Mechanism Of Metformin Improving Insulin Resistance By Reducing The Expression Of Socs3

In recent years,with the development of society and the improvement of life style,the incidence of diabetes is increasing day by day,which seriously threatens human health and quality of life.Metformin,as the first-line drug for patients with type 2 diabetes has been attracted widespread attention in recent years.Metformin’s mechanism is mainly to improve insulin resistance,and its target is mainly in the liver,especially the inhibition of hepatic gluconeogenesis and the reduction of fasting blood glucose have been familiar to everyone.Recently,the research on metformin has become more and more in-depth,and the understanding of its mechanism in regulating blood glucose has become more and more abundant.In addition,with the in-depth understanding of the pharmacological effects of metformin,the effects other than improving glucose metabolism are also more recognized,such as weight loss,anti-tumor,anti-aging,etc.Therefore,it is also necessary to explore new mechanisms of metformin.Suppressor of cytokine signalling 3(Socs3)is one of the members of the cytokine signaling suppressor family.Studies have shown that Socs3 is closely related to insulin resistance,and metformin can improve insulin resistance by inhibiting the expression of Socs3.However,the specific mechanism of metformin in improving insulin resistance by acting on Socs3 is unclear.lncRNA is a non-codingRNA molecule with a length of more than 200 bp,generally without protein coding ability and low conservation.With the understanding of lncRNAs recently,it is recognized that such small molecules are involved in regulatory processes such as chromatin modification,transcriptional activation,and intranuclear transport,and play an important role in human inflammation and tumor diseases.Recent studies have shown that lncRNAs also play an important role in the regulation of glucose and lipid metabolism.Therefore,whether metformin regulates the expression of Socs3 by regulating a certain lncRNA,thereby improving insulin resistance is still unclear and needs to be further studied.In this study,an insulin resistance animal model with high-fat diet was established firstly,and then metformin was given.The liver tissues of the mice were taken for high-throughput sequencing analysis,and the differentially expressed lncRNAs and mRNAs were used to find new targets of metformin in improving insulin resistance.Then,the insulin resistance cell model was established,and the cell transfection technology was used to further explore whether metformin could improve insulin resistance by regulating the expression of lncRNA and acting on Socs3.Finally,through clinical research,the relationship between Socs3、inflammatory factors and insulin resistance,and the effect of metformin on glucose and lipid metabolism and the expression of Socs3、inflammatory factors in diabetic patients were observed.This provided a basis for the discovery of a new mechanism of metformin in improving insulin resistance.Part One Effects of metformin on hepatic lncRNA and mRNA expression profiles of insulin resistance in high-fat diet miceObjective: Through high-throughput sequencing analysis of the liver tissue of insulin-resistant mice treated with metformin,the differentially expressed lncRNAs and mRNAs were analyzed to explore new regulatory targets of metformin in improving insulin resistance.Methods: 36 male C57BL/6J mice were randomly divided into CON group with 12 mice and HFD group with 24 mice after 1 week of adaptive feeding.After 8 weeks of high-fat diet,intraperitoneal glucose tolerance test(IPGTT)was performed and the area under the glucose curve(AUC)was calculated to confirm whether the insulin resistance model was established.In the HFD group,12 mice were selected to be given metformin 200 mg/kg/d by gavage(HFD + MET)randomly,while the CON group and the HFD group were given the same volume of 0.9% Nacl by gavage.At the end of the 6th week,the IPGTT test was performed again.Blood was collected from eyeballs and centrifuged,and serum was collected.The blood lipid level was measured by microplate reader colorimetric method,and the fasting insulin level was measured by enzyme-linked immunosorbent assay(ELISA).The liver tissue was