P0986NO/CGMP PATHWAY MODULATES GLUCOSE UPTAKE VIA REGULATION OF SIRT1 DEACETYLASE EXPRESSION AND ACTIVITY IN PODOCYTES EXPOSED TO HIGH GLUCOSE CONCENTRATIONS

Abstract

Abstract Background and Aims Glomerular podocytes have become the aim of the extensive research in diabetes because of their importance in the development of diabetic nephropathy. High glucose concentration affects podocyte metabolism and function leading to the development of insulin resistance. Moreover, it decreases deacetylase SIRT1 protein amount and activity, leading to abolition of insulin effect on glucose uptake into the cells. The aim of our study was to examine the involvement of NO/cGMP pathway in the regulation of sirtuin 1 (SIRT1) protein expression and activity in podocytes, in the context of high-glucose induced insulin resistance. This work was supported by grant from the National Science Centre (2016/23/B/NZ4/03448). Method Experiments were performed in primary rat podocytes cultured with normal (NG, 11.1 mM) or high (HG, 30 mM) glucose concentrations for 5 days. RT-PCR and immunodetection methods were used to detect mRNA and protein expressions. Glucose uptake was measured by the addition of 1 µCi/well of 2-deoxy-(1,2-3H)-D-glucose uptake diluted in non-radioactive dglucose (50 µM final concentration) with or without 300 nM insulin. SIRT1 activity was determined with a SIRT1 Fluorometric Kit according to the manufacturer’s instructions. SIRT1 expression was modified by transfection with SIRT1 siRNA. To examine the effect of NO/cGMP pathway activity on SIRT1, cells were incubated in the presence of SNAP, a mimetic of nitric oxide synthase activity (NOS), and 8-Br-cGMP, protein kinase G (PKG) activator. We also used L-NAME – NOS inhibitor, and Rp-8-Br-cGMPs, PKG inhibitor. Results SIRT1 protein expression and activity were decreased in podocytes exposed to high glucose concentrations. We showed that in the presence of SNAP and 8-Br-cGMP, SIRT1 protein amount and activity were increased, leading also to enhancement of basal and insulin-dependent glucose uptake. In podocytes, transfected with SIRT1 siRNA, treatment with NO/cGMP pathway activators SNAP or 8-Br-cGMP increased glucose uptake. NO/cGMP pathway inhibitors exerted opposite effects on SIRT1 protein level and activity as well as on glucose uptake. Insulin action on glucose uptake into podocytes cultivated in the presence of normal glucose concentrations was abolished after NO/cGMP pathway inhibition. Conclusion We found that impairment of insulin effect on glucose uptake in HG-exposed podocytes, which was associated with SIRT1 downregulation, may be prevented by NO-dependent signaling modulation.