Mechanism Of Insulin Resistance Improvement By Resveratrol Through Regulation Of Skeletal Muscle LncRNA-NONMMUT044897.2

Insulin resistance is pathological of many chronic metabolic disorders,such as obesity,cardiovascular disease,type 2 diabetes,and certain cancers.Phosphatidylinositol-3-kinase(PI3K)/protein kinase B(AKT)is the most important signaling pathway of insulin in glucose metabolism.In recent years,researchers have discovered that the suppressor of cytokine signaling 1(SOCS1)is an important negative regulator of the PI3K/AKT pathway.As for skeletal muscle,an important target organ responsible for glucose metabolism,SOCS1 and its association with insulin resistance has been less studied,and the search for targeting SOCS1 pharmacologically might provide a new vision for the prevention and treatment of type 2 diabetes.Resveratrol is a naturally-occurring polyphenolic compound composed of two phenyl rings.Resveratrol has been found in more than 70 different plants,with grapes(skins and seeds),red wine,peanuts,and soybeans being important food sources.Resveratrol possesses diverse biological activities,including antioxidant,anti-inflammatory,anti-obesity,and anti-cancer properties,while its effects on improving insulin sensitivity,lowering blood lipids,and regulating blood glucose have received growing attention.Whether or not resveratrol improves insulin sensitivity by regulating SOCS1 expression in skeletal muscle is currently unknown.Long non-coding RNA(lncRNA)is a class of RNA molecules with transcripts more than 200 bp in length that participate in the development of diseases such as coronary artery disease,cancer,metabolic diseases,and insulin resistance.Previous studies from our group found that resveratrol led to improvements in hepatic insulin sensitivity by regulating lncRNAs in mouse models.However,it remains unclear whether resveratrol affects on insulin resistance by regulating the expression of lncRNAs in skeletal muscle.Recently,lncRNAs have been shown to inhibit miRNA activity,increase miRNA target gene expression,and serve as competing endogenous RNAs(ceRNAs)of miRNAs.Then whether lncRNAs affect SOCS1 through the ceRNA mechanism and whether resveratrol improves insulin sensitivity by regulating lncRNAs to affect SOCS1 expression still need further study.Therefore,in this study,we validated the relationship between resveratrol,IncRNA,and SOCS1 by establishing in vivo and in vitro skeletal muscle insulin resistance models,and determined the correlation between SOCS1 expression and insulin resistance through analyzing peripheral blood mononuclear cells SOCS1 mRNA expression and serum inflammatory factors in type 2 diabetic patients,to provide a new potential therapeutic target for type 2 diabetes.Part One Effect of resveratrol intervention on IncRNA expression profiles of skeletal muscle in high-fat diet-induced insulin resistance miceObiective:To investigate the effects of resveratrol on glycolipid metabolic indexes and skeletal muscle lipid deposition in C57BL/6J mice induced by a high-fat diet,and to apply high-throughput sequencing analysis to detect changes in skeletal muscle IncRNA expression profiles in high-fat mice after the application of resveratrol.Methods:C57BL/6J mice were randomly divided into two groups:the control group(CON)and the high-fat diet group(HFD).After 8 weeks of feed,the intraperitoneal glucose tolerance test(IPGTT)in mice was performed.14 mice fed with HFD were selected randomly as the intervention group(HFD+RSV).HFD+RSV group was given resveratrol 100 mg/kg/d by gavage,and the CON and HFD groups were given an equal volume of saline containing 0.1%dimethyl sulfoxide(DMSO).After 6 weeks of intervention,the second IPGTT test was performed.Detection of glucose metabolism:fasting blood glucose(FPG),fasting insulin levels,and calculation of the insulin sensitivity index(QUICKI).Detection of lipid metabolism:total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C).H&E staining and oil red O staining were performed