collected and weighed,and the morphological changes of liver tissue in different groups were observed.After the establishment of the insulin resistance model,4 mice in each group(CON,HFD,HFD + MET)were randomly selected to obtain liver tissue for high-throughput sequencing analysis,and the differentially expressed lncRNAs and mRNAs were screened.GO and KEGG enrichment analysis of differentially expressed mRNAs were performed to identify new targets and signal transduction pathways for metformin in improving hepatic insulin resistance.The key molecules expression levels of related signaling pathway were detected by RT-q PCR and Western blot.Results:1.Establishment of a mouse model of insulin resistance after a high-fat dietFrom the second week of intervention with high-fat diet,the body weight was significantly higher compared with the CON group.The IPGTT test was performed after 8 weeks.Compared with the CON group,the blood glucose level and the area under the glucose curve of the mice in the HFD group were significantly increased,suggesting that the mice had abnormal glucose tolerance and insulin resistance.2.Changes of general indexes of mice in each group after 6 weeks of metformin interventionThe weight of the mice in the HFD group was significantly higher than that in the CON group,and after 3 weeks of metformin intervention,the weight was significantly decreased compared with the HFD group.The IPGTT was performed after 6 weeks of metformin intervention.Compared with the CON group,the blood glucose level of the HFD group increased at 0min,30 min,60 min,and 120 min.The blood glucose level at the above time decreased significantly after metformin intervention.There was no significant difference in the blood glucose level at 15 min.The AUC in the HFD group increased significantly than that of the CON group,and compared with the HFD group,the AUC decreased significantly after metformin intervention.The fasting blood glucose and insulin levels of the mice in the HFD group were significantly increased,and the QUICKI value was significantly decreased than that of the CON group.However,metformin could reverse this results.3.Comparison of blood lipid levels in different groups of miceThe levels of TC,TG and LDL-C in mice after 8 weeks of high-fat diet were significantly increased,and HDL-C was significantly decreased than the CON group.After 6 weeks of metformin intervention,the levels of TC and TG were significantly decreased,and HDL-C was significantly increased,while the LDL-C had no significant change.4.Comparison of liver tissue morphology of mice in different groupsAccording to the H&E staining results,the structure of the hepatic cord in the CON group was clearly visible,the hepatocytes were arranged regularly,and lipid droplet vacuoles were rare.Hepatocytes in the HFD group were swollen and disordered,and a large number of lipid droplet vacuoles were seen in the cytoplasm;after metformin intervention,the lipid droplet vacuoles in the hepatocytes were significantly reduced.Oil red O staining showed that the liver cells in the CON group were not stained with red,and there were fewer orange-red lipid droplets;the hepatocytes in the HFD group were significantly red-stained,and a large number of orange-red lipid droplets were seen;after metformin intervention,the orange-red lipid droplets were significantly reduced.5.Expression profiles of lncRNAs and mRNAs in mouse liverCompared with the CON group,1823 upregulated and 1253 downregulated lncRNAs and 892 upregulated and 511 downregulated mRNAs were screened in the HFD group.Compared with the HFD group,702 upregulated and 867 downregulated lncRNAs and 216 upregulated and 307 downregulated mRNAs were screened in the HFD + MET group.Scatter plots showed the distribution of differentially expressed lncRNAs and mRNAs between groups.6.Expression profiles of lncRNAs and mRNAs reversed by metformin in mouse liverVenn diagrams showed that among the 3076 differentially expressed lncRNAs and 1403 differentially expressed mRNAs in the HFD group compared with the CON group,588 lncRNAs and 209 mRNAs could be reversed by metformin.7.RT-qPCR verified that 5 lncRNAs had significant expression differences among the three groupsHigh-throughput sequencing analysis revealed that some lncRNAs in HFD group