Resveratrol Improves Insulin Resistance And Disorders Of Glucose And Lipid Metabolism In Skeletal Muscle Induced By High-fat Through ATM

Epidemiological data shows that metabolic diseases such as obesity and type 2 diabetes have become one of the global public health problems.The common pathological basis of these metabolic diseases is insulin resistance,and skeletal muscle insulin resistance is an important cause of systemic insulin resistance.In recent years,China's eating habits have been affected by Western culture.Type 2 diabetes and obesity are associated with inappropriate diets and lifestyles,especially the consumption of high fat diets.However,the specific mechanism is not yet clear.Therefore,insulin resistance or lipid accumulation caused by a high fat diet is a good model to explore its mechanism and to seek related treatments.The previous study by our research group found that insulin resistance models of rats and mice with skeletal muscle lipid accumulation can be induced by high fat diets.In this study,high fat-induced insulin resistance model with skeletal muscle lipid accumulation was selected.Resveratrol(Rsv)is a polyphenolic phytoalexin known as the natural "heat-restricted analogue".In recent years resveratrol has been shown to be anti-inflammatory,anti-oxidant,anti-tumor and to improve insulin sensitivity.The atiaxia-telangiectasia mutated(ATM)gene,is a mutated gene of ataxia-telangiectasia(A-T)disease with its protein expressed with the same name,belonging to the family of phosphoinositide 3-kinase(PI3K)-related kinases(PIKKs),is a serine threonine kinase.Studies have found that homozygous mice lacking ATM have abnormal glucose metabolism and serious insulin resistance.Many anti-cancer studies have found that resveratrol can work through ATM.However,whether resveratrol can improve fat-induced insulin resistance and skeletal muscle lipid accumulation through ATM and then prevent the occurrence and development of type 2 diabetes and obesity has been few reported.Therefore,this study intends to observe the changes of glycogen synthase,fatty acid oxidation of mitochondrial and ATM expression in skeletal muscle in high fat-induced insulin resistance mice after resveratrol treatment,and to investigate whether resveratrol can improve lipid accumulation and insulin resistance induced by palmitic acid in L6 cells through ATM.Our study try to reveal the role of ATM in the development of metabolic diseases and to provide a theoretical basis for a new medicine on metabolic diseases such as type 2 diabetes and obesity.Part one Effects of resveratrol on insulin resistance and skeletal musclelipid accumulation in mice fed with high fat dietObjective: Effects of resveratrol on biochemical indicators and histopathology in skeletal muscle of mice induced by high fat will be observed.To investigate the effects of resveratrol on lipid accumulation and mitochondrial morphology in mice induced by high fat diet.Methods: 42 six-week-old C57BL6/J mice were randomly divided into two groups after one week of adaptive feeding: 10 in the normal diet(ND)group and 26 in the high fat diet(HFD)group.The ND group was fed with common diet feeding,and HFD group given high fat diet feeding.Mice of each group were available to water and food freely with the same daily calories.Food intake was recorded daily and fasting weights were measured weekly.After 8 weeks,6 mice were randomly respectively selected from ND group and HFD group for identification of the insulin resistant model.1% pentobarbital anesthesia was performed before euthanizing them.The skeletal muscles were taken for transmission electron microscopy to verify the skeletal muscle lipid accumulation model.Mice of HFD group were then randomly divided into two groups: 10 in the HFD group and the others in the group of high-fat diet with resveratrol intervention(HFD+Rsv).In HFD+Rsv group,resveratrol 100 mg/kg/d was administered once a day.The ND group and HFD group were orally given equal volume of normal saline with 0.1% DMSO in it.During the 8-week resveratrol intervention,the dose was recalculated based on body weights weekly.The intraperitoneal glucose tolerance test(IPGTT)was used to measure blood glucose and area under the curve(AUCglu)at each time point at the end of 8-week high-fat feeding and 8-week resveratrol intervention.Serum samples were collected for fasting blood glucose(FBG),total cholesterol(TC),triglycerides(TG)detection.ELISA were used to detect serum fasting insulin(FINS)and free fatty acid(FFA).The quantitative insulin sensitivity index(QUICKI)was calculated to assess insulin resistance.The skeletal muscle tissue samples were collected for TG content detection.Electron microscopy was used to observe the ultrastructure of skeletal muscle cells.Results:1.Insulin resistance model induced by high-fat diet1)Comparison of general indicators of mice.Compared with ND group,body weights,FBG,FINS,serum TG,TC and FFA were all significantly increased in HFD group(P<0.05).There was no significant difference in calorie intake between the two groups(P>0.05).2)The QUICKI results of mice in two groups were compared.The QUICKI value of HFD group was significantly lower than that of ND group(P<0.05).3)Comparison of the results of glucose tolerance between two