to observe the histomorphological changes in skeletal muscle and fat deposition.Total RNA from skeletal muscle tissues was extracted to construct sequencing libraries.Transcriptome sequencing was performed using high-throughput sequencing technology and differentially expressed genes were identified.The relative expression levels of the differentially expressed genes in each group of mice were then verified by real-time polymerase chain reaction(RT-qPCR).Results:1.Establishment of a high-fat diet-induced insulin resistance miceThere were no significant differences in body weight between the CON and HFD groups at enrolment(P>0.05).After feeding with a high-fat diet for 1 week,the body weight of mice in the HFD group increased significantly(P<0.05).The IPGTT revealed that the blood glucose of mice in the HFD group was higher than that in the CON group before,30 min,60 min,and 120 min after glucose injection(P<0.05),and the AUC of the HFD group was significantly higher than that of the CON(P<0.05),indicating that high-fat diet can lead to abnormal glucose metabolism in mice.2.Improvements of body weight and insulin resistance in mice after 6 weeks of resveratrol intervention.Compared to mice in the CON group,HFD group mice showed higher body weight at all time points(P<0.05),and there was a trend toward decreased body weight after resveratrol application,with statistical differences after 3weeks(P<0.05).Daily caloric intake was not different between the groups.At the end of the 6-week IPGTT experiment,the blood glucose level of the mice in the HFD group was significantly higher at all time points compared with the CON group,and the blood glucose of the HFD+RS V group decreased significantly at Omin,30 min,60 min,and 120 min compared with the HFD group(P<0.05).The AUC was significantly increased in the HFD group and marginally decreased in the HFD+RSV group(P<0.05).Compared with the CON group,fasting blood glucose and insulin were significantly higher in the HFD group(P<0.05)and reduced after resveratrol intervention(P<0.05).HFD reduced QUICKI compared with the CON group(P<0.05)and increased significantly after resveratrol intervention(P<0.05),indicating that resveratrol treatment significantly improves insulin sensitivity in high-fat mice.3.Improvements of blood lipid levels in mice after 6 weeks of resveratrol intervention.TG,TC,and LDL-C levels were significantly higher in the HFD group than in the CON group(P<0.05),TG and LDL-C levels were significantly lower in the HFD+RSV group compared with the HFD group(P<0.05),TC levels no obvious decrease appeared(P>0.05).No significant difference in HDL-C levels between the three groups.4.Histomorphological comparison of skeletal muscle in three groups of mice.The H&E staining results demonstrated that the skeletal muscle cells in the CON group were structurally intact with a close arrangement between myocytes;in the HFD group,the skeletal muscle was loosely structured with widened muscle gaps,varying nuclei sizes,vacuolation-like changes in some myocytes,and a small amount of inflammatory cell infiltration was seen in the muscle gaps.The damage to myocytes was reduced after resveratrol intervention.Oil red O staining showed a small number of orange-red lipid droplets in the CON group;a large number of orange-red lipid droplets were observed in the HFD group;the number of lipid droplets was significantly reduced after resveratrol treatment.5.LncRNA expression profiles in mice skeletal muscleA total of 58245 lncRNAs sequences were detected in the skeletal muscles of mice.On comparing the HFD group with the CON group,we found that there were 3,276 differentially expressed lncRNAs(1,192 upregulated and 2,084 downregulated).Simultaneously,there were 1,640 differentially expressed lncRNAs(921 upregulated and 719 downregulated)in the HFD+RSV group compared with those in the HFD group.Of the upregulated lncRNAs in the HFD group,270 lncRNAs were downregulated in the HFD+RSV group.Of the downregulated lncRNAs in the HFD