could be reversed by metformin.To verify the sequencing results,according to the expression of lncRNAs,3 lncRNAs with upregulated in HFD group and downregulated in HFD + MET group(NONMMUT153838.1,NONMMUT031874.2 and NONMMUT119418.1)and 2 lncRNAs with downregulated in HFD group and upregulated in HFD+MET group(NONMMUT051032.2,NONMMUT153848.1)were selected.Furthermore,RT-q PCR was performed.As shown by RT-q PCR,the expression of the 5lncRNAs were confirmed to be consistent with the sequencing analysis results.8.GO and KEGG analysis of metformin reversed mRNAsGO enrichment analysis was performed on the differentially expressed mRNAs,and it was found that the biological process(BP)enriched by GO mainly included: ‘triglyceride metabolic process’,‘p38MAPK cascade’,‘positive regulation of chemotaxis’;Molecular function(MF)mainly included:‘monooxygenase activity’,‘chemoattractant activity’;cellular component(CC)mainly included: ‘protein-lipid complex’,‘lipoprotein particle’.KEGG analysis showed that,differentially expressed mRNAs were enriched in ’small cell lung cancer’,’MAPK signaling pathway’,’cellular senescence’ and ’insulin signaling pathway’.Both the insulin signaling pathway and the MAPK signaling pathway were related to the insulin resistance model in mice,and it was found that Socs3 in the differentially expressed mRNAs was closely related to the insulin signaling pathway.9.Determination of Socs3 mRNA and insulin signaling pathway related genes PEPCK mRNA,PI3 K mRNA,AKT mRNART-qPCR results showed that,compared with the CON group,the mRNA expression levels of Socs3 and PEPCK in the liver of the mice in the HFD group were significantly increased,and the above indexes were significantly decreased after metformin intervention.The differences were statistically significant.The mRNA expression levels of PI3 K and AKT were not significantly different.10.Determination of Socs3 and insulin signaling pathway related protein PEPCK,p-AKT,p-PI3KWestern blot results showed that,the protein expression levels of Socs3 and PEPCK in the HFD group were significantly increased compared with those in the CON group,however the levels of p-AKT/AKT and p-PI3K/PI3 K were obviously decreased.Compared with the HFD group,the expression levels of Socs3 and PEPCK in the HFD + MET group were significantly decreased,while the levels of p-AKT/AKT and p-PI3K/PI3 K were significantly increased.This result suggested that metformin improved insulin resistance in high-fat diet fed mice.Summary: lncRNA may play an important role in metformin improving insulin resistance.The mRNAs reversed by metformin in the high-fat group were mainly enriched in MAPK and insulin signaling pathways.The differential expression gene Socs3 was closely related to the insulin signaling pathway,and metformin could improve insulin resistance by inhibiting the expression of Socs3 and acting on the PI3K/AKT signaling pathway.Part Two Cellular level validation of metformin inhibition of Socs3 by lncRNA NONMMUT031874.2Objective: AML12 cells were cultured and treated with PA to establish insulin resistance model,and further studies at the cellular level to perform and explore how metformin could improve hepatic insulin resistance by regulating NONMMUT031874.2 and inhibiting Socs3 expression.Methods: AML12 was mouse normal liver cell line,PA was used to establish insulin resistance model.According to high-throughput sequencing results,lncRNA-miRNA-mRNA co-expression network and pathway analysis of differential mRNAs enrichment,lncRNA NONMMUT031874.2 and Socs3 were predicited that closely related to the insulin signaling pathway.NONMMUT031874.2 specific siRNA sequences were designed and synthesized and then transfected into AML12 cells.Determination of NONMMUT031874.2 mRNA level after transfection was used as an evaluation of transfection efficiency.AML12 cells were divided into: control group(CON),PA group(PA),PA + MET 1m M group(PA + MET),PA +siRNA-NONMMUT031874.2 negative control group(PA + siRNA-NC)and PA + siRNA-NONMMUT031874.2 knockdown group(PA + siRNANONMMUT031874.2).After 24 h of transfection,PA and metformin were given to the corresponding group.After 24 h,5.7 μl of insulin stock solution was added to each well,the protein was extracted after 40 min,and the expression of the target gene