Effects of SIRT1 on the expression and activity of eNOS in high fat and high glucose cultured islet endothelial cells

Abstract

Objective To investigate the effects of Sirtuin 1 (SIRT1) on expression and activity of islet endothelial cells (IEC) cultured by palmitic acid and high glucose. Methods Recombinant mouse SIRTl plasmid including green fluorescent protein report gene was synthesized and transfected to islet endothelial cells using Lipofectamine 2000. Then, cells were treated by high level lipid with or without high level glucose for 48 hours. Cells in high glucose and lipid group were cultured in 33.3 mmol/L glucose and 0.5 mmol/L palmitic acid for 48 hours.Cells in high lipid group were cultured in 0.5 mmol/L palmitic acid for 48 hours. Western blot was used to test the transfecting efficacy of SIRTl recombinant plasmid. Real time quantitative-PCR and Western blot were used to exam the expression of endothelial nitric synthases (eNOS) mRNA and protein, respectively. Nitric oxide reductase method was used to test NO level in supernatant of cell culture. Results Compared with the control group, there was no change on eNOS mRNA and protein level in high lipid group; however, the expression of eNOS mRNA and protein level decreased significantly in cells cultured in high level lipid and glucose group. SIRT1 overexpression increased the protein level of eNOS in high lipid group, and also increased mRNA and protein level in high lipid and glucose group. Compared with the control group, NO decreased significantly in high level lipid cultured endothelial cells, and decreased further in high lipid and high glucose cultured group. SIRT1 overexpression increased NO not only in normal cells but also in high level lipid with or without high glucose cells. Conclusions SIRT1 improves the expression and activity of eNOS in IEC and can increase the release of NO by endothelial cells, which may be helpful in improving the function of islet beta cells. SIRT1 may have great promise in new medicine development, gene therapy and islet transplantation in diabetes. Key words: SIRT1; Type 2 diabetes; Islet endothelial cell; Endothelial nitric oxide synthases; Nitric oxide; Islet B cell