| With the influence of western eating habits in civilians,the intake of fructose-containing soft drinks such as cola and fruit juice increases year by year,resulting in an increase in the intake of fructose.Harm of high-fructose diets is gradually coming into light.Excessive fructose intake can cause metabolic syndrome manifestations such as dyslipidemia,fatty liver,insulin resistance,increased body mass,high blood pressure and hyperuricemia.The mechanism by which a high-fructose diet causes insulin resistance is still unclear.Skeletal muscle is the main organ of insulin resistance.Lipid deposition and impaired mitochondrial structure in skeletal muscle are closely related to insulin resistance.Studies have shown that oxidative stress is an important pathological mechanism leading to mitochondrial dysfunction,tissue damage,insulin resistance and the development of type 2 diabetes.Thioredoxin(TRX)is the main mercaptan reducing agent in cells.It is involved in a variety of signal pathway transduction processes in cells by regulating the REDOX state in cells.It can act as disulfide reductase.Thioredoxin-interacting protein(TXNIP)is an endogenous inhibitor of TRX.By binding/inhibiting the activity of TRX,it breaks the intracellular REDOX balance and increases oxidative stress,while inhibiting or knocking out TXNIP exhibits tissue protection effect.Recent studies have shown that TRX/TXNIP was involved in variety of glycolipid metabolism related pathophysiological processes.Glucagon like peptide 1(GLP-1)analogues is a novel oral glucoselowering drug,which can promote the biological synthesis and secretion of insulin,protect islet beta cells,improve insulin sensitivity,inhibit glucagon secretion,suppress appetite and feeding,delay the stomach emptying,reduce blood sugar and maintain it at a constant level.So far,few researches aboutGLP-1 agonists in relieving insulin resistance and impaired skeletal muscle mitochondrial function induced by high fructose has been reported.Our study firstly established a rat model with insulin resistance induced by high fructose.Various methods were used to further investigate insulin resistance and oxidative stress in skeletal muscle of high fructose diet fed rats.The effects of GLP-1 agonists on insulin resistance in skeletal muscle of high fructose fed rats and its corresponding mechanisms were studied as well,providing theoretical basis for clinical medication.Part one Effect of GLP-1RA on glucose and lipid metabolism in skeletal muscle of insulin-resistant rats induced by high fructose and its mechanismObjective: The present study aims to establish a high fructose diet induced insulin resistance rat model,and to investigate the effects of GLP-1receptor agonists on glycolipid metabolism and mitochondrial function.Methods: 60 male SD rats(weight 180g-200g),after 1 week of adaptive feeding,were randomly divided into 2 groups: 18 normal control(ND)and 42 high frctose diet(HFD).The normal control group was fed with normal feed,and the high-fructose group was fed with equal-calorie high-fructose feed.After 8 weeks of feeding,6 rats were randomly selected from each group for high-insulin-positive glucose clamping experiment.The insulin sensitivity was evaluated by calculating glucose infusion rate(GIR).Intraperitoneal glucose tolerance test(IPGTT)detect the blood glucose value and the area under the glucose curve(AUCglu)at each time point,then kill the rats and take blood and skeletal muscle tissue samples.After high fructose induced insulin resistance rats were successfully modeled,the remaining high fructose group rats were randomly divided into 3 groups: high fructose diet model group(HFD),high fructose + pioglitazone group(Pio),high fructose + exenatide group(HFG),and continued high fructose feed and give the corresponding drug intervention for 6 weeks,weigh each week and recalculate the dose;after6 weeks of drug intervention,detect IPGTT,blood glucose value at each time point and calculate AUCglu,perform high insulin-normal glucose clamp test,the rats were sacrificed after GIR,and serum and skeletal muscle specimens were taken to measure fasting glucose(GLU),total cholesterol(TC),triglyceride(TG),and low-density lipoprotein cholesterol(LDL-C).Observation of mitochondrial ultrastructure of skeletal muscle cells by electron microscope;detection of carnitine palmitoyl transterase-1?