Mechanistic Investigation Of Toll Like Receptor 3 On High-Fat Diet Induced Insulin Resistance

Objective:The aim of this study was to evaluate the role of Toll like receptor 3(TLR3)on high-fat diet mediated insulin resistance,and to further explore the mechanism of TLR3 on systemic insulin resistance,primarily in the adipose tissues and liver.Methods:Wild-type C57BL/6 and TLR3-/-male mice were fed normal diet(ND)or high fat diet(HFD)for 15 weeks.Each group has 8 mice.In each week,body weight was weighed and food intake was recorded.Fasting blood glucose was detected at week 10 and 15.The glucose tolerance and insulin sensitivity were evaluated by intraperitoneal glucose tolerance test(IPGTT)and insulin tolerance test(ITT).At euthanasia,blood was collected to measure insulin,circulatory adipokines and biochemical parameters.Hematoxylin and eosin(H.E.)staining,quantitative real-time RCR,and flow cytometry were used to assess the morphology and function of visceral adipose tissue(VAT);The morphology,lipid accumulation and function of liver were detected by H.E.staining,oil red O staining and automatic biochemical analyzer(ACA);Western blotting was used to assess pathways of insulin signaling,inflammation,and lipid and glucose metabolism inVAT and liver.Results:1.Each group of mice showed a time-dependent increase in body weight.HFD induced increase in body weight,blood glucose,insulin levels and HOMA-IR,with impaired whole-body glucose tolerance and decreased insulin sensitivity,which were ameliorated by TLR3 deletion.2.In the visceral adipose tissue:HFD feeding induced abnormal structure and function of the visceral adipose tissue,demonstrated by defective insulin signaling,increased adipose weight and adipocyte area,and altered adipokines,all of which were blocked by TLR3 deficiency.Moreover,HFD inhibited expression of hormone-sensitive lipase(HSL)and adipose triglyceride lipase(ATGL),as well as HSL phosphorylation(ser563,ser565 and ser660),which were ameliorated by TLR3 deletion.In addition,HFD feeding induced enhancement of macrophages infiltration,M1 macrophages polarization,and pro-inflammatory cytokines(F4/80,IL-6 and TNFa)secretion,all of which were corrected by TLR3 deletion.Finally,TLR3 activated by HFD induced insulin resistance through activation of inflammatory response by the TBK1/IKKβNF-κB signaling pathway.3.In the liver,HFD evoked liver dysfunction,dyslipidemia,and increased hepatic lipid accumulation,which were corrected by TLR3 deficiency.Moreover,HFD incuced increase infatty acid intake related proteins(FABP1,FABP2,FABP5,and D36)and lipid synthesis related enzymes(ACL,ACC 1,ACC2,DGAT2,SCD1,GPAT,FAS,SREBP1 and SREBP1c),some of which were completely or partially inhibited in TLR3-/-mice.However,TLR3 deficiency failed to correct increased fatty acidβ-oxidation mediators(ACO and PPARα)in response to HFD.In addition,HFD feeding increased the rate-limiting enzyme invlved in gluconeogenesis(PEPCK,FBPase,G6Pase and PC),which were ameliorated by TLR3 deficiency.However,the increased expression of enzymes involved in glycolysis and glucose transporter(GK,L-PK and GLUT2)in response to HFD were not corrected in TLR3-/-mice.Finally,decreased phosphorylation of AMPKa at Thr172 in response to HFD was reversed by TLR3deficiency.Conclusion:1.TLR3 deficiency ameliorates HFD induced impairment in glucose tolerance and insulin sensitivity.2.TLR3 modulates obesity and insulin resistance in the visceral adipose tissue by adipose accumulation which is induced by out-of-balance between adipose synthesis and lipolysis,and VAT inflammation which is demonstrated by macrophages infiltration and M1 polarization.These pathologic process may be mediated by TLR3/TBK1/IKKβ/NF-κB signaling pathway.3.TLR3 modulates liver dysfunction and glucose/lipid metabolism in the liver through inhibiting AMPKa activity.Inhibition of AMPKa evoked glucose metabolism disorder of enhanced gluconeogenesis and hepatic lipid accumulation,which is induced by out-of-balance between adipose synthesis and lipolysis.