Dataset Information

Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor.

ABSTRACT: Nephrin belongs to a family of highly conserved proteins with a well characterized function as modulators of cell adhesion and guidance, and nephrin may have a role in metabolic pathways linked to podocyte and pancreatic ?-cell survival. However, this role is incompletely characterized. In this study, we developed floxed nephrin mice for pancreatic ?-cell-specific deletion of nephrin, which had no effect on islet size and glycemia. Nephrin deficiency, however, resulted in glucose intolerance in vivo and impaired glucose-stimulated insulin release ex vivo Glucose intolerance was also observed in eight patients with nephrin mutations compared with three patients with other genetic forms of nephrotic syndrome or nine healthy controls.In vitro experiments were conducted to investigate if nephrin affects autocrine signaling through insulin receptor A (IRA) and B (IRB), which are both expressed in human podocytes and pancreatic islets. Coimmunoprecipitation of nephrin and IRB but not IRA was observed and required IR phosphorylation. Nephrin per se was sufficient to induce phosphorylation of p70S6K in an phosphatidylinositol 3-kinase-dependent but IR/Src-independent manner, which was not augmented by exogenous insulin. These results suggest a role for nephrin as an independent modulator of podocyte and pancreatic ?-cell nutrient sensing in the fasting state and the potential of nephrin as a drug target in diabetes.

SUBMITTER: Villarreal R

PROVIDER: S-EPMC4814188 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor.

Villarreal Rodrigo R Mitrofanova Alla A Maiguel Dony D Morales Ximena X Jeon Jongmin J Grahammer Florian F Leibiger Ingo B IB Guzman Johanna J Fachado Alberto A Yoo Tae H TH Busher Katin Anja A Gellermann Jutta J Merscher Sandra S Burke George W GW Berggren Per-Olof PO Oh Jun J Huber Tobias B TB Fornoni Alessia A

Journal of the American Society of Nephrology : JASN 20150923 4

Nephrin belongs to a family of highly conserved proteins with a well characterized function as modulators of cell adhesion and guidance, and nephrin may have a role in metabolic pathways linked to podocyte and pancreatic β-cell survival. However, this role is incompletely characterized. In this study, we developed floxed nephrin mice for pancreatic β-cell-specific deletion of nephrin, which had no effect on islet size and glycemia. Nephrin deficiency, however, resulted in glucose intolerance in ...[more]

PMID: 26400569

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Project description:ObjectivesThe mammalian target of Rapamycin (mTOR) is a metabolic master regulator of both innate and adaptive immunity; however, its exact role in stromal cell biology is unknown. In this study we explored the role of the mTOR pathway on Rheumatoid Arthritis synovial fibroblast (RASF) metabolism and activation and determined if crosstalk with the Hippo-YAP pathway mediates their effects.MethodsPrimary RA synovial fibroblasts (RASF) were cultured with TNFα alone or in combination with the mTOR inhibitor Rapamycin or YAP inhibitor Verteporfin. Chemokine production, matrix metalloproteinase (MMP) production, and adhesion marker expression were quantified by real-time PCR, ELISA, and/or Flow Cytometry. Invasion assays were performed using Transwell invasion chambers, while wound repair assays were used to assess RASF migration. Cellular bioenergetics was assessed using the Seahorse XFe96 Analyzer. Key metabolic genes (GLUT-1, HK2, G6PD) were measured using real-time PCR. Reanalysis of RNA-Seq analysis was performed on RA (n = 151) and healthy control (HC) (n = 28) synovial tissue biopsies to detect differential gene and pathway expression. The expression of YAP was measured by Western Blot.ResultsTranscriptomic analysis of healthy donor and RA synovial tissue revealed dysregulated expression of several key components of the mTOR pathway in RA. Moreover, the expression of phospho-ribosomal protein S6 (pS6), the major downstream target of mTOR is specifically increased in RA synovial fibroblasts compared to healthy tissue. In the presence of TNFα, RASF display heightened phosphorylation of S6 and are responsive to mTOR inhibition via Rapamycin. Rapamycin effectively alters RASF cellular bioenergetics by inhibiting glycolysis and the expression of rate limiting glycolytic enzymes. Furthermore, we demonstrate a key role for mTOR signaling in uniquely mediating RASF migratory and invasive mechanisms, which are significantly abrogated in the presence of Rapamycin. Finally, we report a significant upregulation in several genes involved in the Hippo-YAP pathway in RA synovial tissue, which are predicted to converge with the mTOR pathway. We demonstrate crosstalk between the mTOR and YAP pathways in mediating RASF invasive mechanism whereby Rapamycin significantly abrogates YAP expression and YAP inhibition significantly inhibits RASF invasiveness.ConclusionmTOR drives pathogenic mechanisms in RASF an effect which is in part mediated via crosstalk with the Hippo-YAP pathway.

Dataset Information

Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor.

Publications

Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor.

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