was detected by Western blot.Results:1.lncRNA(NONMMUT031874.2)-miRNA-mRNA co-expression networkAmong the 5 lncRNAs screened and validated,the expression of NONMMUT031874.2 was higher and could be detected by mi R-7054-5p,mi R-7669-3p,mi R-1894-3p,mi R-7076-5p and mi R-7016-3p to regulate mRNA Socs3.2.Establishment of insulin resistance model in AML12 cellGlucose concentrations in the medium were measured at 0 h,8 h,16 h and 24 h after PA intervention in AML12 cells.The results showed that the glucose concentration in the medium of the PA group increased significantly than the CON group at 16 h and 24 h,which indicated that the insulin resistance cell model was successfully established.3.Transfection efficiencyAfter three siRNAs with different sequences were transfected into AML12 cells for 24 h,RT-q PCR showed that,compared with the CON group,the expression of NONMMUT031874.2 mRNA in the three siRNA groups decreased.Compared with the siRNA1 and siRNA2 groups,the NONMMUT031874.2 mRNA expression in the siRNA3 group was lower,with the difference being significant when compared with siRNA2 group.This suggested that the knockdown efficiency of siRNA3 was the most significant,indicating its value for use as intervention siRNA.4.miR-7054-5p mRNA expression after silencing of NONMMUT031874.2After siRNA transfection into AML12 cells,the expression of mi R-7054-5p mRNA in PA group and PA + siRNA-NC group were significantly decreased than that of CON group;the expression of mi R-7054-5p mRNA in PA + siRNA-NONMMUT031874.2 group and PA + MET group expression were significantly increased than that of PA group.In contrast,the expression of mi R-7054-5p mRNA in PA + siRNA-NONMMUT031874.2 group and PA + MET group showed no significant change.5.Change of glucose concentration after silencing of NONMMUT0-31874.2After siRNA transfection into AML12 cells,compared with the CON group,the glucose concentrations in the medium of the PA and PA +siRNA-NC groups were increased.Additionally,compared with the PA group,the glucose concentrations in the medium of the PA + siRNANONMMUT031874.2 and PA + MET groups were decreased.6.The expression of Socs3 and related indicators of insulin signaling pathway after silencing of NONMMUT031874.2According to the Western blot results,compared with the CON group,the expression of Socs3 and PEPCK in the PA and PA + siRNA-NC groups were higher,while the expression of p-AKT/AKT and p-PI3K/PI3 K were decreased.Compared with the PA group,the expression of Socs3 and PEPCK in the PA +siRNA-NONMMUT031874.2 were decreased,while the expression of p-AKT/AKT and p-PI3K/PI3 K were increased.After metformin intervention,the expression of PEPCK and Socs3 were significantly decreased,the p-AKT/AKT and p-PI3K/PI3 K were significantly increased compared with PA group.Moreover,the expressions of p-PI3K/PI3 K and p-AKT/AKT increased and PEPCK decreased in the PA + MET group than the PA + siRNANONMMUT031874.2 group.However,the differences had no statistical significance.The expression of Socs3 in the PA + MET group was decreased significantly,although there were no significant differences in the expression of AKT and PI3 K in the different groups.The above results indicated that metformin improved insulin resistance by down-regulating NONMMUT031874.2 and inhibiting the expression of Socs3.Summary: Metformin improved PA-induced insulin resistance in AML12 cells by inhibiting Socs3 expression and acting on PI3K/AKT signaling pathway,while silencing NONMMUT031874.2 had a similar effect to metformin.Metformin improved insulin resistance by down-regulating NONMMUT031874.2,inhibiting Socs3 expression and acting on PI3K/AKT signaling pathway.Part Three Effects of metformin on the expression of Socs3 and inflammatory factors CRP,IL-6 in patients with type 2 diabetes mellitusObjective: This part mainly studied the relationship between Socs3 and insulin resistance,and the effects of metformin on glucose and lipid metabolism,insulin resistance,Socs3 and serum inflammatory factors in diabetic patients,so as to provide new targets for the clinical effect of metformin.Methods: A total of 110 patients with newly diagnosed type 2 diabetes who were in the endocrinology clinic of our hospital from December 2019 to March 2021 were selected as the type 2 diabetes