Objective: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein.Homozygous WFS1 gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic b-cells are selectively lost from the patient's islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses. Design and methods: Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic b-cell-derived MIN6 cells, and Akita mouse-derived Ins2 96Y/Y insulinoma cells. The human WFS1 gene promoter-luciferase reporter analysis was also conducted. Result: Islet b-cells were the major site of Wfs1 expression. This expression was also found in d-cells, but not in a-cells. WFS1 expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increased WFS1 mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells. WFS1 gene expression was also increased in Ins2 96Y/Y insulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. The WFS1 gene promoter-luciferase reporter system revealed that the human WFS1 promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active in Ins2 96Y/Y cells than Ins2 wild/wild cells. Conclusion: Wfs1 expression, which is localized to b-and d-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link between Wfs1 and ER stress.

Troglitazone is one of the thiazolidinediones, a new class of oral antidiabetic compounds that are ligands of peroxisome proliferator-activated receptor-␥. This study on vascular endothelial growth factor (VEGF), also known as vascular permeability factor, was prompted by our clinical observation that the characteristics of troglitazone-induced edema were very similar to those caused by vascular hyperpermeability. When Japanese diabetic patients were screened for plasma VEGF, we found levels to be significantly (P < 0.001) increased in troglitazone-treated subjects (120.1 ؎ 135.0 pg/ml, n ‫؍‬ 30) compared with those treated with diet alone (29.2 ؎ 36.1 pg/ml, n ‫؍‬ 10), sulfonylurea (25.8 ؎ 22.2 pg/ml, n ‫؍‬ 10), or insulin (24.6 ؎ 19.0 pg/ml, n ‫؍‬ 10). Involvement of troglitazone in increased VEGF levels was further supported by the plasma VEGF levels in five patients before treatment (20.2 ؎ 7.0 pg/ml), after 3 months of troglitazone treatment (83.6 ؎ 65.9 pg/ml), and 3 months after discontinuation (28.0 ؎ 11.6 pg/ml). We further demonstrated that troglitazone, as well as rosiglitazone, at the plasma concentrations observed in patients, increased VEGF mRNA levels in 3T3-L1 adipocytes. VEGF is an angiogenic and mitogenic factor and is currently considered the most likely cause of neovascularization and hyperpermeability in diabetic proliferative retinopathy. Although increased VEGF may be beneficial for subjects with macroangiopathy and troglitazone is currently not available for clinical use, vascular complications, especially diabetic retinopathy, must be followed with great caution in subjects treated with thiazolidinediones.

Glutamate dehydrogenase (GDH) is important in normal glucose homeostasis. Mutations of GDH result in hyperinsulinism/hyperammonemia syndrome. Using PCR/single-strand conformation polymorphism analysis of the gene encoding GDH in 12 Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), we found a mutation (Y266C) in one PHHI patient. This mutation was not found in any of the control or type 2 diabetic subjects. The activity of the mutant GDH (GDH266C), expressed in COS-7 cells, was constitutively elevated, and allosteric regulations by ADP and GTP were severely impaired. The effect of the unregulated increase in GDH activity on insulin secretion was examined by overexpressing GDH266C in an insulinoma cell line, MIN6. Although glutamine alone did not stimulate insulin secretion from control MIN6-lacZ, it remarkably stimulated insulin secretion from MIN6-GDH266C. This finding suggests that constitutively activated GDH enhances oxidation of glutamate, which is intracellularly converted from glutamine to ␣-ketoglutarate, a tricarboxylic acid cycle substrate, which thereby stimulates insulin secretion. Interestingly, insulin secretion is also exaggerated significantly at low glucose concentrations (2 and 5 mmol/l) but not at higher glucose concentrations (8 -25 mmol/l). Our results directly illustrate the importance of GDH in the regulation of insulin secretion from pancreatic ␤-cells.

Leptin production by adipose cells in vivo is increased after feeding and decreased by food deprivation. However, molecular mechanisms that control leptin expression in response to food intake remain unknown. Here, we test the hypothesis that leptin expression in adipose cells is regulated by nutrient- and insulin-sensitive mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. The activity of mTORC1 in 3T3-L1 adipocytes was up-regulated by stable expression of either constitutively active Rheb or dominant-negative AMP-activated protein kinase. In both cases, expression of endogenous leptin was significantly elevated at the level of translation. To investigate the role of leptin 5'-untranslated region (UTR) in the regulation of protein expression, we created bicistronic reporter constructs with and without the 5'-UTR. We found that the presence of leptin 5'-UTR renders mRNA resistant to regulation by mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that does not require the presence of either the 5'-terminal oligopyrimidine tract or the 5'-UTR.

Objective To examine the association between glycemic control and the new onset of macroangiopathy in Japanese subjects with type 2 diabetes. Methods We examined seven-year follow-up data for 572 patients. We divided the subjects by the average of seven-year glycemic control based on the guidelines. First, we excluded the subjects with a past history of macroangiopathy and then examined the incidence of the new onset of macroangiopathy. Results The incidence of ischemic heart disease (IHD) was 1.0% per year, and that of cerebral vascular disease (CVD) was 1.0% per year. However, IHD events were not observed at all for five years in the most intensive glycemic control group (HbA1c<6%). Similarly, CVD events were not observed at all for seven years in the most intensive glycemic control group (HbA1c<6%). In addition, the cumulative incidence rate of IHD tended to increase as the glycemic control became poorer (HbA1c<6%, 4.5%; 6 ! HbA1c<7%, 6.0%; 7 ! HbA1c<8%, 7.2%; HbA1c"8%, 10.7%). Furthermore, a logistic regression analysis showed that the duration of diabetes and HbA1c level were independent risk factors contributing to the onset of IHD, but not to the onset of CVD. Conclusion This seven-year observational study showed the possible association between glycemic control and the onset of macroangiopathy in a total of 572 Japanese subjects with type 2 diabetes.