The Effect Of N1-methylnicotinamide On Hepatic Insulin Sensitivity And Glucose Metabolism In Ob/ob Obese Diabetic Mice And Its Regulatory Mechanism

Objective: In recent years,with the aging of the population,economic development and urbanization,people are more sedentary lifestyle and the intake of unhealthy food related to obesity,Diabetes mellitus(DM),with its rising incidence and prevalence,is one of the major threats to health and human development.Type 2 diabetes mellitus(T2DM)is the most common type of diabetes,accounting for about 90% of all diabetes.The main pathogenesis of T2 DM is insulin resistance(IR)and relative insufficiency of insulin secretion.The liver is one of the important target organs of insulin action,and about 90% of endogenous glucose production comes from the liver.The liver plays an important role in maintaining the production and output of fasting endogenous glucose,as well as the absorption,utilization and storage of glucose after eating.Hepatic insulin resistance is a decrease in the ability of insulin to inhibit the hepatic glucose production,the main pathophysiological features involved are abnormal insulin signaling pathways in the liver,increased gluconeogenesis and glycogen decomposition,which lead to increased hepatic glucose production.At the same time,insulin resistance leads to liver lipid deposition,which further reduces insulin sensitivity and forms a vicious cycle.The contribution of hepatic IR to the pathophysiological mechanism of T2 DM is significant,especially to fasting glucose and basal insulin levels.The therapeutic methods of alleviating hepatic IR play an important role in the treatment of T2 DM.N1-methylnicotinamide(MNAM)is a methylated product of nicotinamide,a form of vitamin B3.Recently,the relationship between MNAM and metabolic diseases has attracted much attention.Currently,animal and human studies have suggested that MNAM is related to T2 DM insulin resistance,but the conclusions are controversial and the mechanism is unknown.Recent studies have shown that MNAM intervention reduced fasting glucose and insulin levels in mice with a high-fat diet,and improved insulin sensitivity,and it was found that SIRT1 protein expression was increased in liver of mice treated with MNAM.SIRT1 is a NAD+ dependent protein deacetylase,which can regulate its activity by deacetylation of a variety of transcription factors that control metabolic and endocrine signals,thus extensively participating in regulating glucose metabolism balance,insulin secretion and other metabolic pathways.The purpose of this study was to explore the effect of MNAM on insulin resistance and glucose metabolism in the liver of T2 DM,and to further investigate its possible regulatory mechanism.Methods:1.To study the effect of MNAM on glucose metabolism and insulin resistance in ob/ob obese diabetic mice: 4 weeks old male ob/ob and C57BL6 mice,after 4 weeks of feeding(about 8 weeks old of the animals),the ob/ob mouse model of T2 DM was identified to be stable,they were randomly divided into 3 groups: ob/ob group,low-dose MNAM group(ob/ob+0.3%MNAM group),high-dose MNAM group(ob/ob+1%MNAM group);Normal wild-type C57BL6 mice were randomly divided into two groups: WT group and high-dose MNAM group(WT+1%MNAM group).Mice were fed with MNAM or normal diet continuously for 8 weeks.IPGTT and insulin release level were detected.The blood glucose and insulin of each group at each time were measured,and the curve was drawn,and the area under the curve and the relevant indexes of insulin resistance were calculated.2.To study the effect of MNAM on hepatic insulin sensitivity in ob/ob obese diabetic mice: The effects of MNAM intervention on hepatic lipid deposition were observed by HE staining,oil red staining and triglyceride detecting in liver tissues.Real-time PCR and Western Blot were used to detect the mRNA expression of the key glycogenic enzymes PEPCK and G-6-Pase,and the protein expression and phosphorylation levels of IRS-2,PI3 K,AKT,and GSK3?,which are key molecules of the insulin signaling pathway.3.To investigate the potential mechanism of MNAM in regulating insulin sensitivity and glucose metabolism in liver: mRNA and protein expressions of SIRT1,FOXO1 and PGC-1? in liver tissues were detected by real-time PCR and Western Blot.The hepatocyte insulin resistance model was established by PA treatment with human normal hepatocyte LO2.After MNAM treatment,the glucose residue in the medium was determined.We detected the expression of key glycogenic enzymes and molecules of insulin signaling pathway.SIRT1 inhibitors were applied to treat IR hepatocytes interfered by MNAM to verify the effect of SIRT1 in MNAM on hepatic IR.Results: 1.After 8 weeks of MNAM intervention,the body weight and fasting blood glucose of mice in ob/ob+1%MNAM group were significantly lower than those in ob/ob group(p<0.05);IPGTT results showed that the fasting blood glucose of mice in ob/ob+1%MNAM group decreased,but there was no significant difference between the two groups after glucose stimulation(p>0.05);The insulin secretion level indicated that the fasting and overall insulin secretion levels in the MNAM intervention group were lower than those in the ob/ob group(p<0.05);Calculation of insulin resistance related indicators showed that HOMA-IR of each MNAM intervention group was lower than that of ob/ob group(p<0.05),and QUICKI and Matsuda index are higher than ob/ob group(p<0.05).2.Histopathological examination of the liver revealed that compared with the WT group,the liver of ob/ob group showed obvious steatosis and lipid drip infiltration,and the TG content of liver was also significantly increased(p<0.05).Intervention of MNAM could alleviate the above situation to some extent;Compared with the control group,the mRNA expression of glycogenic PEPCK and G-6-Pase in liver tissues of mice in ob/ob group was significantly increased(p<0.05).However,the above gene expression in the two groups of mice treated with MNAM was significantly lower than that of ob/ob mice(p<0.05).In the ob/ob group,the liver p-IRS-2/IRS-2,p-PI3 K /PI3 K,p-AKT/AKT,p-GSK3?/GSK3? ratio were significantly lower than the control WT groups(p<0.05).However,the phosphorylation level of the above proteins in each MNAM intervention group was higher than that in ob/ob group(p<0.05).Compared with the control group,the mRNA and protein expression of SIRT1 in the liver of ob/ob mice decreased,the expression of PGC-1? was up-regulated,and the acetylation level of FOXO1 was increased(p<0.05).1%MNAM intervention can increase the mRNA and protein expression of SIRT1,decrease the expression of PGC-1?,and decrease the acetylation level of FOXO1 in the liver of ob/ob mice(p<0.05).IR model of hepatocytes was established by PA intervention on LO2 cells.After MNAM treatment,the glucose residue in the medium was determined,the amount of glucose residual in insulin resistant PA group was higher than that in other groups(p<0.05).The relative mRNA expression levels of PEPCK and G-6-Pase in PA group were significantly higher than those in control group and MNAM intervention group(p<0.05).The phosphorylation levels of IRS-2,PI3 K,AKT and GSK3? proteins in the PA group were significantly lower than those in the control group and the MNAM intervention group(p<0.05).After treatment with SIRT1 inhibitor,MNAM's effect of reducing PA-induced gluconeogenesis in IR hepatocytes and regulating the insulin signaling pathway in IR hepatocytes disappeared.Conclusion: By regulating liver SIRT1,FOXO1 and PGC-1?,MNAM can affect insulin signaling pathway conduction and hepatic gluconeogenesis,reduce hepatic lipid deposition,regulate hepatic insulin sensitivity,reduce hepatic glucose production,reduce fasting blood glucose and body weight in T2 DM mice,and improve insulin resistance in T2 DM.