| Insulin resistance is one of the main characteristics of type 2 diabetes,which is related to high lipid load and obesity.Skeletal muscle accounts for nearly 80% of the total body glucose intake and plays an important role in insulin resistance.The Mammalian target of rapamycinm(TOR)pathway is related to a variety of human diseases and pathological conditions,including diabetes,obesity,cancer,neurodegenerative diseases and shortened life span.Studies have shown that m TOR signaling pathway was activated by nutrient overload for a long time,which might lead to skeletal muscle insulin resistance.DNA damage inducible transcription factor 4(DDIT4)plays a role in DNA damage,insulin signal transduction,oxidative metabolism,nutrient deprivation,hypoxia or endoplasmic reticulum stress,and can negatively regulate m TOR activity.Persistent overexpression of DDIT4 results in m TOR inhibition,which is involved in the survival of lung cancer cells.DDIT4 and m TOR are involved in adipocyte insulin signaling pathway.However,the mechanism of DDIT4 in insulin resistance of skeletal muscle has not been completely determined.Resveratrol(RSV),also known as stilbene/stilbene triol,is a polyphenolic plant antitoxin,mainly derived from peanut,grape,polygonum cuspidatum,mulberry and other plants.Resveratrol can have beneficial effects on metabolism and improve insulin resistance and related metabolic abnormalities,including dyslipidemia,hyperglycemia and hyperinsulinemia.Among them,the anti-insulin resistance effect of resveratrol has been confirmed in many studies.This study aimed to explore the role and mechanism of DDIT4 and m TOR signaling pathway in resveratrol improving insulin resistance by establishing insulin resistance models in vivo(animal)and in vitro(cell),and to provide new ideas and new targets for the treatment of type 2 diabetes.Part One Effects of resveratrol on serum and skeletal muscle morpho-logy in high-fat diet-induced insulin resistance miceObjective: C57BL/6J mice were fed with high-fat diet to establish animal model with insulin resistance,and to observe the effects of high-fat diet(HFD)and resveratrol intervention on blood glucose,blood lipid,insulin,insulin sensitivity,pathological morphology and lipid deposition of skeletal muscle.Methods: C57BL/6J mice were randomly divided into the control(CON)group and the HFD group.At the end of 8-week high-fat feeding,an intraperitoneal glucose tolerance test(IPGTT)was performed to evaluate whether the insulin resistance model was established successfully.Eleven mice in the HFD group were randomly selected and given resveratrol 100mg/kg/d by gavage as a HFD+resveratrol(HFD+RSV)group.After 6 weeks of intervention,fasting blood glucose was measured,IPGTT was performed and to calculate the area under the curve of glucose(AUC),serum and skeletal muscle tissue samples were taken,blood lipid was measured by biochemical method,and insulin level was measured by enzyme-linked immunosorbent assay.H&E staining and oil red O staining were used to observe the changes of skeletal muscle histomorphology and lipid deposition.The food intake and body weight of the mice were measured every week.Results:1.The establishment of insulin resistance model mice by feeding HFDThere was no difference in the initial body weight between the CON group and the HFD group.The weight of mice in the HFD group was significantly increased compared with the CON group from the second week of HFD.The IPGTT experiment was conducted after 8 weeks of HFD.Compared with the CON group,the blood glucose of the HFD group increased significantly at 0,30,60 and 120 min,and the AUC increased significantly,which confirmed that the establishment of insulin resistant model was successful.2.General indexes after 6 weeks of resveratrol intervention in each groupCompared with the CON group,the body weights of the HFD group were significantly heavier.Compared with the HFD group,the body weights of the HFD+RSV group were significantly lower from 4 weeks of resveratrol intervention.IPGTT experiment