(CPT-1?)and medium-chain fatty acyl-coenzyme dehydrogenase related factors of skeletal muscle glucose and lipid metabolism in rats medium chain acyl-Co A dehydrogenase(MCAD),long-chain acyl-Co A dehydrogenase(LCAD),fatty acid translocase(FAT/CD36),skeletal muscle glucose transporter 4(Glucose transporter 4,GLUT-4),peroxisome proliferator-activated receptor ?coactivator-1?(PGC-1?),mitochondrial transcription factor A,mtTFA),nuclear respiratory factors 1(nuclear respiratory factors-1,Nrf1)expression and skeletal muscle tissue malondialdehyde(malondialdehyde,MDA)content and glutathione(GSH),catalase(CAT),superoxide dismutase(SOD)activity changes.Results:1.Compared with the ND group,the HFD group's body weight increased significantly after 8 weeks of feeding(P<0.05);the HFD group's IPGTT result AUCglu was significantly higher than that of the ND group(P<0.05),suggesting that glucose tolerance was reduced;while the GIR was significantly lower In the HFD group(P<0.05),it was suggested that a model of insulin resistance induced by high fructose was successfully established.2.Comparison of body weight changes in rats: the body weight of rats in HFD group was significantly higher than that in ND group at 2 weeks,4weeks,and 6 weeks(P<0.05);rats after intervention in Ex-4 and Pio groups,the body weight of 2 weeks,4 weeks and 6 weeks was lower than that of HFD group(P<0.05).3.Comparison of biochemical indexes of rats in each group: GLU,TC,TG,LDL-C in the HFD group were significantly higher than those in the ND group,the difference was statistically significant(P<0.05);GLU,TC,TG,LDL-C was lower than that of HFD and Pio groups(P<0.05).4.Comparison of glucose tolerance and glucose infusion in rats: at the end of 6 weeks of drugs intervention,AUCglu in the HFD group was significantly higher than that in the ND group(P<0.05),and AUCglu in the HFG group and Pio group was lower than that in the HFD group(P<0.05).The GIR of the HFD group was significantly lower than that of the ND group(P<0.05),and the GIR of the HFD group and Pio group was higher than that of the HFD group(P<0.05).5.Comparison of skeletal muscle mitochondrial morphological changes in rats: under electron microscope,there was no obvious lipid droplet deposition in the skeletal muscle cytoplasm of the ND group.The mitochondria were clear and rich,and the muscle fibers and sarcomere were arranged regularly;more fat was seen in the skeletal muscle cells of the HFD group Droplet deposition,the nucleus is located under the sarcolemma,mitochondrial swelling,and vacuolization are obvious,suggesting that muscle cell lipid deposition and mitochondrial damage;lipid droplets in the HFG and Pio groups are significantly reduced compared with the HFD group,mitochondrial vacuolization is reduced,and the sarcoplasmic reticulum is not obvious.Glycogen,granules and lipid droplets were significantly reduced,and mitochondrial swelling was improved.6.Changes in the activities of mitochondrial fatty acid oxidation-related enzymes CPT-1?,MCAD,and LCAD in rats: the activities of CPT-1?,MCAD,and LCAD in HFD group were significantly lower than those in ND group(P<0.05).The activities of CPT-1?,MCAD and LCAD in the HFG and Pio groups were significantly higher than those of HFD group(P<0.05).7.Compared with ND group rats,HFD group skeletal muscle FAT/CD36,PGC-1?,Nrf1 and mitochondrial transcription factor A(mtTFA)protein levels were significantly reduced(P<0.05);compared with HFD group,skeletal muscle FAT/CD36,PGC-1?,Nrf-1 and mtTFA protein levels in the HFG and Pio groups were significantly increased(P<0.05).Compared with the ND group,the expression of GLUT-4 in skeletal muscle of the HFD group was significantly lower than that of the ND group,and the GLUT-4 protein ofthe HFG group and Pio group was increased compared with the HFD group(P<0.05).8.Skeletal muscle tissue MDA content and GSH,CAT,SOD activity changes: HFD group compared with ND group,skeletal muscle tissue MDA content increased,GSH,SOD,CAT activity was significantly reduced,the differences were statistically significant(P<0.05);compared with HFD,rats in the HFG group and Pio group can significantly reduce MDA levels and increase the activity of GSH,SOD,and CAT(P<0.05).Summary:1.A high-fructose diet can induce hyperlipidemia,insulin resistance and lipid deposition in skeletal