groups: IPGTT results showed that blood glucose levels of 0,30,and 60 min in HFD group were higher than those in ND group(P<0.05),and AUCglu in HFD group was also significantly increased(P<0.05).2.Lipid accumulation model induced by high-fat dietComparison of ultrastructure of skeletal muscle cells in two groups.No cytoplasmic lipid droplets,abundant mitochondria and rough endoplasmic reticulum in skeletal muscle cells of ND mice were displayed under electron microscope.The rough endoplasmic reticulum is arranged neatly.However,more lipid droplets and mitochondrial swelling were seen in the skeletal muscle cells of the HFD group.These suggest lipid accumulation and mitochondrial damage muscle cells.3.Changes in various indicators after resveratrol intervention1)Comparison of general indicators of mice in three groups.Body weights,FBG,FINS,TG,FFA,TC in HFD group were significantly higher than that in ND group(P<0.05).After the intervention of resveratrol,weights,FBG,FINS TG and FFA in HFD+Rsv group were significantly lower than those of HFD group(P<0.05).TC had no significant difference(P>0.05).There was no significant difference in caloric intake between groups(P>0.05).2)Comparison of QUICKI results of mice in three groups: The QUICKI value in the HFD group was significantly lower than that in the ND group(P<0.05).The QUICKI value in the HFD+Rsv group was significantly higher than that in the HFD group after intervention of resveratrol(P<0.05).3)Comparison of glucose tolerance in three groups: IPGTT results showed that blood glucose levels in 0min,30 min,60min,and 120 min groups in HFD group were higher than those in ND group(P<0.05),and HFD+Rsv group was 0 min and 30 min after intervention with resveratrol.Blood glucose at 60 min and 120 min was lower than that in HFD group(P<0.05).The AUCglu in HFD group was higher than that in ND group(P<0.05).After treatment with resveratrol,the AUCglu in HFD+Rsv group was significantly lower than that in HFD group(P<0.05).4)Comparison of ultrastructure of skeletal muscle cells in three groups: Under SEM,no cytoplasmic lipid droplets,abundant mitochondria and rough endoplasmic reticulum in skeletal muscle cells of ND mice were displayed under electron microscope.The rough endoplasmic reticulum is arranged neatly.However,more lipid droplets and mitochondrial swelling were seen in the skeletal muscle cells of the HFD group.These suggest lipid accumulation and mitochondrial damage muscle cells.After intervention with resveratrol,lipid droplets in the HFD+Rsv group decreased compared with the HFD group,and mitochondrial swelling was improved.Part two Effect of resveratrol on ATM expression and glycogensynthase of skeletal muscle in insulin resistant mice inducedby high fat dietObjective: By detecting the changes in protein expression of ATM,glycogen synthase upstream transcription factors and key enzymes,we explored whether ATM is used as a downstream target to participate in the improvement of glycogen synthase in skeletal muscle by resveratrol.Methods: The groups and sample collection are the same as the first part.Western blot was used to detect the protein expression of ATM,phosphorylation of glucose metabolism upstream transcription factor Akt and glycogen synthase key enzyme GSK-3?.Results:1.All Three groups of mouse skeletal muscle ATM protein expression.Compared with ND group,protein expression of ATM in skeletal muscle tissue of HFD group decreased(P<0.05).Compared with HFD group,resveratrol interfered with HFD+Rsv group after intervention.ATM protein expression increased(P<0.05).2.The phosphorylation of the upstream transcription factor,Akt,in skeletal muscle in three groups of mice.Compared with ND group,the phosphorylation of Akt at serine 473 in skeletal muscle tissue decreased(P<0.05).Compared with the HFD group,the level of p-Akt(Ser473)and increased in the HFD+Rsv group after intervention of resveratrol(P<0.05).3.The phosphorylation of the key enzymes,GSK-3?,in skeletal muscle in three groups of mice.Compared with ND group,the phosphorylation of GSK-3? at serine 9 in skeletal muscle tissue decreased(P<0.05).Compared with the HFD group,the level of p-GSK-3?(Ser9)increased in the HFD+Rsv group after intervention of resveratrol(P<0.05).Part three Effect of resveratrol on mitochondrial fatty acid metabolismin skeletal muscle of insulin resistant mice induced by highfat dietObjective: By detecting the changes of the upstream transcription factor AMPK,mitochondrial metabolism factors PGC1?,COXIV and the key enzymes CD36 and CPT1,we aim to investigate whether ATM acts as a downstream target involved in the improvement of skeletal muscle mitochondrial dysfunction and fatty acid oxidation by resveratrol.Methods: Animal groups and tissue sample collection are the same as the first part.Western blot was used to detect the protein expression of PGC1?,COXIV,CD36 and CPT1 and the phosphorylation of AMPK.Results:1.The TG content in mice skeletal muscle.Compared with the ND group,the skeletal muscle TG content of mice in the HFD group was increased(P<0.05).Resveratrol intervention reduces TG content in skeletal muscle of HFD mice(P<0.05).2.The skeletal muscle protein expression of fatty acid oxidation upstream transcription factors and mitochondrial metabolism factors in