group(T2DM).Diabetes diagnosis was in line with the diagnostic criteria of the "China Guidelines for the Prevention and Treatment of Type 2 Diabetes"(2017 edition).The diabetic patients were with the 7%≤Hb A1 c ≤10%,the course of disease 6-40 months,and the age between 18-65 years old.Exclusion criteria: 1)Taking any hypoglycemic drugs and hormones which affected the blood glucose;2)Acute and serious chronic complications of diabetes,acute and chronic infectious diseases,serious systemic diseases,major trauma,surgery,malignant tumors and so on within 6 months;3)Pregnant or breastfeeding women;4)Severe liver and kidney dysfunction,cardio-cerebrovascular disease or suffering from mental illness.During the same period,57 healthy controls(NC)were selected who matched age,gender and diabetes group in the physical examination center of our hospital.110 patients with newly diagnosed type 2 diabetes were randomly divided into 55 cases of type 2 diabetes with diet control + exercise for 3 months,and 55 cases with oral metformin treatment for 3 months on this basis.Collected the general information of the subjects,such as age,sex,course of disease,smoking history,drinking history,family history;the height and weight were measured after 8 h of fasting the next morning,and the body mass index was calculated.The levels of FBG,Hb A1 c,blood lipids,fasting insulin,TNF-α,IL-6,CRP,MCP-1 and Socs3 were determined.The correlation analysis was performed between Socs3,inflammatory factors and insulin resistance and the Binary logistic regression analysis was used to identify independent risk factors for insulin resistance.At last,the above indicators were measured after the treatment with metformin for 3 months.Results:1.Comparison of general indicators、Socs3 and inflammatory factors between normal control group and type 2 diabetes mellitusThere were no significant difference in age and gender between the NC group and the T2 DM group.Compared with the NC group,the BMI,TC,TG,LDL-C,Hb A1 c,FBG,HOMA-IR,Socs3 and the levels of the above inflammatory factors in the T2 DM group were significantly increased.2.Correlation analysis of Socs3 and inflammatory factors and insulin resistance indexSpearman correlation analysis showed that,Socs3 and inflammatory factors CRP、IL-6、TNF-α、MCP-1 were positively correlated with HOMA-IR.3.Binary logistic regression analysis of influencing factors of insulin resistanceThe result of binary logistic regression analysis showed that after correcting for confounding factors such as gender and age,Socs3,TNF-α,TG,LDL-C were independent risk factors for insulin resistance.4.Changes of general indicators and inflammatory factors before and after metformin treatmentThere were no significant difference in age,Hb A1 c,BMI,course of disease,TC,TG,Socs3,CRP,IL-6 and other inflammatory factors between the two groups before metformin treatment.After 3 months of metformin treatment,compared with the T2 DM group with diet control + exercise for 3months,the levels of BMI,Hb A1 c,TC,TG,Socs3,IL-6,TNF-α and MCP-1in the T2 DM + MET group were significantly decreased.However,there was no significant change in CRP level.Summary:1.The levels of blood glucose,blood lipids,HOMA-IR,Socs3 and inflammatory factors IL-6,TNF-α,CRP and MCP-1 in patients with type 2diabetes were significantly increased.2.The levels of Socs3 and inflammatory factors IL-6,TNF-α,CRP,and MCP-1 were positively correlated with the HOMA-IR,and binary logistic regression analysis suggested that correcting age and gender confounding factors,Socs3,TNF-α,TG,LDL-C were still the independent risk factor for insulin resistance.3.After metformin treatment,the levels of blood glucose,blood lipids,Socs3 and the inflammatory factors decreased,and insulin resistance improved.Conclusions:1.The hypoglycemic mechanism of metformin was partly through the regulation of hepatic lncRNA and mRNA expression.The lncRNANONMMUT031874.2 could inhibit the expression of hepatic Socs3,which was a new pathway for metformin to improve hepatic insulin resistance.2.Metformin could reduce the levels of Socs3 and serum inflammatory factors IL-6,TNF-α,CRP,and MCP-1 while improving blood glucose,and the changes in the levels of Socs3 and inflammatory factors were closely related to the improvement of insulin resistance.