muscle of rats.2.The intervention of GLP-1 receptor agonist Ex-4 and pioglitazone can regulate mitochondrial glycolipid metabolism and relieve skeletal muscle lipid deposition,improve glucose tolerance,increase glucose infusion and increased insulin sensitivity and reduce mitochondrial oxidative stress in high fructose fed rats.Part two Effect of GLP-1RA on TRX/TXNIP expression in skeletal muscle of insulin resistant ratsObjective: To study the effect of GLP-1 receptor agonist on TRX/TXNIP expression and its related pathways in insulin-resistant rats by using a new protein signal complex TRX/TXNIP as a redox signal transduction agent.Methods: Established rat insulin resistance model,randomly divided into four groups: normal control group(ND),high fructose diet model group(HFD),high fructose+pioglitazone group(Pio),high fructose+Ex-4 group(HFG),after 6 weeks of drug intervention,RT-PCR and Western blot detection of thioredoxin-interacting protein(TXNIP),thioredoxin(thioredoxin1,TRX1),NF-E2 related factor 1(nuclear factor erythroid 2 related factor 1,Nrf1),protein kinase/phosphorylated protein kinase B(Akt/p-Akt),mitogenactivated protein kinase/phosphorylated mitogen-activated protein kinase(MAPK/p-MAPK)mRNA and protein expression levels.Results:1.RT-PCR results showed that compared with the ND group,the TXNIP mRNA,Akt,and MAPK mRNA of skeletal muscle in the HFD group were significantly increased;while the expression of TRX1 and Nrf1 mRNA were significantly reduced compared with the ND group;MAPK mRNA was lower than the HFD group(P<0.05),while TRX1,Nrf1 mRNA expression was significantly higher than that of the HFD group and Pio group(P<0.05).2.Western Blot results showed that compared with the ND group,the expression of TXNIP,Akt and MAPK protein in skeletal muscle of the HFD group was significantly increased(P<0.05);while the expression of TRX1,Nrf1,p-Akt and p-MAPK protein was decreased than that of the ND group(P<0.05);TXNIP,Akt and MAPK in HFG group and Pio group were lower than that of HFD group(P<0.05);while TRX1,Nrf1,p-Akt,p-MAPK protein was significantly higher than that of the HFD group(P<0.05).Summary:GLP-1 receptor agonists Ex-4 and pioglitazone can improve insulin resistance in skeletal muscle induced by high fructose.The mechanism is related to their regulation in TRX/TXNIP,Nrf1,Akt and MAPK signaling pathways,which improved mitochondrial oxidative stress response,glycogen synthesis and fatty acid oxidation in skeletal muscle.Part three The mechanism by which GLP-1RA affects the mitochondrial function of skeletal muscle cells by regulating the expression of TRX/TXNIPObjective: To further confirm the effect of GLP-1 receptor agonist on TRX/TXNIP expression in insulin-resistant rats and explore related pathways in vitro in cell experiments.Methods: L6 skeletal muscle cells were cultured and induced to differentiate into myotubes.L6 cell differentiation was observed under a microscope to construct and identify L6 cell insulin resistance models.The groups are as follows: normal group(ND),high fructose group(HFD),high fructose + Ex-4 group(HFG),high fructose + sh TXNIP group(HFD + TXNIP(-)),high sugar + TXNIP lentivirus transfection + Ex-4 group(HFG + TXNIP(+));measure the concentration of GLU and TG in cell culture medium of each group;observe the morphological changes of mitochondria of skeletal muscle cells under electron microscope;RT-PCR detect the expression levels of CPT-1?,MCAD,LCAD,and Western blot FAT/CD36,GLUT-4,PGC-1?,mtTFA,NQO1 and HO-1 protein expression in skeletal muscle;RT-PCR detection of skeletal muscle tissue TXNIP,TRX1,Nrf1,Akt,MAPK mRNA levels;Western blot detection of skeletal muscle Tissue protein levels of TXNIP,TRX1,Nrf1,Akt,MAPK,p-Akt,p-MAPK.Results:1 Myoblasts induce differentiation into myotubes after inducing L6 cell differentiation,the microscope observes that fusiform myocytes aggregate with each other and gradually form myotubes;the mRNA expression of desmin and myogenin are significantly increased(P<0.05),indicating that the induction of L6 cell differentiation was successful.2 Insulin resistance model identificationAfter 24 hours of differentiated L6 cell intervention with high fructose,the cell culture medium GLU of the HFD group was significantly higher than that of the ND group(as shown in Figure 3),the difference was statistically