was conducted at the end of 6 weeks after resveratrol intervention.Compared with the CON group,the blood glucose levels of the HFD group were significantly higher at 0,15,30,60 and 120 min,and the blood glucose levels in the HFD+RSV group were lower than those of the HFD group at each time point.The AUC of the HFD+RSV group was significantly reduced after the administration of RSV.Compared with the CON group,the fasting blood glucose and insulin were significantly higher in the HFD group,and those decreased in the HFD+RSV group compared with the HFD group.Compared with the CON group,the QUICKI value in the HFD group was lower,and that in HFD+RSV group was higher compared with the HFD group.3.The effect of resveratrol on blood lipids in miceCompared with the CON group,the levels of TG,TC and LDL-C in the HFD group were significantly higher.Compared with the HFD group,the levels of TG and LDL-C in the HFD+RSV group decreased significantly,and the levels of HDL-C increased significantly.4.Histomorphology of skeletal muscle of mice in each groupH&E staining showed that the cell structure of skeletal muscle tissue in the CON group was clear and complete,and the cytoplasm was uniform red stained,with fewer lipid droplets.While in the HFD group,the cell structure of skeletal muscle tissue was disordered with different sizes of lipid droplet vacuoles in the cytoplasm.The morphology of skeletal muscle cells in the HFD+RSV group was between CON group and HFD group,and the lipid droplet vacuoles were significantly reduced.The results of oil red O staining of skeletal muscle tissues showed that blue nuclei were visible in the CON group,and the number of orange-red lipid droplets was small.The cells in the HFD group contained a lot of red lipid droplets.The number of lipid droplets in the HFD+RSV group was significantly lower after the intervention of RSV.Summary: Resveratrol reduced the blood glucose and blood lipid,improved insulin sensitivity,and relieved lipid deposition in skeletal muscle of mice with insulin resistance induced by HFD.Part Two The effects of resveratrol on insulin pathway,DDIT4 and m-TOR pathway in skeletal muscle of mice with high-fat-induced insulin resistanceObjective: To investigate the effects of DNA-damage-inducible transcript 4(DDIT4)and m TOR(Mammalian target of rapamycin)pathway in insulin resistance in skeletal muscle of high-fat diet mice after resveratrol intervention,and further to clarify the mechanism of resveratrol in improving high-fat induced insulin resistance in mice.Methods: Animal grouping and specimen collection were the same as the first part.The m RNA and protein expression levels of IRS-1,PI3 K,AKT,GLUT4,m TOR,p70S6 K and DDIT4 were detected by real-time polymerase chain reaction(RT-PCR)and western blot.Results:1.The m RNA and protein expressions of insulin signaling pathway indicators in skeletal muscle of each groupThere were no difference in the m RNA expressions of IRS-1,PI3 K and AKT in skeletal muscle among the CON,HFD and HFD+RSV groups.Compared with the CON group,the m RNA expression of GLUT4 in the HFD group decreased.Compared with the HFD group,the m RNA expression of GLUT4 in HFD+RSV group increased.The total protein expression levels of IRS-1,PI3 K and AKT had no difference among all groups.Compared with the CON group,the protein expressions of p-PI3 K,p-AKT,GLUT4 in the HFD group decreased,the protein expression of p-IRS-1 increased,and the phosphorylated protein-tototal protein ratios for PI3 K and AKT were decreased,while that for IRS-1was increased in the PA group.Compared with the HFD group,the phosphorylated protein expressions of PI3 K,AKT and GLUT4 protein in the HFD+RSV group increased,the protein expression of p-IRS-1 decreased.Furthermore,the phosphorylated protein-to-total protein ratios for PI3 K and AKT were increased,while that for IRS-1 was decreased in the PA+RSV group.2.The expressions of DDIT4 in skeletal muscle in each groupCompared with CON group,the m RNA and protein expressions of DDIT4 in skeletal muscle of HFD group