group,359 lncRNAs were upregulated in the HFD+RSV group.The Venn diagram showed that 629 lncRNAs were reversed between the HFD and HFD+RSV groups.6.RT-qPCR of mice skeletal muscle tissue to validate 4 lncRNAsLncRNA of resveratrol reversing insulin resistance induced by high fat was selected from the screened sequencing results for quantitative RT-qPCR detection.Two upregulated lncRNAs in HFD,a downregulated HFD+RSV(NONMMUT005295.2,NONMMUT044897.2),and two lncRNAs with a reverse trend(NONMMUT128951.1,NONMMUT 145909.1).The expression levels of the selected lncRNAs were consistent with those of the sequencing results,but there was no statistical difference in the increase of NONMMUT 128951.1 in the HFD+RSV group(P>0.05).Summary:High-fat diet-induced insulin resistance and skeletal muscle fat deposition in mice,and application of resveratrol improved insulin sensitivity and reduced fat deposition.The reversal of some lncRNA expression levels after resveratrol application suggests that lncRNA may play an important role in the improvement of high-fat-induced insulin resistance in mice.Part Two Effect of resveratrol intervention on the mRNA expression profiles of skeletal muscle in high-fat diet-induced insulin resistance miceObjective:High-throughput sequencing analysis was performed to explore the effect of resveratrol on the mRNA expression profiles of skeletal muscle in high-fat diet mice.The biological functions and signaling pathways mainly associated with the differentially expressed mRNAs after resveratrol application were identified by GO and KEGG enrichment analysis.Methods:Three groups of mice skeletal muscle transcriptome expression were analyzed using high-throughput sequencing and differentially expressed genes were identified.GO and KEGG enrichment analysis of differentially expressed genes was performed using the R package clusterProfiler.The expression levels of insulin signaling pathway-related molecules SOCS1,AKT,GSK3β,and GLUT4 mRNA were detected by RT-qPCR.SOCS1,AKT,p-AKT,GSK3β,and p-GSK3β protein expression levels were detected by Western blot.Results:1.Expression profile of mRNA in mouse skeletal muscleA total of 83,089 mRNA sequences were detected in mouse skeletal muscle tissue.2118 differentially expressed mRNAs(314 up-regulated and 1804 down-regulated)were found in the HFD group compared to the CON group.The HFD+RSV group had 604 differentially expressed mRNAs(444 up-regulated and 160 down-regulated)compared to the HFD group.Of the mRNA up-regulated in the HFD group,58 mRNAs were down-regulated in the HFD+RSV group.Among the mRNA down-regulated in the HFD group,280 mRNAs were up-regulated in the HFD+RSV group.The Venn diagram showed that 338 mRNAs were reversed between the HFD and HFD+RSV groups.The SOCS1 sequencing results also showed an upregulation in the HFD group and a decrease after resveratrol application.2.GO and KEGG analysis of resveratrol reversed differentially expressed mRNAThe functions of these differentially expressed mRNAs were investigated by enrichment analysis.The most enriched GO terms are "myelination,ensheathment of neurons,axon ensheathment,cellular component assembly involved in morphogenesis,transition between fast and slow fiber(biological process),myofibril,sarcomere,contractile fiber,compact myelin,contractile fiber part(cellular component)",and "structural constituent assembly",and"structural constituent of myelin sheath,actin binding,fatty acid synthase activity,ion gated channel activity,gated channel activity(molecular function)".KEGG analysis showed that differentially expressed mRNAs were mainly involved in JAK-STAT signaling pathway,Chemokine signaling pathyway,Histidine metabolism,type 2 diabetes mellitus,and Fatty acid metabolism.KEGG analysis showed that SOCS1 plays an important role in the JAK-STAT signaling pathway and type 2 diabetes mellitus.3.Comparison of mRNA levels of SOCS1 and insulin signaling pathway-related genesRT-qPCR results showed that the mRNA expression level of SOCS1 was higher in