Effect And Mechanism Of Low-grade Increase Of Palmitate And Lipopolysaccharide On Pancreatic ?-cells

Background: The pathogenesis of type 2 diabetes mellitus is very complex,in which bacteria and lipotoxicity play an important role.Lipopolysaccharide is the main component of the cell wall of Gram-negative bacteria.Studies have shown that patients with type 2 diabetes have a low-grade increase of plasma lipopolysaccharide due to intestinal flora imbalance and dyslipidemia leading to impaired intestinal mucosal permeability.A systematic review shows that trace amounts of lipopolysaccharide could be detected in normal human blood,ranging from 0.39 to 6.60 ng/mL.Different from the lipopolysaccharide increased by 10 to 100 times in severe acute inflammation,the level of lipopolysaccharide in type 2 diabetic patients is significantly higher than that of the general population,but the average increase is about 66.4%.And its absolute value is slightly higher,so it is called "metabolic endotoxemia".Previous studies have focused more on lipopolysaccharide-induced insulin resistance.Studies have shown that low-grade increase of lipopolysaccharide can bind to the specific toll-like receptor 4(TLR4)on the surface of immune cells such as macrophages,and stimulate the release of inflammatory factors to interfere with insulin signaling pathway to induce insulin resistance.In addition to insulin resistance,pancreatic ?-cell dysfunction is another key link in the pathogenesis of type 2 diabetes.TLR4 is also present on the islet beta-cell membrane,and lipopolysaccharide can bind to ?-cells via TLR4.Previous studies have shown that although high concentrations of lipopolysaccharide(1 ug/mL)significantly induce pancreatic ?-cell damage,lipopolysaccharide at metabolic endotoxemia concentration does not affect ?-cell function.Thus,it is not clear that low-grade increase of lipopolysaccharide,which is common in chronic metabolic diseases such as type 2 diabetes,has an effect on ?-cells.Increased free fatty acids are often present in people with type 2 diabetes.Similar to lipopolysaccharide,although the free fatty acid level in type 2 diabetic patients is significantly higher than that of the general population,it is mostly at low-grade level of increase,with an average of 0.40~0.66 mmol/L.Palmitate,which accounts for about a quarter of free fatty acids,is an important component of free fatty acids associated with metabolic diseases.Previous studies have shown that high concentrations of palmitate(?0.3 mmol/L)can induce ?-cell apoptosis and inhibit insulin synthesis and secretion by promoting ceramide synthesis,increasing the level of endoplasmic reticulum and oxidative stress.However,studies have also find that a low-grade increase of palmitate(0.1-0.2 mmol/L),which is more common in people with disorders of glucose metabolism,does not affect ?-cell function.The low-grade increase of palmitate and lipopolysaccharide are widely co-existing in the type 2 diabetic population.Related studies have shown that the combination of low-grade increase of free fatty acid and lipopolysaccharide synergistically promote the secretion of inflammatory factors in immune cells by regulating the metabolism of sphingolipid signaling molecules.Due to the presence of co-receptor TLR4 for palmitate and lipopolysaccharide on the islet beta-cell membrane,we hypothesize that low-grade increase of palmitate or lipopolysaccharide alone do not cause ?-cell damage,but the combination may have a synergistic effect similar to macrophages.The synergistic effect further affects ?-cell function.In addition,our previous studies report that neutral ceramidase,a key enzyme in the sphingolipid metabolic pathway,plays an important role in the regulation of ?-cell damage in type 2 diabetes.Therefore,we further study the role of neutral ceramidase in the synergistic effect of low-grade increase of palmitate and lipopolysaccharide on ?-cell function.In summary,this study is the first to observe the role of low-grade increase of palmitate and lipopolysaccharide,which are widely co-existing in type 2 diabetes,in impairing pancreatic ?-cells synergistically,and to explore related mechanisms.Objective: To explore the effect of low-grade increase of free fatty acid and lipopolysaccharide on pancreatic ?-cells and its possible mechanisms.Methods:(1)Pancreatic ?-cell line rat insulinoma cells(INS-1)were treated with various concentrations of palmitate(0.1?0.125?0.2?0.25?0.3 mmol/L)for 24 h.Cell viability was determined using the cell counting kit-8(CCK-8)assay.(2)After INS-1 cells were incubated with different concentrations of lipopolysaccharide(1?2?5?10?50 ng/mL)for 24 h,CCK-8 assay was used to measure the viability of the INS-1 cells and Annexin V-FITC / PI double staining flow cytometry was used to detect apoptosis.(3)Then INS-1 cells were incubated with 0.125 mmol/L palmitate and different concentrations of lipopolysaccharide(1?5?10?50 ng/mL)for 24 h.The cell viability was detected by CCK8 assay and the apoptosis was determined by flow cytometry.Based on these results,we finally chose 0.125 mmol/L palmitate and 10 ng/mL lipopolysaccharide as stimulatory concentration for subsequent experiments.(4)After INS-1 cells were treated with 0.125 mmol/L palmitate and 10 ng/mL lipopolysaccharide,the expression of NCDase protein in INS-1 cells was determined by Western blot assay.(5)Next,stable clones of INS-1 cell line transfected with recombinant plasmids pEGFP-C3-NCDase and pEGFP-C3 vector were established.Then they were incubated with palmitate and lipopolysaccharide for 24 h.CCK-8 assay was used to detect the viability of the INS-1 cells and flow cytometry was used to measure apoptosis.Results:(1)The viability of INS-1 cells was inhibited by palmitate in a dosedependent manner.Palmitiate significantly inhibited the viability of INS-1 cells in the concentration range of 0.2-0.3 mmol/L(P<0.05),while there was no statistically significant effect on the viability of INS-1 cells in the concentration range of 0.1-0.125 mmol/L.(2)Lipopolysaccharide had no significant effect on INS-1 cell viability and apoptosis in the concentration range of 1-50 ng/mL.(3)Palmitate(0.125 mmol/L)combined with lipopolysaccharide(10 ng/mL?50 ng/mL)significantly inhibited INS-1 cell viability and induced apoptosis(P<0.05).(4)Palmitate(0.125 mmol/L)and lipopolysaccharide(10 ng/mL)could synergistically down-regulate the expression of NCDase in INS-1 cells(P<0.05).(5)Overexpression of NCDase attenuates the cytotoxicity which was induced by palmitate combined with lipopolysaccharide in INS-1 cells(P<0.05).Conclusion: The synergistic effect of low-grade increase of palmitate and lipopolysaccharide on the cytotoxicity of pancreatic ?-cells is related to the down-regulation of NCDase protein in ?-cells.