decreased significantly.Compared with the HFD group,the m RNA and protein expressions of DDIT4 in skeletal muscle of the HFD+RSV group increased.3.The expressions of m TOR,p70S6 K in skeletal muscle in each groupCompared with the CON group,the m RNA expressions of m TOR and p70S6 K in skeletal muscle of HFD group were significantly increased.Compared with the HFD group,the m RNA expressions of m TOR and p70S6 K in skeletal muscle of the HFD+RSV group decreased significantly.Compared with the CON group,the total and phosphorylated protein expressions of m TOR and p70S6 K in skeletal muscle tissue of the HFD group increased significantly,and the phosphorylated protein-to-total protein ratios for m TOR and p70S6 K increased.Compared with the HFD group,the total and phosphorylated protein expressions of m TOR and p70S6 K in skeletal muscle of the HFD+RSV group decreased significantly,and the phosphorylated protein-to-total protein ratios for m TOR and p70S6 K decreased.Summary: In the skeletal muscle of mice with insulin resistance induced by HFD,insulin signaling pathway was inhibited,the expression of DDIT4 was reduced,the expressions of m TOR and p70S6 K in m TOR pathway were increased,and the administration of resveratrol reversed the above effects of HFD.DDIT4 and m TOR pathway played a role in resveratrol improving the insulin sensitivity in skeletal muscle of HFD-induced insulin resistant mice.Part Three Mechanism of resveratrol improving palmitic acid induced insulin resistance through DDIT4/m TOR pathway in C2-C12 cellsObjective: Insulin resistance is one of the main characteristics of type 2diabetes mellitus,which is related to high lipid load and obesity.This experiment established C2C12 cell model of insulin resistance through palmitic acid(PA)incubation,and explored whether resveratrol(RSV)could regulate insulin signal through DDIT4/m TOR pathway.Methods: The glucose content in the cell culture medium was measured at 0,12,24 h after the 0.5 mmol/L PA incubation,respectively,to establish an insulin resistant model of C2C12 cells.After 24 hours of PA incubation,the TG content of the cells was measured,and oil red O staining was performed.Cell survival rates were calculated by CCK-8 method after 24 hours of resveratrol intervention at different concentrations(100,50,30,20,10 μ M).C2C12 cells were divided into the control(CON)group,the PA(0.5 mmol/L)group,the 0.5 mmol/L PA + 30 μmol/L resveratrol(PA+RSV)group.Then the glucose content in the culture medium and TG content of the cells were measured after 24 h of the intervention.The m RNA and protein expressions of IRS-1,PI3 K,AKT,GLUT4,DDIT4,m TOR and p70S6 K were measured by real-time polymerase chain reaction(RT-q PCR)and western blot,respectively,and the oil red O staining was performed.After inhibiting DDIT4 with DDIT4-si RNA transfection or activating m TOR with MHY1485 intervention,the western blot was used to measure the expressions of the above indicators.Results:1.PA induced lipid deposition and insulin resistance in C2C12 cellsCompared with the CON group,the glucose concentration in the medium of 0.5 mmol/L PA group was significantly higher.The oil red O staining of C2C12 cells showed that the cytoplasm of the CON group was light blue,and there were a large number of red lipid droplets in 0.5 mmol/L PA group compared with the CON group,suggesting that C2C12 cells had lipid deposition.Compared with the CON group,the TG content of cells in 0.5mmol/L PA group was significantly increased.The insulin resistance model of C2C12 cells was successfully established.Compared with the CON group,the m RNA expression of GLUT4 decreased in the PA group,but there were no difference in the m RNA expressions of IRS-1,PI3 K and AKT between the two groups.The protein expressions of phosphorylated(p)-PI3 K,p-AKT and GLUT4 in C2C12 cells decreased,but the protein expression of p-IRS-1 increased in the PA group.The phosphorylated protein-to-total protein ratios for PI3 K and AKT decreased,while that of IRS-1 increased in the PA group.2.RSV