| In recent years the etiological study of insulin resistance(IR)has become a hot spot,because insulin resistance is a common soil of these metabolic diseases,such as hyperlipidemia,diabetes,hypertension,gout,etc.Numerous studies show that inflammation is an important mechanism of insulin resistance.Since the found of the first inflammatory cytokines tumor necrosis factor alpha(TNF?)associated with insulin resistance,inflammation mechanism has become hot spot of obesity-induced insulin resistance.Inflammation in obesity is a low-grade chronic inflammation.As a pattern recognition receptors,Toll-like receptors(TLRs)not only plays an important role in infection immune and injury-induced inflammatory response,but also participates in the development of metabolism related disease.In the cloned toll-like receptors family,toll-like receptor 2(TLR2)is the most widely expressed member and the one of identifying the most pathogenic microorganism species.TLR2 express mainly in the mammalian lungs,heart,brain and muscle tissues,and its most prominent biological function is directly or indirectly to promote synthesis and release of inflammatory cytokines.As about 60% ~ 70% plasma glucose metabolized in skeletal muscle of humans and rodents,skeletal muscle is the main site of insulin stimulated glucose uptake.Lipid metabolism plays an important role in the formation of skeletal muscle insulin resistance.As fatty acids oxidation occurred in mitochondrial,it showed that mitochondrial function plays an important role in the formation of skeletal muscle insulin resistance.To investigate the effect of TLR2 on skeletal muscle mitochondrial biogenesis and insulin resistance will exert great influence to the prevention and treatment of metabolic disease,and provide scientific evidences for seeking new targets in treating diabetes.In the free fatty acids,palmitic acid(PA)is the main saturated fatty acid.In vitro,cells incubated with palmitic acid may be insulin resistance.This method is one of the commonly used methods of establishing model of insulin resistance in vitro cell.This subject adopts palmitic acid incubation L6 muscle cellsto constructed insulin resistance model.The content of TLR2 was adjusted by plasmid up-regulated and siRNA down-regulated in skeletal muscle,respectively.To explore the saturated fatty acids affected mitochondrial biogenesis of skeletal muscle and led to insulin resistance whether through the expression of TLR2.Objective: To study the role of TLR2 on insulin resistance induced by palmitic acid in skeletal muscle cells,and the potential mechanisms by modulating the expression of TLR2.Methods: L6 rat myoblasts were cultured and differentiated into skeletal muscle cells.With palmitic acid(PA)incubation,the model of insulin resistance was established.Then the following group: the control group(control),palmitic acid group(PA),PA group with transfected with negative control siRNA(PA + NC-siRNA),PA group with TLR2 knockdown by small infecting RNA(PA + si-TLR2),palmitic acid with empty plasmid group(PA+ pcDNA),palmitic acid with TLR2 overexpression group(PA + pcDNA /TLR2).The expression level of TLR2 mRNA and protein were detected by RT-PCR and Western methods to determine whether the transfection is successful or not.After the success of the confirm transfection,to determine glucose concentration in nutrient solution in each group with glucose oxidase method and evaluation insulin sensitivity.The expression of inflammation factors TNF? and IL-1? was performed by using ellisa method in six groups of samples.The mRNA and protein expression of TLR2 in each group were detected by Real-Time PCR and western-blotting,respectively.the protein expression of PGC1?,MFN2,NRF-1 related to mitochondrial biogenesis and insulin signaling pathway PI3 K,P-AKT,GLUT4 protein expression level in each group were detected by western-blotting,respectively.Significance of differences among the groups were determined using One-way ANOVA,andtwo groups were determined using Student's t-test.Results:1 Successful differentiation of skeletal muscle cells: Compared with undifferentiated L6 myoblasts,mRNA expression of differentiation marker genes(Desmin and Myogenin)in differentiation group were significantly increased(P <0.05);2 Establish the insulin resistance model: After the success of the skeletal muscle cell differentiation,cells were divided into normal control group and palmitic acid intervention group.Glucose concentrations were measured at12 h,16h,20 h,24h respectively.Compared with control group,the glucose concentration was significantly increased(P<0.05)at 24 h in palmitic acid intervention group;3 After the success of the transfection,concentrations of glucose and the changes of Inflammatory cytokines TNF? and IL-1? in six groups of samples:Compared with control group,the glucose concentration and inflammatory factor TNF? and IL-1? in the medium were significantly increased(P <0.05)in PA group;No statistically signifcant differences were seen between the PA group and PA+NC-siRNA group and PA+pcDNA group with regard to the glucose concentration and inflammatory factor TNF? and IL-1? in the medium;Compared with PA+NC-siRNA group,the glucose concentration and inflammatory factor TNF? and IL-1? in the medium were significantly decreased(P <0.05)in PA+si-TLR2 group;Compared with PA+pcDNA group,the glucose concentration and inflammatory factor TNF? and IL-1? in the medium were significantly increased(P <0.05)in PA+pcDNA/TLR2 group;4 The expression of TLR2 after the successful transfection in six groups of samples:Compared with control group,the protein expression of TLR2 was significantly increased(P < 0.05)in PA group;No statistically signifcant differences were seen between the PA groupand PA+NC-siRNA group and PA+pcDNA group with regard to the protein expression of TLR2;Compared with PA+NC-siRNA group,the protein expression of TLR2 was significantly decreased(P<0.05)in PA+si-TLR2 group,suggesting si-TLR2 transfected successfully;Compared with PA+pcDNA group,the protein expression of TLR2 was significantly increased(P < 0.05)in PA+pcDNA/TLR2 group,suggesting that TLR2 plasmid transfected successfully;5 The protein expression related to mitochondrial biogenesis and insulin signaling pathway after the success of the transfection in six groups of samples:Compared with control group,the related protein expression of mitochondrial biogenesis(PGC1??MFN2?NRF-1)and insulin signaling pathway(PI3K?P-AKT?GLUT4)were significantly decreased(P<0.05)in PA group;No statistically signifcant differences were seen between the PA group and PA+NC-siRNA group and PA+pcDNA group with regard to the related protein expression of mitochondrial biogenesis(PGC1??MFN2?NRF-1)and insulin signaling pathway(PI3K?P-AKT?GLUT4);Compared with PA+NC-siRNA group,the related protein expression of mitochondrial biogenesis(PGC1? ? MFN2 ? NRF-1)and insulin signaling pathway(PI3K?P-AKT?GLUT4)were significantly increased(P<0.05)in PA+si-TLR2 group;Compared with PA+pcDNA group,the related protein expression of mitochondrial biogenesis(PGC1? ? MFN2 ? NRF-1)and insulin signaling pathway(PI3K?P-AKT?GLUT4)were significantly decreased(P<0.05)in PA+pcDNA/TLR2 group;Conclusions:1 High-fat incubation skeletal muscle cells could be induced to insulin resistance,it characterized by reducing glucose intake,at the same time increasing TLR2 expression and inflammatory factor TNF? and IL-1? release.2 As silence or overexpression of TLR2 could affect glucose uptake,inflammatory cytokines secretion,the protein expression of PGC1?,MFN2,NRF-1 related to mitochondrial biogenesis and insulin signaling pathway PI3 K,P-AKT,GLUT4 protein expression level.This suggests that increase the expression of TLR2 can cause insulin resistance by damaging the proteins involved in mitochondrial biogenesis. |
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