the skeletal muscle of mice in the HFD group than in the CON group(P<0.05),and resveratrol intervention reversed the SOCS1 mRNA level(P<0.05).Compared with the CON group,GLUT4 mRNA expression level in the HFD group significantly decreased and increased after resveratrol intervention(P<0.05).There were no significant differences in the mRNA expression levels of AKT and GSK3β between groups(P>0.05).4.Comparison of protein expressions levels of SOCS1 and insulin signaling pathway-related genesWestern blot showed that SOCS1 expression was abnormally elevated in the HFD group but decreased following RSV treatment(P<0.05).Between the CON,HFD,and HFD+RSV groups,no differences were found in the protein levels of AKT and GSK3β.The HFD group showed dramatic repression of p-AKT and p-GSK3β protein levels compared with those in the CON group,while the HFD+RSV group showed a marked increase in p-AKT and p-GSK3β protein expression(P<0.05).These results demonstrate that high lipid activates SOCS1 expression in mouse skeletal muscle and inhibits insulin signaling pathways and that resveratrol application improves insulin sensitivity.Summary:Resveratrol reversed mRNA expression in skeletal muscle of insulin resistant mice induced by a high-fat diet.SOCS1 is a major differential gene in the type 2 diabetes pathway.The large effect of resveratrol on SOCS1 gene expression in skeletal muscle of high-fat diet mice provides a direction for further study on the improvement of insulin sensitivity by resveratrol through affecting the SOCS1/AKT/GSK3β pathway.Part Three Validation of resveratrol inhibition of SOCS1 via lncRNANONMMUT 044897.2 in Cytological levelObjective:The role of the screened lncRNAs in the improvement of insulin resistance in skeletal muscle by resveratrol was explored at the cellular level by constructing a lncRNA-miRNA-mRNA co-expression network.Methods:The lncRNA NONMMUT044897.2,which is closely related to SOCS1,was screened by lncRNA-miRNA-mRNA co-expression network and KEGG analysis,combined with our sequencing results.The levels of NONMMUT044897.2 in C2C12 cells transfected with lentiviral vectors were measured using RT-qPCR to assess the effect of transfection.C2C12 cells were divided into five groups:control group(CON),PA group(PA),PA+shRNA-NONMMUT044897.2 negative control group(shRNA-NC),PA+shRNA-NONMMUT044897.2 knockdown group(shRNA-NONMMUT044897.2)and PA+RSV 30 μM group(RSV).The corresponding groups were given PA and resveratrol incubation,and total protein was extracted after 24 h.5.7 μl of insulin storage solution was added to each well,and cellular protein was extracted after 20 min.Protein expression was determined by western blotting.Results:1.LncRNA-miRNA-mRNA co-expression networkAmong the validated lncRNAs,NONMMUT044897.2 was highly expressed.To elucidate the relationship between NONMMUT044897.2 and SOCS1,a ceRNA network map was constructed using Cytoscape software.The results showed that NONMMUT044897.2 functions as miR-7051-5p and miR-762 sponge,regulating SOCS1 expression.Using miRcode and starBase databases,it was demonstrated that NONMMUT044897.2 contains the binding sequence of miR-7051-5p.Based on the information from TargetScan and the starBase database,SOCS1 may be the target gene of miR-7051-5p.2.Successful cell differentiationFour days after the induction of C2C12 cell differentiation by 2%horse serum,fusiform myoblasts aggregated with each other and gradually formed bundles of myotubes;the mRNA expression of desmin and myogenin increased significantly,and the difference was statistically significant(P<0.05),suggesting that the induction of C2C12 cell differentiation was successful.3.Effect of NONMMUT044897.2 knockdown on insulin signaling pathwayKnockdown of NONMMUT044897.2 distinctively upregulated the p-AKT,p-GSK3β,and GLUT4 protein levels,and greatly reduced the SOCS1 protein level,compared with those of the PA group.RSV treatment had a similar effect in terms of NONMMUT044897.2 silencing on p-AKT,p-GSK3β,GLUT4,and SOCS1 protein