Effect of oral nitrite administration on gene expression of SNARE proteins involved in insulin secretion from pancreatic islets of male type 2 diabetic rats

Abstract

Background: Nitrite stimulates insulin secretion from pancreatic β-cells; however, the underlying mechanisms have not been completely addressed. The aim of this study is to determine effect of nitrite on gene expression of SNARE proteins involved in insulin secretion from isolated pancreatic islets in Type 2 diabetic Wistar rats.

Methods: Three groups of rats were studied (n = 10/group): Control, diabetes, and diabetes + nitrite, which treated with sodium nitrite (50 mg/L) for 8 weeks. Type 2 diabetes was induced using a low-dose of streptozotocin (25 mg/kg) combined with high-fat diet. At the end of the study, pancreatic islets were isolated and mRNA expressions of interested genes were measured; in addition, protein expression of proinsulin and C-peptide in pancreatic tissue was assessed using immunofluorescence staining.

Results: Compared with controls, in the isolated pancreatic islets of Type 2 diabetic rats, mRNA expression of glucokinase (59%), syntaxin1A (49%), SNAP25 (70%), Munc18b (48%), insulin1 (56%), and insulin2 (52%) as well as protein expression of proinsulin and C-peptide were lower. In diabetic rats, nitrite administration significantly increased gene expression of glucokinase, synaptotagmin III, syntaxin1A, SNAP25, Munc18b, and insulin genes as well as increased protein expression of proinsulin and C-peptide.

Conclusion: Stimulatory effect of nitrite on insulin secretion in Type 2 diabetic rats is at least in part due to increased gene expression of molecules involved in glucose sensing (glucokinase), calcium sensing (synaptotagmin III), and exocytosis of insulin vesicles (syntaxin1A, SNAP25, and Munc18b) as well as increased expression of insulin genes.

Keywords: Gene expression; Insulin secretion; Nitrite; Rat; Type 2 diabetes.

Fig. 1

Experimental design of the study. Abbreviations: STZ: streptozotocin; GLUT2: glucose transporter 2; Munc18: mammalian homologue of the unc (uncoordinated mutant)-18; SNAP-25: synaptosome associated protein-25.

Fig. 2

Effect of nitrite on expression of proinsulin (A) and C-peptide (B) in the pancreatic tissues of Type 2 diabetic rats. Representative images of pancreatic tissue immune fluorescence stained with DAPI (blue, DNA stain), proinsulin, and C-peptide (green) as well as quantification of immune fluorescence staining by imageJ analysis are shown. Scale bar = 100 mm. Values are mean ± SEM (n = 6/group). ∗, † significant difference (p values < 0.05) compared with control and diabetes groups, respectively.

Fig. 3

Effect of nitrite administration on mRNA expression of insulin1 (A) and insulin2 (B) in pancreatic isolated islets in Type 2 diabetic rats. ∗, † significant difference compared with control and diabetes, respectively. n = 10/group.

Fig. 4

Effect of nitrite administration of mRNA expression of glucose transporter 2 (GLUT2) and glucokinase in pancreatic isolated islets in Type 2 diabetic rats. ∗, † significant difference compared with control and diabetes, respectively. n = 10/group.

Fig. 5

Effect of nitrite administration of mRNA expression of calcium sensor synaptotagmin III in pancreatic isolated islets in Type 2 diabetic rats. ∗, † significant difference compared with control and diabetes, respectively. n = 10/group.

Fig. 6

Effect of nitrite administration of mRNA expression of syntaxin1A, synaptosome-associated protein of 25 kDa (SNAP25), and mammalian homologue of the unc (uncoordinated mutant)-18 gene (Munc18b) in pancreatic isolated islets in Type 2 diabetic rats. ∗, † significant difference compared with control and diabetes, respectively. n = 10/group.