1747-P: Glucolipotoxicity Inhibits Proinsulin Processing through Disrupting V-ATPase Function Leading to Impaired Acidification within Secretory Granules

Increase in serum proinsulin level reflects dysfunction of pancreatic beta cells. Insulin is secreted by pancreatic beta cells after the precursor proinsulin is processed by prohormone convertase (PC) 1/3 in secretory granules (SGs). In the progression of diabetes where pancreatic beta cells are under excess fatty acids and high glucose, insulin biosynthesis is impaired and proinsulin is secreted as an unprocessed form of insulin. However, the exact mechanism of the impairment of proinsulin processing is unclear. As a first step to elucidate its mechanism, we used islets isolated from db/db mice and islets treated with high glucose and palmitate (HGPal), as a model of glucolipotoxicity. As PC1/3 enzyme function is known to be dependent on the luminal acidity in the SGs, we investigated the pH in both islets and found decrease in the acidity of the entire cells by labeling cells with fluorogenic pH indicator with flow cytometry. We also detected decrease in the protein level of insulin and change in the protein expression pattern of PC1/3 by immunoblotting. Morphological analysis with electron microscope revealed that accumulation of immature SGs in these islets probably due to the accumulation of proinsulin inside. Given that the V-ATPase plays an essential role to maintain the acidity of SGs as a proton pump, both fractionation assay and proximity ligation assay revealed that HGPal stimuli inhibit the assembly formation of two major domain V1 and V0 in of V-ATPase Our results demonstrate that glucolipotoxicity altered the luminal acidity in SGs and impaired the proinsulin processing through disrupting V-ATPase function. We anticipate these results may explain underlying molecular mechanisms of the increase in the serum proinsulin level reflecting dysfunction of pancreatic beta cells.