Nephrin Is Expressed on the Surface of Insulin Vesicles and Facilitates Glucose-Stimulated Insulin Release

Abstract

OBJECTIVENephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic β-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in β-cell function.RESEARCH DESIGN AND METHODSNephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin–transfected cells was used to study nephrin endocytosis.RESULTSNephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin–transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release.CONCLUSIONSOur data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes.

Prostaglandin I2 Receptor Agonism Preserves β-Cell Function and Attenuates Albuminuria Through Nephrin-Dependent Mechanisms

“…However, by contrast, the additional phosphorylation of Tyr-1217 promoted nephrin degradation (10). Nephrin is found both on the plasma membrane and on insulin vesicles in b-cells (8), and it regulates insulin exocytosis through interaction with the actin cytoskeleton and phosphorylation-dependent trafficking (10). In the current study, we found that IP receptor agonism phosphorylated nephrin at Tyr-1176/1193 in podocytes and in MIN6 cells.…”

“…Although preservation of islet b-cell mass may explain some of the reduction in blood glucose levels with selexipag treatment in STZ-C57BL/6 mice, a separate secretagogue-like effect is supported by the findings in MIN6 cells. However, unlike secretagogue therapies currently available in the clinic, IP receptor agonism alone is not sufficient to induce insulin release, emphasizing that its effects occur downstream of glucose-sensing, plausibly by priming b-cells through nephrin phosphorylation (8).…”

“…Experiments were conducted in the MIN6 B1 clone (Addex Biotechnologies, San Diego, CA), except nephrin overexpression experiments, which were conducted in MIN6 C3 cells (18), previously reported to constitutively express lower levels of nephrin than B1 cells (8). MIN6 C3 cells were provided by Dr. Philippe Halban (University of Geneva, Switzerland) with permission from Dr. Jun-ichi Miyazaki, University of Osaka, who produced the maternal MIN6 cell line (19).…”

“…1G). To determine whether nephrin phosphorylation and GSIS are causatively related, we overexpressed nephrin in the MIN6 C3 clone (which constitutively expresses lower levels of nephrin [8]) using mutant constructs in which the phosphorylated tyrosine residues 1176 or 1193, or both, had been replaced (Fig. 1H).…”

“…The presence of nephrin in pancreatic b-cells has been appreciated for more than a decade (7), but only more recently has the protein been identified as being a key regulator of glucose-stimulated insulin secretion (GSIS) (8) and b-cell viability (9). Because nephrin does not possess a natural ligand (10), efforts to therapeutically alter its actions are best addressed through posttranslational modification of the protein.…”