significant(P<0.05),oil red O staining showed that the red stained part of HFD group was significantly increased than that of ND group,indicating that the high fructose-induced IR in vitro cell model has been successfully established.3 Glucose concentration in L6 cell culture mediumCompared with ND group,glucose concentration in HFD group increased significantly(P<0.05);compared with HFD group,glucose concentration in HHF group and HFD + sh TXNIP group decreased significantly(P<0.05),compared with HFG Compared with the group,the glucose content of the HFG + TXNIP overexpression group increased significantly(P<0.05).4 Cell TG concentrationCompared with the ND group,the TG content of the HFD group was significantly increased(P<0.05);compared with the HFD group,the TG content of the HHF group and HFD + sh TXNIP group was significantly reduced(P<0.05),compared with the HFG group,HFG + The TG content in the TXNIP overexpression group increased significantly(P<0.05).5 Effects of glucose and lipid metabolism in L6 cells5.1 RT-PCR detection of L6 cells CPT-1?,MCAD,LCAD mRNA expression changescompared with ND group,HFD group CPT-1?,MCAD,LCAD mRNA expression levels were significantly reduced(P<0.05);compared with HFD,HFG CPT-1?,MCAD and LCAD mRNA of group and HFD + TXNIP(-)group were significantly increased(P<0.05);compared with HFG group,CPT-1?,MCAD and LCAD mRNA expression levels of HFG + TXNIP(+)group were significant The decrease was significant(P<0.05).5.2 Western blot detection of L6 cell lipid transport and mitochondrial biogenesis related genes PGC-1?,Nrf-1,mtTFA and FAT/CD36 expression changescompared with ND group,HFD group PGC-1?,Nrf-1,mtTFA,FAT/CD36 protein levels were significantly reduced(P<0.05);compared with HFD group,PGC-1?,Nrf-1,mtTFA and PGC-1? protein levels were significantly increased in HHF group(P<0.05).After sh TXNIP transfection in the HFD group,the protein expression of PGC-1?,Nrf-1,mtTFA,and FAT/CD36 in the HFD group was significantly increased(P<0.05).After intervention with Ex-4,the PGC-1?,Nrf in the HGF group cells-1.The levels of mtTFA and FAT/CD36 were significantly higher than those in the HFD group(P<0.05).5.3 Comparison of glucose transporter 4,GLUT-4 expression levels in each groupCompared with the ND group,the GLUT-4 mRNA expression level in the HFD group was significantly reduced(P<0.05);compared with the HFD,the HFG group and GLUT-4 protein levels in HFD+TXNIP(-)group increasedsignificantly(P<0.05);compared with HFG group,GLUT-4 cells in HFG +TXNIP(+)group decreased significantly(P<0.05).6 Comparison of NQO1 and HO-1 protein expression: Compared with ND group,the content of antioxidant enzymes NQO1 and HO-1 in HFD group decreased(P<0.05);compared with HFD group,the antioxidant proteases NQO1 and HO-1 in the cells of the HFG group and HFD+TXNIP(-)group were significantly increased(P<0.05);compared with the HFG group,the NQO1 and HO-1 in the HFG+TXNIP(+)group were significantly decreased(P<0.05).7 The effect of GLP-1 receptor agonist Ex-4 on the expression of TRX/TXNIP in insulin resistance rats and the related pathways7.1 TXNIP,TRX1,Nrf1,Akt,MAPK mRNA levels: compared with the ND group,the TXNIP,Akt,MAPK mRNA in the HFD group cells was significantly increased(P<0.05),TRX1,Nrf1 were significantly reduced(P<0.05);and compared with HFD group,TXNIP,Akt and MAPK mRNA in HFG group and HFD+TXNIP(-)group were significantly decreased(P<0.05),TRX1 and Nrf1 mRNA were significantly increased(P<0.05);compared with HFG group,the mRNA levels of TXNIP,Akt,and MAPK in the HFG+TXNIP(+)group were significantly increased(P<0.05),and TRX1 and Nrf1 were significantly decreased(P<0.05).7.2 TXNIP,TRX1,Nrf1,Akt,p-Akt,MAPK,p-MAPK protein levels:compared with the ND group,the expression of TXNIP,Akt,MAPK protein in HFD group cells was significantly increased(P<0.05),TRX1 The expression of Nrf1,p-Akt and p-MAPK protein was significantly reduced(P<0.05);compared with the HFD group,the expression of TXNIP,Akt and MAPK protein in the HGF group and HFD+TXNIP(-)group were significantly reduced(P<0.05),TRX1,Nrf1,p-Akt and p-MAPK protein expression were significantly increased(P<0.05);compared with HFG group,the expression levels of TXNIP,Akt and MAPK protein in HFG+TXNIP(+)group cells were significantly increased(P<0.05),TRX1,Nrf1,p-Akt,p-MAPK protein expression levels were significantly