Effect of Altered Glycemic Levels on Synaptogenic Adhesion Molecules Mediated Insulin Secretion

Abstract

Background: Extreme glycemic fluctuations are known to disrupt insulin secretion. Islet degeneration underlying diabetes further deteriorates the already dampened insulin secretion. Insulin during diabetic treatment, often administered to maintain tight glycemic regulation leads to hypoglycemia that hampers insulin secretion. Insulin vesicular exocytosis from pancreatic β-cell is governed by SAMs that are involved in cellular machinery for timely vesicular exocytosis as required. It is hypothesized that discrepancies in expression of SAMs molecules may cause gap in communication which may fail to signal timely insulin secretion even in healthier islets. Methods: Adult male Wistar rats were categorized into experimental groups which are control (C), diabetic (D), insulin induced hypoglycemia in - control (C+IIH) and diabetes (D+IIH). Diabetes was induced using streptozotocin (50 mg/kg body weight i.v.), while hypoglycemia was induced by daily two shots of insulin to C (1.5 IU/kg body weight) and D (10 IU/kg body weight), s.c. for six weeks. Food, water consumption and physiological parameters were regularly monitored. Insulin, glucagon and C-peptide levels were assessed from serum of experimental rats, during fasting as well as post prandial (20’ and 150’) states. ROS levels were assessed from pancreatic homogenate, while GPx expression pattern was also assessed. Histopathological analysis was carried out by Masson’s trichrome staining for studying alterations in morphometry and functional insulin using immunofluorescence. Alterations in genomic expressions were studied using qPCR with gene specific primers for ZnT8, Neurexin-1, Neuroligin-2, Mint-1, Syntaxin-1, Munc-18, SNAP25, VAMP2, Synaptotagmin-7, CADPS1 and CaV1.2 genes, and protein expression studies with immuno-labelled antibodies for selected molecules. Effect of hyper and hypoglycemic conditions on gene expressions were also assessed from MIN6 cell-line. Insulin secretion was studied from MIN6 cell line. Results: During in vivo studies, D condition demonstrated decreased levels of serum insulin, while both C+IIH and D+IIH showed increased circulating insulin levels most likely due to the insulin induced model, however, the levels of C-Peptide were observed to be decreased in all the experimental conditions. This was further supported by the levels of serum glucagon, which accordingly showed increased levels in D, C+IIH and D+IIH conditions. Further, the histopathological and morphometric analysis showed degenerated islets in D and hypertrophic islets in hypoglycemic conditions, which is backed by the data suggesting degeneration of islets, while showing a decline in functional levels of insulin by immunofluorescence assay. A significantly increased ROS and deteriorated GPx expression is suggestive of the prevalent oxidative stress owing to the underlying glycemic fluctuations. The gene expression patterns of docking proteins Nrxn-1, Nlgn-2 and Mnt-1, along with priming proteins Munc-18, Stx-1, SNAP25 and fusion proteins VAMP2 and CAPS1 shows decreased expressional patterns in both diabetic and hypoglycemic conditions. However, Syt-7 demonstrated an increase in the expression pattern for all three experimental conditions suggestive of a possible compensatory mechanism. Conclusion: Thus, our study demonstrates the alteration of SAMs molecules during diabetes, which gets dampened due to hypoglycemic events.