that induces diabetes. To investigate this possibility, the prevalence of SHP mutations in Japanese with and without type 2 diabetes mellitus and the functional properties of the mutant proteins were evaluated. Direct sequencing of two exons and flanking sequences of SHP in 805 diabetic patients and 752 non-diabetic controls identified 15 different mutations in 44 subjects, including 6 novel mutations. Functional analyses of the mutant proteins revealed significantly reduced activity of nine of the mutations. Mutations with reduced activity were found in 19 patients (2.4%) in the diabetic group and in 6 subjects (0.8%) in the control group. The frequency difference between DM and control subjects adjusted for sex and age was statistically significant (P=0.029, odds ratio 2.67, 95% CI 1.05-6.81, 1-beta=0.91). We conclude that SHP mutations associated with mild obesity in childhood increase susceptibility to type 2 diabetes in later life in Japanese.

Glutathione peroxidase protein expression and activity in human islets isolated for transplantation.

BACKGROUND: Overexpression of antioxidant enzymes has been reported to protect rodent beta cells from oxidative stress. However, very little is known about protein expression and activity of antioxidant enzymes in human islets. METHOD/RESULTS: Human islet protein levels by Western analysis and enzymatic activity for the key antioxidant enzymes superoxide dismutases (SODs), catalase, and glutathione peroxidase-1 (GPx) were examined. Enzyme protein expression and activity were in the order SODs > catalase > GPx. Human islet GPx protein expression was significantly less than that found for catalase (p < 0.0001) and levels of GPx activity were virtually undetectable. As glucose and estrogens have been proposed to alter antioxidant enzyme levels, we examined islet data from male and female donors separately and under varying glucose concentrations. We found significantly less (p < 0.001) GPx protein expression in islets from females compared to males, but no significant regulation by glucose in either gender. CONCLUSIONS: Human islets have very low protein and activity levels for GPx, the essential enzyme for protection against excessive levels of intracellular lipid peroxides. GPx mimetics may be especially valuable in providing human islets with the broadest spectrum of protection against oxidative stress during isolation and transplantation.

Reversal of defective glucagon responses to hypoglycemia in insulin-dependent autoimmune diabetic BB rats.

The intraislet insulin hypothesis has been proposed to explain absent glucagon responses to hypoglycemia. Recently we directly confirmed this hypothesis by restoring glucagon secretion via provision of a pancreatic artery insulin infusion, which was switched off at the time of hypoglycemia in Wistar rats made diabetic by streptozotocin. The current study examined this hypothesis in a model of spontaneous, autoimmune diabetes, the insulin-dependent diabetic BB rat. The insulin switch-off signal restored the defective glucagon responses to hypoglycemia. However, the magnitude of the restored response was markedly less than that observed in control nondiabetic BB rats (4- to 5-month-old diabetic BB rats = 147 +/- 27; 2-month-old nondiabetic BB rats = 1038 +/- 112 pg/ml, peak delta; P < 0.0001). Because time was required for the BB rat to spontaneously develop diabetes, we asked whether the incomplete restoration of the glucagon response might be related to the animals' growth and development. This led us to compare the glucagon response to hypoglycemia in nondiabetic BB and Wistar rats at 2 and 4-5 months of age. We observed age-related deterioration of not only glucose tolerance and insulin sensitivity but also glucagon responses to hypoglycemia in both strains. There was no significant difference between the glucagon responses to hypoglycemia in age-matched nondiabetic BB rats and diabetic BB rats provided with the insulin switch-off signal. We conclude that defective glucagon responses to hypoglycemia in BB rats can be corrected by restoring regulation of alpha-cell function by insulin.

Mutation analysis of NR0B2 among 1545 Danish men identifies a novel c.278G>A (p.G93D) variant with reduced functional activity.

Variations of the small heterodimer partner (SHP, NR0B2) gene, an atypical nuclear receptor that inhibits transactivation by hepatocyte nuclear factor (HNF)-4alpha, are associated with obesity among Japanese. The purpose of the study was to evaluate the prevalence of SHP variants among obese Danish men. Using combined SSCP and heteroduplex analysis, we analyzed the entire coding region of SHP for variants in a cohort of 750 Danish men with early-onset obesity and genotyped a cohort of 795 nonobese control subjects using PCR-RFLP. Functional analyses of the identified coding region variants were performed in both MIN6-m9 and HepG2 cell lines. A total of five novel variants, including three missense variants (c.100C>G [p.R34G], c.278G>A [p.G93D], and c.415C>A [p.P139H]) and two silent variants (c.65C>T [p.Y22Y] and c.339G>A [p.P113P]) were identified. Moreover, the previously reported c.512G>C [p.G171A] polymorphism was identified. The 171A allele was not associated with obesity (p = 0.07). The 34G, 93D, and 139H-alleles were rare variants, which were found only among obese subjects. Among the four coding region variants, the 93D-allele showed a reduced in vitro inhibition of the HNF-4alpha transactivation of the HNF-1alpha promoter expression when expressed in MIN6-m9 and HepG2 cell lines (p<0.01). In contrast to reported findings among obese Japanese, functional variants are rare among Danish men. A functional 93D variant of SHP was identified in 1 out of 750 obese and in none of 795 nonobese control subjects. Further large-scale population studies are necessary to assess the clinical impact of this rare variant on obesity risk among European subjects.