Recent studies have implicated epigenetics in the pathophysiology of diabetes. Furthermore, DNA methylation, which irreversibly deactivates gene transcription, of the insulin promoter, particularly the cAMP response element, is increased in diabetes patients. However, the underlying mechanism remains unclear. We aimed to investigate insulin promoter DNA methylation in an over-nutrition state. INS-1 cells, the rat pancreatic beta cell line, were cultured under normal-culture-glucose (11.2 mmol/l) or experimental-high-glucose (22.4 mmol/l) conditions for 14 days, with or without 0.4 mmol/l palmitate. DNA methylation of the rat insulin 1 gene (Ins1) promoter was investigated using bisulfite sequencing and pyrosequencing analysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and increased DNA methylation at all five CpG sites within the Ins1 promoter, including the cAMP response element, in a time-dependent and glucose concentration-dependent manner. DNA methylation under experimental-high-glucose conditions was unique to the Ins1 promoter; however, palmitate did not affect DNA methylation. Artificial methylation of Ins1 promoter significantly suppressed promoter-driven luciferase activity, and a DNA methylation inhibitor significantly improved insulin mRNA suppression by experimental-high-glucose conditions. Experimental-high-glucose conditions significantly increased DNA methyltransferase activity and decreased ten-eleven-translocation methylcytosine dioxygenase activity. Oxidative stress and endoplasmic reticulum stress did not affect DNA methylation of the Ins1 promoter. High glucose but not palmitate increased ectopic triacylglycerol accumulation parallel to DNA methylation. Metformin upregulated insulin gene expression and suppressed DNA methylation and ectopic triacylglycerol accumulation. Finally, DNA methylation of the Ins1 promoter increased in isolated islets from Zucker diabetic fatty rats. This study helps to clarify the effect of an over-nutrition state on DNA methylation of the Ins1 promoter in pancreatic beta cells. It provides new insights into the irreversible pathophysiology of diabetes.

Glucose-dependent insulinotropic polypeptide (GIP), a gut hormone secreted from intestinal K-cells, potentiates insulin secretion. Both K-cells and pancreatic b-cells are glucoseresponsive and equipped with a similar glucose-sensing apparatus that includes glucokinase and an ATP-sensitive K C (K ATP ) channel comprising KIR6.2 and sulfonylurea receptor 1. In absorptive epithelial cells and enteroendocrine cells, sodium glucose co-transporter 1 (SGLT1) is also known to play an important role in glucose absorption and glucose-induced incretin secretion. However, the glucose-sensing mechanism in K-cells is not fully understood. In this study, we examined the involvement of SGLT1 (SLC5A1) and the K ATP channels in glucose sensing in GIP secretion in both normal and streptozotocin-induced diabetic mice. Glimepiride, a sulfonylurea, did not induce GIP secretion and pretreatment with diazoxide, a K ATP channel activator, did not affect glucose-induced GIP secretion in the normal state. In mice lacking K ATP channels (Kir6.2 K/K mice), glucose-induced GIP secretion was enhanced compared with control (Kir6.2 C/C ) mice, but was completely blocked by the SGLT1 inhibitor phlorizin. In Kir6.2 K/K mice, intestinal glucose absorption through SGLT1 was enhanced compared with that in Kir6.2 C/C mice. On the other hand, glucose-induced GIP secretion was enhanced in the diabetic state in Kir6.2 C/C mice. This GIP secretion was partially blocked by phlorizin, but was completely blocked by pretreatment with diazoxide in addition to phlorizin administration. These results demonstrate that glucose-induced GIP secretion depends primarily on SGLT1 in the normal state, whereas the K ATP channel as well as SGLT1 is involved in GIP secretion in the diabetic state in vivo.

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Objective: The S100 calcium binding protein B (S100B) implicated in brain inflammation acts via the receptor of advanced glycation end products (RAGE) and is also secreted from adipocytes. We investigated the role of S100B in the interaction between adipocytes and macrophages using a cell-culture model. Design and Methods: RAW264.7 macrophages (RAW) were stimulated by recombinant S100B to observe alterations in TNF-a and M1 markers; 3T3-L1 adipocytes (L1) were stimulated by TNF-a to examine S100B secretion. RAW and L1 were then mutually stimulated with conditioned media of each other, or co-cultured. The effects of S100B silencing or a RAGE-neutralizing antibody were also investigated. Results: S100B upregulated TNF-a and M1 markers in RAW, and TNF-a augmented S100B secretion from L1. L1 conditioned media stimulated TNF-a secretion from RAW, and RAW conditioned media increased S100B secretion from L1. The co-culture of RAW and L1 increased TNF-a, S100B, and the expression of M1 markers and the MCP-1 receptor CCR2. The silencing of S100B or RAGE neutralization significantly ameliorated TNF-a hypersecretion from RAW that were stimulated with L1 conditioned media. Conclusions: Thus, S100B as an adipokine may play a role in the interaction between adipocytes and macrophages to establish a vicious paracrine loop.

Background: Actin dynamics is involved in insulin secretion, but the mechanism is unknown. Results: The G-actin predominant or F-actin remodeling state in pancreatic ␤-cells, which is regulated by the balance of N-WASP and cofilin activities, determines the biphasic glucose-induced insulin secretion (GIIS). Conclusion: Actin dynamics regulated by N-WASP and cofilin underlie the biphasic GIIS. Significance: The regulation of actin dynamics in ␤-cells and its role in GIIS are clarified.