mice.Compared with ND group,the protein expression of p-AMPK PGC1? and COXIV were decreased with statistically significant difference(P<0.05).Compared with HFD group,after intervention with resveratrol,the protein expression of was decreased in HFD+Rsv group,and the protein expression of p-AMPK,PGC1? and COXIV were increased(P<0.05).3.The protein expression of CD36 and CPT1,which are key enzymes for fatty acid oxidation in mitochondria,in mice skeletal muscle.Compared with the ND group,the protein expression of CD36 in mice skeletal muscle in the HFD group was increased,and the protein expression of the CPT1 was decreased(P<0.05).Compared with HFD group,the protein expression of CD36 was significantly decreased and the protein expression of CPT1 was significantly increased in the HFD+Rsv group after intervention with resveratrol(P<0.05).Part four Explore the role target of resveratrol on glucose and lipidmetabolism by regulating ATM expressionObjective: ATM inhibitors and agonists were used to regulate the expression of ATM and observe the changes of glycogen synthase and lipid accumulation in L6 cells.To investigate whether resveratrol can improve the insulin resistance through ATM-regulated skeletal muscle cell fatty acid metabolism and glycogen synthase,reveals that The relationship between alcohol and ATM in the process of sugar uptake and abnormal lipid accumulation explores the mechanism by which resveratrol improves glucose and lipid metabolism and insulin resistance.Methods: To observe the lipid accumulation,the glucose concentration in the culture medium,the intracellular TG levels in L6 cells,and oil red O staining were tested.Resveratrol,an ATM agonist chloroquine(Chq),and an ATM inhibitor Ku-55933 were administered to differentiated L6 cells cultured in palmitic acid(PA).Then western blot was used to detect the protein expression of ATM,glycogen synthase and fatty acid oxidation upstream transcription factors and their related enzymes.Results:1.Differentiation into myotubes from L6 myoblastsCompared with undifferentiated L6 cells,2% fetal bovine serum induced differentiated L6 cells for 5 days.Inverted microscope observation of dispersed shuttle-forming myoblasts accumulates and gradually forms myotubes;The mRNA expression of desmin and myogenin was significantly increased(P<0.05),suggesting that L6 cells were successfully differentiated.2.Model establishment in vitro of lipid accumulation and insulin resistance in L6 cellsThe levels of glucose in cultured cells were significantly lower in differentiated L6 cells after PA intervention for 24 hours than that in Con group with the statistically significant difference(P<0.05),suggesting that PA can reduce glycogen synthase in L6 cells.In the Con group,oil red O staining showed that the spot deposition staining of the PA group was greater than that of the Con group,indicating that the saturated fatty acid-induced lipid accumulation and insulin resistance in vitro cell model was successfully established.3.Effects of the ATM agonist and inhibitor on ATM protein expression in L6 cellsAfter intervention with resveratrol and ATM agonist chloroquine in lipid accumulation in vitro models,the expression of ATM protein was increased,suggesting that resveratrol can be used as an agonist of ATM.The intervention of ATM inhibitor Ku-55933 was used in an in vitro model of lipid accumulation.After that,the expression of ATM protein was significantly reduced(P<0.05).4.Effects of regulation of ATM on L6 cell glycogen synthaseAfter intervention with resveratrol in lipid accumulation in vitro model,compared with PA group,the protein expression of glycogen synthase related factor p-GSK-3? and p-Akt in PA+Rsv group was increased.After intervention with the ATM inhibitor Ku-55933 in an in vitro model,there was no significant difference in the expression of p-Akt and p-GSK-3? between PA+Ku group and PA group(P>0.05).5.Effects of regulation of ATM on mitochondrial fatty acid oxidation in L6 cellsAfter intervention of resveratrol on lipid accumulation,compared with PA group,protein expression of CD36 decreased in PA+Rsv group,and the protein expression of p-AMPK,CPT1 and COXIV increased.After intervention with the ATM inhibitor Ku-55933,the expression of COXIV,CD36,CPT1,and p-AMPK in the PA+Ku group was not significantly different from that in the PA group(P>0.05).Conclusion:1.High-fat diet can affect the protein expression of ATM,mitochondrial metabolism factor PGC1?,COXIV and key fatty acid oxidation enzymes CD36,CPT1 and the level of phosphorylation of the upstream transcription factor of glucose metabolism Akt,the key enzyme of glycogen synthesis GSK-3?,and upstream transcription factor of fatty acid oxidation AMPK in skeletal muscle indicates that high fat can reduce glycogen synthesis and inhibit fatty acid oxidation,leading to disorders of glucose and lipid metabolism and insulin resistance in skeletal muscle.2.Resveratrol regulates levels of ATM/Akt/GSK-3? and ATM/AMPK/CPT1 signaling pathway,by increasing the expression of ATM,improving glucose and lipid metabolism disorders in skeletal muscles of insulin resistance models.ATM is the key factor mediating the effects of resveratrol on improving insulin resistance and glucose and lipid metabolism in skeletal muscle.