The Role Of TLR4 Signaling And Cholinergic Receptor α7nAChR In High-fat Diet-induced Insulin Resistance

Diabetes mellitus is a chronic metabolic disease caused by disorders of glucose and lipid metabolism.Currently,type 2 diabetes mellitus（T2DM）,characterized by insulin resistance（IR）,accounts for about 90%of patients.A high-fat diet and chronic low-grade inflammation are important factors influencing the development and progression of IR.The inflammatory signaling pathway mediated by Toll-like receptor4（TLR4）is one of the important pathways to generate pro-inflammatory responses,and free fat acid（FFA）is one of its important ligands.When the body is obese,FFA secreted by adipocytes will activate the TLR4 pro-inflammatory pathway to secrete inflammatory factors,which can directly or indirectly inhibit insulin by When the body is obese,FFA secreted by adipocytes will activate the TLR4 pro-inflammatory pathway to secrete inflammatory factors and inhibit insulin signaling in a direct or indirect manner,ultimately leading to IR.Under normal conditions,the body’s immune system maintains a dynamic balance of immune function through the interaction of pro-and anti-inflammatory systems.The cholinergic anti-inflammatory pathway（CAP）,composed mainly of the vagus nerve,splenic and splenic sympathetic nerves,acetylcholine andα7 nicotinic acetylcholine receptor（α7n ACh R）,has It is a rapid,reversible and accurate pathway that is essential for anti-inflammation in the body.Activation of this pathway ameliorates obesity-induced inflammation and IR.To observe the role of TLR4 andα7n ACh R in high-fat diet-induced IR,9-10-week-old wild-type mice and TLR4 gene deletion(TLR4^-/-)mice were randomly divided into two groups:WT basal diet mice,WT high-fat diet mice,TLR4^-/-basal diet mice and TLR4^-/-high-fat diet mice,respectively.After 8 weeks,the mice were subjected to glucose tolerance test（GTT）and insulin tolerance test（ITT）at an interval of one week;the body fat mass of the mice was measured after anesthesia and execution.The levels of tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）in mice serum were measured by enzyme-linked immunosorbent assay（ELISA）,and the key targets of insulin action were detected by Western Blotting.The levels of protein kinase B（PKB or Akt）and insulin receptor substrate-1（IRS1）,the key proteins of insulin signaling pathway,in liver,the key target tissue of insulin action,were detected by western blotting.receptor substrate 1（IRS-1）,and its phosphorylated protein kinase B（p-Akt）,phosphorylated insulin receptor substrate 1（p-IRS-1）expression levels;α7n ACh R expression levels in liver and spleen were examined to analyze the effect of TLR4 onα7n ACh R expression in liver and spleen under IR conditions caused by obesity.After the high-fat diet-induced construction of IR model,the changes in body weight of mice were evaluated,and the results showed that the body weight of WT mice in the high-fat diet group was significantly increased（P<0.0001）compared with WT mice in the basal diet group,and the body weight of TLR4^-/-mice in the basal diet group was significantly decreased（P<0.01）,and the body weight of TLR4^-/-mice in the high-fat diet group was significantly increased（P<0.05）;compared with TLR4^-/-mice in the high-fat diet group had significantly lower body weight compared with WT mice in the high-fat diet group（P<0.0001）.Assessment of whole-body fat mass in mice showed that compared with WT mice in the high-fat diet group,whole-body fat mass was significantly lower in WT mice in the basal diet group and TLR4^-/-mice in the basal diet group（P<0.001）,and TLR4^-/-mice in the high-fat diet group had significantly lower whole-body fat mass（P<0.01）,suggesting that high-fat diet leads to increased body weight and fat mass in mice The deletion of TLR4 gene significantly improved the increase of body weight and fat mass in mice.Glucose tolerance and insulin sensitivity were significantly lower in the high-fat diet group compared with the basal diet group（P<0.001）,suggesting that the high-fat diet leads to the development of insulin resistance;glucose tolerance and insulin sensitivity were significantly higher in the high-fat diet group compared with the high-fat diet group（P<0.05 and P<0.01）,suggesting that TLR4 gene deletion could significantly improve glucose tolerance and insulin sensitivity in mice.The experimental results of the effect of TLR4 on serum inflammatory factor levels in high-fat diet-induced insulin-resistant mice showed that compared with the basal diet group of WT mice,TNF-αand IL-6 levels were significantly higher in the high-fat diet group of WT mice（P<0.05）,and TNF-αand IL-6 levels were significantly lower in the basal diet group of TLR4^-/-mice（P<0.05）;compared with the high-fat diet group of TNF-αand IL-6 levels were significantly lower in TLR4^-/-mice in the high-fat diet group compared with WT mice（P<0.01 and P<0.05）,suggesting that high-fat diet leads to elevated levels of inflammation;it also suggests that TLR4 gene deletion can significantly ameliorate high-fat diet-induced inflammation levels.Experimental results on the effects of high-fat diet and TLR4signaling on insulin signaling pathway showed that compared with WT mice in the basal diet group,p-Akt protein expression was decreased（P<0.05）,total Akt protein expression was not different（P>0.05）,and p-Akt/Akt ratio was decreased（P<0.05）in WT mice in the high-fat diet group;compared with WT mice in the basal diet group,p-IRS-1 diet group WT mice showed elevated p-IRS-1 protein expression（P<0.05）and decreased total IRS-1 protein expression（P<0.05）p-IRS-1/IRS-1 ratio（P<0.05）compared to WT mice in the basal diet group;p-Akt,Akt,p-IRS-1 and The expression levels of IRS-1 were not statistically significant（P>0.05）.The above results suggest that the deletion of TLR4gene decreases the effect of high-fat diet on the expression of Akt and IRS-1 proteins,key signaling molecules of insulin signaling pathway and their phosphorylated proteins in liver tissues.To explore the link between TLR4 pro-inflammatory pathway and CAP in regulating high-fat diet-induced insulin resistance,we examined the expression of TLR4 protein andα7n ACh R in the spleen,an important organ of CAP,and liver,an important target tissue for insulin action.The results showed that compared with WT mice in the basal diet group,TLR4protein expression levels were increased in both liver and spleen of WT mice in the high-fat diet group（P<0.05）,α7n ACh R protein expression levels were decreased in both liver and spleen of WT mice in the high-fat diet group（P<0.05）,andα7n ACh R protein expression levels in both TLR4^-/-mice in the basal and high-fat diet groups（P<0.001）;there was no difference in the expression levels ofα7n ACh R protein in TLR4^-/-mice in the basal diet and high-fat diet groups.That is,overactivation of TLR4inflammatory pathway decreasedα7n ACh R expression in liver and spleen,and deletion of TLR4 gene also decreasedα7n ACh R expression in liver and spleen.In summary,high-fat diet caused IR by altering the phosphorylation levels of Akt and IRS-1,key signaling molecules of the insulin signaling pathway,in liver tissues;high-fat diet led to decreased expression ofα7n ACh R,a key role protein of CAP,in liver and spleen tissues,suggesting that high-fat diet damaged CAP in peripheral tissues;TLR4 gene deletion protected mice from high-fat diet-induced IR;Both TLR4 hyperactivation and TLR4 deletion resulted in decreased hepatic and splenicα7n ACh R expression,suggesting an interaction between TLR4 signaling and cholinergic anti-inflammatory pathways,and that TLR4 may be involved in high-fat diet-induced insulin resistance through regulation of splenic and hepaticα7n ACh R receptor expression and/or interactions with them.