improved the lipid deposition and insulin resistance of C2C12cellsCompared with the CON group,the glucose concentration in cell culture medium of the PA group was significantly higher after 24 h of incubation.Compared with the PA group,the glucose concentration in the culture medium was significantly reduced in the PA+RSV group.The oil red O staining of C2C12 cells showed that the number of orange red lipid droplets were reduced,and the TG content was significantly decreased in the PA+RSV group.It suggested that RSV could alleviate the lipid deposition of C2C12 cells induced by PA.There were no difference in the m RNA expressions of IRS-1,PI3 K and AKT among the CON,PA and PA+RSV groups.Compared with the PA group,the m RNA expression of GLUT4 in the PA+RSV group increased.The protein expressions of p-PI3 K,p-AKT and GLUT4 increased,and that of p-IRS-1 decreased in the PA+RSV group.The phosphorylated protein-to-total protein ratios for PI3 K and AKT increased,while that of IRS-1 decreased in the PA+RSV group.It was suggested that RSV was able to alleviate PA-induced insulin resistance of C2C12 cells.3.Effects of RSV on DDIT4 and m TOR pathway in C2C12 cellsCompared with the CON group,the m RNA and protein expression levels of DDIT4 in the PA group were significantly lower.Compared with the PA group,the m RNA and protein expression levels of DDIT4 increased in the PA+RSV group.Compared with the CON group,the m RNA expressions of m TOR and p70S6 K in the PA group increased significantly,and the expressions of phosphorylated protein and total protein of m TOR and p70S6 K as well as their ratios of phosphorylated protein to total protein increased significantly.Compared with the PA group,the m RNA expressions of m TOR and p70S6 K decreased significantly,and the expres-sions of phosphorylated protein and total protein of m TOR and p70S6 K and their ratios also decreased significantly in the PA+RSV group.4.Effects of silencing DDIT4 on insulin signaling pathway and m TOR pathwayCompared with the PA+RSV group,the glucose concentration in the cell culture medium was higher,and the number of lipid droplets and the content of TG in the cells were also increased,but the m RNA and protein expressions of DDIT4 was significantly inhibited in the PA+RSV+DDIT4-si RNA group.The expressions of total and phosphorylated protein of m TOR and p70S6 K and their ratios of phosphorylated protein to total protein were also increased in the PA+RSV+DDIT4-si RNA group.The protein expressions of p-PI3 K,p-AKT,GLUT4 decreased,the protein expression of p-IRS-1 increased,the phosphorylated protein-to-total protein ratios for PI3 K and AKT decreased,and that of IRS-1 increased in the PA+RSV+DDIT4-si RNA group compared with the PA+RSV group.Any intervention did not affect the total protein expressions of PI3 K,AKT and IRS-1.5.Effects of MHY1485 on DDIT4,m TOR pathway and insulin signaling pathway indicatorsCompared with the PA+RSV group,the glucose concentration in the culture medium,the TG content of the cells and the number of lipid droplets of the cells were up-regulated in the PA+RSV+MHY1485 group significantly.However,there were no difference in the m RNA and protein expressions of DDIT4 between the two groups.The phosphorylated protein and total protein of m TOR and p70S6 K and their ratios of phosphorylated protein to total protein were up-regulated,while the protein expressions of p-PI3 K,p-AKT,GLUT4 were down-regulated,and the protein expression of p-IRS-1 was up-regulated in the PA+RSV+MHY1485 group.Moreover,the phosphorylated protein-to-total protein ratios for PI3 K and AKT were down-regulated,while that for IRS-1 was up-regulated in PA+RSV+MHY1485 group.Summary: PA decreased the glucose processing ability in C2C12 cells,increased cell lipid deposition,reduced DDIT4 expression,activated m TOR pathway,inhibited insulin pathway transmission,and promoted insulin resistance and metabolism disorder of glucose and lipid.The above effects of PA were reversed after RSV intervention.After further inhibiting DDIT4 with