The Role And Mechanism Of Neutral Ceramidase In Palmitate-induced Lipotoxicity In Pancreatic β Cells

Background:Lipotoxicity of pancreatic β cells plays a vital role in the pathogenesis of type 2 diabetes mellitus.The phenomenon that excessive fatty acidslead to triglyceride,diacylglycerol and ceramide aberrant deposition in islets or pancreaticβcells,thereby damaging the cells function and inducing apoptosis,is known as lipotoxicity.As living and eating habit change,the population of obesity and type 2 diabetes mellitus is increasing.Clinical studies show that free fatty acid level was elevated in obesity and type 2 diabetic patients.Increasing evidence show that elevated free fatty acids induced damage in several different types of cell,such as pancreatic β cells,endothelial cells,skeletal muscle cell and hepatocyte,et al.Ceramides are bioactive lipids that mediate cell apoptosis,proliferation and differentiation.Ceramide was closely related to saturated fatty acids-induced apoptosis in pancreatic P cells by exogenous death receptors pathway,endoplasmic reticulum stress or mitochondrial dysfunction.Saturated fatty acids such as palmitate(Palm)as precursor of palmitoyl-CoA increased ceramide content by promoting ceramide de novo synthesis.Furthurmore,total ceramide content was regulated dependent on ceramide catabolism,apart from ceramide anabolism pathway.Ceramide was hydrolyzed by ceramidase to product sphingosine.Ceramidases were clarified into acidic,neutral and alkaline ceramidase at optimal pH value.Moreover,our previous study showed that NCDase can be expressed in rat insulinoma INS-1 cells and secreted from INS-1 cells.Importantly,NCDase can protect INS-1 cells against cytokines-induced toxicity.It has been reported that adiponectin has a protective effect on pancreatic(3 cells by activating NCDase.Therefore,NCDase has a protective effect on pancreatic β cells.Animal study demonstrated that NCDase activity in muscle tissue was inhibited by high-fat-diet.However,the effect of Palm-induced lipotoxicity on NCDase activity in pancreatic(3 cells has not been understood.Here,we investigated the role and mechanism of NCDase in Palm-induced lipotoxicity in pancreatic(3 cells.Objective:To investigate the role and mechanism of NCDase in Palm-induced lipotoxicity in pancreatic(3 cells.Methods:After rat insulinoma INS-1 cells were treated with various concentrations of Palm(0,0.1,0.25,0.3,0.5 and 0.75mM)for 24h and 48h,cell viability of rat insulinoma INS-1 cells was measured by MTT assay.NCDase mRNA and protein levels were determined by RT-qPCR and Western blot analysis,respectively.NCDase activity was detected by HPLC method and sphingolipids(ceramide and sphingosine)were measured by ESI-MS/MS method.Results:(1)Cell viability of INS-1 cells began significantly to be inhibited at the concentration of 0.3mM Palm for 24 h,and inhibition rate of cell viability was around 50%at 0.5mM Palm.Cell viability of INS-1 cells began significantly to be inhibited at the concentration of 0.25mM Palm for 48 h,and the inhibition rate was 68.2%at 0.5mM Palm.(2)Interestingly,we found that Palm inhibited NCDase gene and protein expression in time dependent manner.(3)NCDase activity was inhibited by Palm in time dependent manner.(4)NCDase overexpression played a protective role in Palm-induced apoptosis in INS-1 cells.Conversely,NCDase siRNA exacerbated Palm-induced apoptosis in INS-1 cells.(5)Palm treatment led to an increase in ceramide content,whereas sphingosine level was not increased concomitantly.Conclusion:Inhibition of NCDase activity was involved in free fatty acid-induced lipotoxicity of pancreatic β cells by increasing ceramide levels,and activating NCDase can alleviate free fatty acid-induced lipotoxicity of pancreatic β cells.