Pancreatic B-cell Apoptosis ,Insulin Secretion & Their Modulatory Mechanisms By Natural Compounds In Vitro & In Vivo

Abstract

Insulin secretory dysfunction is major pathophysiology of diabetes which is aggravated by β-cell apoptosis. Metformin and sulfonylureas, two major oral hypoglycemic agents used for the treatment of diabetes, enhance insulin sensitivity and stimulate insulin secretion, respectively. Though some dipeptidyl peptidase-IV inhibitors are reported for β-cell protective activity; however, results are not conclusive yet. Therefore, taking advantage of our in house pure compounds library an attempt was made to identify compounds having β-cell protective as well as insulin secretory activity, and study their mechanism(s) at molecular and cellular levels. In order to evaluate the compounds for this dual activity, we used pancreatic β-cell line MIN6 cells to develop H2O2 mediated apoptosis and glucose stimulated insulin secretory assay systems. Out of 34 tested compounds, seven (7) compounds showed strong β-cell apoptosis inhibitory activity. Amongst them genestein (GS), quercetin (QCT), and cinnamic acid (CA) were evaluated through triple channel immunostaining for mitochondria-actin-nuclei which revealed restoration of mitochondrial membrane potential, preservation of cytoarchitecture of cells and reduced nuclear condensation in MIN6 cells. Oxidative stress mediated activation of cleaved caspase 3 was undetectable in MIN6 cells after treatment with these compounds, further confirming the inhibition of mitochondria mediated apoptosis. Moreover, real time PCR study of mRNA expression showed that QCT and GS both downregulated the expression of apoptotic gene Casp9, and increased expression of both Ins1 and Ins2 genes. GS, QCT and CA also stimulated insulin secretion from MIN6 cells/ mice isolated islets. We found CA significantly inhibited nuclear condensation, decreased TUNEL positivity of pancreatic β-cells and preserved islets cytoarchitecture in Wistar rats. The effect of nicotinamide-cinnamic acid (NA-CA) was also studied in vivo, and we found that NA-CA significantly decreases β-cell apoptosis and induces insulin secretion than these agents alone. Immunohistochemical analysis of NA-CA pre-treated rat pancreas revealed decreased cleaved casp3 levels and increased phosphorylation of ERK½ in β-cells. Moreover, real time PCR anlaysis of NA-CA showed decrease in expression of Casp3 and Casp9 mRNA in MIN6 cells. This suggests that dual effect of NA-CA seems to be mediated via ERK½ signaling pathway and through modulating the mRNA expressions of apoptotic proteins. Intriguingly, we found orobol, tambulin and hispidulin as novel insulin secretagogues in the current study. TM enhanced insulin secretion in a dose dependent manner only at stimulatory glucose concentration in isolated mice islets. Pharmacological inhibition of protein kinase A and calcium channels significantly decreased insulin secretion induced by TM. This suggests that TM exerts an exclusive insulin secretory effect by modulating Ca2+ channels and PKA pathway. From the current study, some compounds with potent insulin secretory activity were identified. Interestingly few lead compounds having dual activity were also discovered. Taken together, these compounds may serve as lead compounds to be further studied for their anti-diabetic activity.