DDIT4-si RNA transfection or activating m TOR with MHY1485,it was confirmed that resveratrol up-regulated the expression of DDIT4,and then inhibited the m TOR pathway,so that improved insulin signal transduction in C2C12 cells,increased glucose uptake,reduced lipid deposition and alleviated insulin resistance.Part Four Circulating levels of DDIT4 and m TOR,and contributions of BMI,inflammation and insulin sensitivity in hyperlipidemiaObiective: Evidence shows a high incidence of insulin resistance,inflammation,and excess body mass index in adults with hyperlipidemia.The aim of this part was to determine circulating levels of DDIT4 and m TOR,and to assess the relevance of DDIT4,m TOR,lipids,inflammatory markers,insulin sensitivity and body mass index in hyperlipidemic adults.Methods: Cases with elevated blood lipid in the physical examination population were randomly selected as the hyperlipidemia(HL)group,and those with normal physical examination results in the physical examination population were randomly selected as the normal control(CON)group,with55 cases in each group.The diagnosis of hyperlipidemia patients met the 2016 Guidelines for the Prevention and Treatment of Dyslipidemia in Chinese Adults.Record general indicators,including sex,age,SBP,DBP,height,waist circumference(WC),body weight,BMI.An automatic biochemical analyzer was used to detect FINS,FBG,TG,TC,LDL-C and HDL-C.Quantitative ELISA kits were used to determine the levels of DDIT4,m TOR,C-reactive protein(CRP),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1),and calculate HOMA-IR.Results:1.Height,gender,age and HDL-C were not significantly different in the CON and HL groups(P>0.05).Compared with the CON group,the SBP,DBP,WC,body weight,BMI,FBG,FINS,HOMA-IR,TC,TG,LDL-C,inflammatory markers(CRP,IL-6,TNF-α,MCP-1)were increased in the HL group(P<0.05).The level of serum DDIT4 in the HL group was lower than that in the CON group,and the level of serum m TOR was higher than that in the CON group(P<0.05).2.Insulin resistance level was positively correlated with blood pressure,BMI,blood lipid(TG,TC,LDL-C),m TOR and inflammatory markers,but negatively correlated with HDL-C and DDIT4 levels(P<0.05).Blood lipid levels were positively correlated with BMI,m TOR,inflammatory markers,but negatively correlated with DDIT4 levels(P<0.05).3.Factor analysis identified four domains that explained 84.629% of the total variance of hyperlipidemia(factor 1: inflammation and lipid 1 domain,44.429%;factor 2: overweight domain,21.695%;factor 3: insulin sensitivity domain,11.782%;factor 4: lipid 2 domain,6.723%).Summary:1.The levels of blood pressure,body weight,blood glucose,blood lipid,insulin resistance,m TOR and inflammatory markers in hyperlipidemia population increased,and the expression of DDIT4 decreased.The level of insulin resistance was positively correlated with blood pressure,BMI,blood lipid,m TOR and inflammatory markers,and negatively correlated with DDIT4.The blood lipid level was positively correlated with BMI,m TOR and inflammatory markers,and negatively correlated with DDIT4.2.Insulin resistance,inflammation,blood lipid level and abnormal weight played a synergistic role in hyperlipidemia.Serum DDIT4 and m TOR levels were closely related to blood lipid levels,insulin resistance and inflammatory status,which might be potential predictors of hyperlipidemia.Conclusions:1.Resveratrol could significantly improve insulin resistance and skeletal muscle lipid deposition induced by high fat.2.Resveratrol up-regulated the expression of DDIT4,and inhibited m TOR and p70S6 K in m TOR pathway,thereby promoted IRS-1/PI3K/AKT/GLUT4 insulin signal transduction,which was one of the mechanisms that resveratrol reduced the lipid deposition in skeletal muscle induced by high fat and improved the insulin resistance of skeletal muscle cells.3.The levels of serum DDIT4 and m TOR in hyperlipidemia population were closely related to blood lipid level,insulin resistance and inflammatory status. |
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