levels.The above results suggest that the mechanism of resveratrol in improving insulin resistance may be related to its downregulation of NONMMUT044897.2 expression.Summary:Resveratrol ameliorates palmitate-induced insulin resistance in C2C12 cells by inhibiting SOCS1 and activating the AKT/GSK3β/GLUT4 signaling pathway;resveratrol reduces the expression level of lncRNA NONMMUT044897.2,thereby inhibiting SOCS1,to improve insulin sensitivity.Part Four Analysis of peripheral blood mononuclear cells SOCS1 mRNA and serum inflammatory factors in patients with type 2 diabetes mellitusObjective:The correlation between peripheral blood mononuclear cells SOCS1 mRNA expression and insulin resistance was determined by measuring the levels of peripheral blood mononuclear cells SOCS1 mRNA expression and serum inflammatory factors in patients with type 2 diabetes.Methods:Fifty patients with first-onset type 2 diabetes mellitus who attended our hospital from December 2019 to March 2021 were selected as study subjects.Inclusion criteria:1)meeting the diagnostic criteria of type 2 diabetes mellitus in the 2017 Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes Mellitus;2)primary patients;3)normal function of liver,kidney,heart,and other important organs;4)age 18-80 years.Exclusion criteria:1)patients with other types of diabetes;2)combined acute and chronic complications of diabetes;3)combined infectious diseases;4)serious systemic diseases;5)major trauma,surgery,or tumor within 6 months;6)long-term use of hormonal drugs.During the same period,50 healthy people who were examined at the medical check-up center were selected as healthy controls(NC).The general information of the subjects,such as age,sex,disease duration,smoking history,drinking history,and family history,were collected;height and weight were measured and body mass index(BMI)was calculated in the early morning of the next day after 8h fasting,and blood was drawn for fasting blood glucose(FBG),glycosylated hemoglobin(HbAlc),blood lipids,fasting insulin,and insulin resistance index.Tumor necrosis factor-a(TNF-α),interleukin-6(IL-6),C-reactive protein(CRP),and monocyte chemotactic protein-1(MCP-1)were also measured.RT-qPCR was performed to measure the level of SOCS1.We also analyzed the correlation between inflammatory factors and insulin resistance.Results:1.Comparison of general clinical data(mean± SD)After statistical analysis,there was no significant difference in age and gender between NC and T2DM groups.Compared with the NC group,BMI,TC,TG,LDL-C,HbA1c,FBG,HOMA-IR,SOCS1,CRP,IL-6,TNF-α,and MCP-1 were significantly higher in the T2DM group,and the difference was statistically significant(P<0.05),there was no significant change in HDL-C level between groups.2.Correlation analysis of inflammatory factors and insulin resistance indexSpearman correlation analysis showed that serum inflammatory factors CRP,IL-6,TNF-α,and MCP-1 were positively correlated with insulin resistance index in type 2 diabetic patients(P<0.01).Expression of PBMC SOCS1 was positively correlated with insulin resistance index in type 2 diabetic patients(P<0.01).The expression of PBMC SOCS1 was positively correlated with CRP,IL-6,TNF-α,and MCP-1(P<0.01).Summary:1.The levels of glucose,lipids,HOMA-IR,SOCS1,IL-6,TNF-α,CRP,and MCP-1 were significantly increased in type 2 diabetic patients.2.Peripheral blood mononuclear cells SOCS1,IL-6,TNF-α,CRP,and MCP-1 were positively correlated with the insulin resistance index.Conclusions:1.High-throughput sequencing revealed that resveratrol improved the gene expression profile of skeletal muscle in mice on a high-fat diet in several ways,and was associated with the insulin signaling pathway.2.Resveratrol can inhibit SOCS1 by reducing skeletal muscle lncRNA NONMMUT044897.2 levels,further activating the AKT/GSK3β/GLUT4 signaling pathway to improve insulin resistance.3.Expression levels of SOCS1 mRNA in peripheral blood mononuclear cells can be intensively studied as a new biomarker for insulin resistance.