Background and Objectives: The deficiency of insulin synthesis in diabetes is attributed to a decrease of mRNA level transcribed in the pancreatic beta cell. Insulin transcription is initiated and controlled by three primary transcriptional factors: MAFA, PDX1 and NKX6 via the direct binding to its promoter regions. Intestinal electrical stimulation (IES) has been previously shown to ameliorate blood glucose homeostasis and enhance the insulin secretion in diabetic rats. However, how this stimulation affects the insulin synthesis and gene expression in the pancreas is unclear. The aim of this study was to explore the effect of chronic IES on several major insulin gene transcriptional factors in type 2 diabetic rats. We hypothesized that IES increases both MAFA and PDX1 expressions, then enhances their bindings on the insulin gene promoter, which leads to the upregulation of insulin gene expression; meantime, IES activates both GLP-1R and GLUT2 systemically, which facilitates indirectly the insulin gene transcription in the beta cells. Methods: Sixteen SD rats with type 2 diabetes induced with high fat diet followed with a low dose of streptozotocin (35mg/kg) were surgically implanted with serosal electrodes on the duodenum for IEA and randomized into two groups: IES and SHAM (same as IES but 0mA stimulation). IES was applied three hours daily for 8 weeks using previously validated parameters (40Hz, 3mA, 0.6s/0.9s ON/OFF). At the end of the experiment the rats were euthanized, and the pancreas tissues were collected for the protein and mRNA assay. The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed before and after the 8-week chronic IES. Quantitative reverse transcription PCR and Immunofluorescence were used to assess gene expressions and protein expressions in the pancreas, respectively. Results: The OGTT data showed that the blood glucose was significantly inhibited with IES, compared with SHAM. ITT data demonstrated significant improvement in insulin sensitivity with IES (Fig.1A). The insulin mRNA level was significantly increased in the IES group compared to the SHAM group (Fig.1B). Accompanied with the insulin, the mRNA levels of Mafa, Pdx1 and Glut2 in the pancreas were increased simultaneously by IES (Fig.1C-E). Similarly, the protein level of MAFA, PDX1, and GLUT2 in the pancreas were all enhanced in the IES group compared to the SHAM group (Fig.2). Conclusions: This study shows for the first time that the IES-induced the activation of MAFA, PDX1 and GLUT2 in the pancreas upregulates insulin gene transcription, which reveals a mechanism in which the amelioration of insulin sensitivity and secretion by IES is attributed to the IES-induced insulin transcription upregulated by MAFA, PDX1 and GLU2. (This work was supported by an NIH grant (R01DK107754)).

Fig.1. Changes of insulin sensitivity and gene expression by IES in diabetic pancreas. A, ITT was performed after insulin subcutaneous injection. B, Insulin mRNA level in pancreas between SHAM and IES groups. C, D, and E, Mafa, Glut2 and Pdx1 mRNA levels in pancreas between SHAM and IES, respectively. * p≤0.05.

Fig.2. Expression of MAFA, PDX1 and GLUT2 in islet beta cells. A,B and C, Immunofluorescence staining using MAFA, PDX1 and GLUT2 antibodies, respectively in diabetic rat islets between SHAM and IES. D, E and F, the positive staining cell numbers of MAFA, PDX1 and GLUT2 in each islet from the A,B and C, respectively (Scale bar:100um).G, the fluorescence intensity of GLUT2 in each islet from the C. * p≤0.05.

Gaojue Wu

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INSULIN SYNTHESIS AND GENE EXPRESSION INDUCED BY INTESTINAL ELECTRICAL STIMULATION IS ASSOCIATED WITH MAFA, PDX1 AND GLUT2 IN TYPE 2 DIABETIC RATS

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