reduced(P<0.05).Summary:1.Ex-4 regulates the expression level of TRX/TXNIP,improves skeletal muscle lipid deposition and mitochondrial oxidative stress response.2.Ex-4 regulates Nrf1/2,Akt,and MAPK signaling pathways to regulate skeletal muscle glycogen synthesis and fatty acid oxidation,thereby improving high fructose-induced insulin resistance in L6 cells.Part four The correlation between serum TXNIP level and islet function and oxidative stress level in people with different glucose toleranceObjective: To compare the expression levels of TXNIP in people with different glucose tolerances,and to further explore the correlation between TXNIP and pancreatic islet function and oxidative stress levels.Methods: 151 volunteers who participated in diabetes screening in the Department of Endocrinology,Hebei Provincial People's Hospital from January 2018 to July 2018 were selected.After informed consent,the subjects underwent oral glucose tolerance test.They were divided into three groups according to fasting blood glucose and 2 hours postprandial blood glucose level.They were normal glucose tolerance group(NGT,fasting glucose <6.1mmol/L,meal after 2h blood glucose<7.8mmol/L),impaired glucose regulation group(impaired glucose regulation,IGR,fasting blood glucose6.1 ? 6.9mmol/L,or 2h postprandial blood glucose 7.8 ? 11.0mmol/L)and type 2 diabetes group(diabetes mellitus,T2 DM,fasting blood glucose?7.0mmol/L and or 2h postprandial blood glucose?11.1mmol/L).Detection of thioredoxin interacting protein(TXNIP),islet function,malondialdehyde(MDA),superoxide dismutase T-SOD,glutathione peroxidase(GSH-Px),catalase(CAT)levels,analysis of the correlation between TXNIP and islet function,oxidative stress related indicators.Results:1.There are no statistically significant differences in age,sex,weight,height,and BMI between the three groups of general data(P<0.05).Compared with the NGT group,TC,TG,LDL-C,FINS,HOMA-?,HOMA-IR in the IGR and T2 DM groups is statistically different(P<0.05).2.The TXNIP level in the T2 DM and IGR groups was higher than that in the NGT group,and the T2 DM was higher than the IGR group.The differences were statistically significant(P<0.05).3.The insulin resistance index of the T2 DM and IGR groups was higher than that of the NGT group,and the islet ?-cell function index was lower than that of the NGT group,and the differences were statistically significant(P<0.05).4.The MDA level in the T2 DM and IGR groups was higher than that in the NGT group,and the T-SOD,GSH-Px,and CAT levels were lower than that in the NGT group.The differences were statistically significant(P<0.05).The levels of T-SOD,GSH-Px,and CAT in the T2 DM group were lower than those in the IGR group(P<0.05).The MDA levels in the T2 DM group were higher than those in the IGR group(P<0.05).5.Pearson correlation analysis showed that TXNIP level was positively correlated with insulin resistance index and negatively correlated with islet?-cell function(P<0.05).6.Pearson correlation analysis showed that TXNIP level was positively correlated with MDA and negatively correlated with T-SOD,GSH-Px and CAT(P<0.05).Summary:1.The level of serum TXNIP is closely related to the disorder of glucose metabolism and increases with the development of the disease;colleagues are accompanied by changes in oxidative stress indicators.2.TXNIP level is positively correlated with insulin resistance index and negatively correlated with islet ? cell function.3.Serum TXNIP levels is associated with oxidative stress in patients with abnormal glucose metabolism.Conclusions:1.High-fructose diet can induce dyslipidemia,lipid deposition in skeletal muscle,glucose and lipid metabolism disorder in skeletal muscle cells,and insulin resistance in rats.2.GLP-1 receptor agonist Ex-4 can relieve lipid deposition in skeletal muscle.By regulating Nrf1/2,Akt,MAPK signaling pathways and related genes,it can affect glucose and lipid metabolism and mitochondrial oxidative stress and thereby improve high-fructose induced insulin resistance in skeletal muscle.3.TRX/TXNIP is an important target of GLP-1 receptor agonist Ex-4 in regulation of skeletal muscle mitochondrial oxidative stress.4.Serum TXNIP level is closely related to the level of oxidative stress in patients with insulin resistance and glucose metabolism disorder. |
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