Role Of MicroRNA Involved In Diabetes Amelioration Induced By Duodenal-Jejunal Bypass And Mechanical Study

Part ? miR-200a regulates Rheb-mediated amelioration of insulin resistance after duodenal-jejunal bypassBackgroundsIn recent years,the incidence of diabetes in China and India as the representative of the various countries in the world are on the rise.There are 415 million adult diabetic patients in the world,of which about 114 million of patients with diabetes in China.Diabetes is characterized by elevated levels of chronic blood glucose,and more than 90%are type 2 diabetes.There are currently 415 million adults with diabetes,and the cost of prevention and treatment of more than $673 billion.By 2040,the total number of adults with diabetes will reach 642 million.Large numbers of patients with diabetes and the huge expenditure of diabetes prevention and treatment has become an urgent public health problem.Traditional treatment of T2DM can improve blood glucose in a short time,but its long-term effect is not ideal.Bariatric/metabolic surgery was initially designed to reduce weight for a severely obese population.A lot of evidence shows that Bariatric/metabolic surgery has a role in the treatment of T2DM,and the effect is better than traditional medical treatment.Currently,Bariatric/metabolic surgery has been considered as one of the treatment of T2DM by the Chinese Medical Association Diabetes Branch,the American Diabetes Association(ADA)and the International Diabetes Federation(IDF).Duodenal jejunal bypass surgery can cause rapid and lasting improvement of T2DM on the premise of no obvious weight loss,but the specific mechanism is unknown.The liver plays a key role in glucose metabolism,which is crucial for the storage,decomposition and regulation of glucose.The basic activity of hepatic insulin signaling pathway is the key to the homeostasis of glucose homeostasis,and Rheb plays an important role in the regulation of insulin pathway.MicroRNAs(miRNAs)is a group of endogenous,functional non-coding small RNA fragments,which regulate gene expression at the post transcriptional level.It has been proved that miRNAs is of great importance in many human diseases including T2DM.The abnormal expression of miRNA in liver tissue may also affect the homeostasis of glucose homeostasis.Therefore,the application of miRNA as a clinical marker will be more significant for patients with T2DM.ObejectivesDJB and SHAM procedures were performed in a type 2 diabetes Wistar rat model.The main objectives included:(1)How does the miRNA expression profile change after DJB?(2)Whether DJB could regulates Rheb mediated hepatic insulin pathway activity or not?(3)Whether miR-200a can directly bind to the mRNA of Rheb leads to its degradation and the expression of its protein level?(4)Whether miR-200a can induce the down-regulation of Rheb expression in insulin-resistant rat hepatocytes,which can increase the activity of IRS 1-mediated hepatic insulin pathway?(5)Whether the T2DM rats after DJB induced by lentiviral vector transfected with miR-200a can cause diabetes recurrence?MethodsHFD/STZ induced T2DM rats undergo ad libitum blood glucose levels,body weight,calorie intake,oral glucose tolerance test,insulin tolerance test.The liver tissue of rats were sacrificed and the key molecules of insulin pathway were examined by Western blotting and immunohistochemistry at 2 weeks,4 weeks and 8 weeks postoperatively.The expression of miRNA in liver of rats after DJB operation was analyzed by high throughput miRNA expression array.The effect of miR-200a on the insulin pathway in insulin-resistant rat hepatocytes was studied by using the rat liver cell line and liposome-mediated siRNA transfection.Transfection of miR-200a using lentiviral expression system was used to study the effect of miR-200a on insulin sensitivity of T2DM rats after DJB.Results1.4 weeks after DJB,the DJB group showed lower ad libitum blood glucose levels than the sham group,and this difference became more evident from 4 weeks after surgery and persisted until 8 weeks after surgery.At week 4 after DJB,the post-prandial blood glucose level at all measuring time points after glucose challenge was significantly lower in the DJB group than in the sham group.Insulin sensitivity(assessed using an ITT)indicated an improvement in systemic insulin sensitivity in the DJB groups at week 4.During the OGTT,no alteration of plasma insulin levels was observed in the DJB group at week 4;in contrast,plasma GLP-1 levels were mildly elevated in the DJB group.2.We collected the liver tissue at fed state 8 weeks after DJB or Sham operation and conducted an initial screening of diabetes-related microRNAs..We found 12 miRNAs that were highly significantly dysregulated in post-DJB diabetic rats compared with sham-operated rats.miR-200a is the one that has been predicted to target Rheb.MiR-200a was decreased by 30%in the liver of after-HFD rats.Three days after STZ injection,hepatic miR-200a expression(n=5)reduced 45%compared with before-HFD rats at fed state.we measured the hepatic miR-200a level at week 2(n=5)and week 4(n=5),showing that both were significantly elevated compared with sham group.3.DJB causes the Rheb-mediated amelioration of insulin resistance.At 8 weeks postoperatively,western blot shows that hepatic Rheb/mTOR cassette was significantly downregulated in the DJB group compared with the sham group;the tyrosine phosphorylation levels of IRS 1/2 and serine phosphorylation of AKT were higher in the DJB group.Serine phophorylation of IRS2,which is believed to be regulate hepatic insulin sensitivity,seems unchanged according to the western blot.Immunohistochemistry(IHC)confirmed an obvious decrease of Rheb in the liver of DJB group 8 weeks after surgery.Phosphoenolpyruvate carboxykinase(PEPCK)and G6Pase,which regulate the hepatic gluconeogenesis,are downregulated in liver of DJB group using qPCR.4.miR-200a regulates Rheb protein levels.MiR-200a over-expression in BRL cells resulted in a 65%reduction of Rheb mRNA and 33%reduction in Rheb protein levels;in contrast,decreased levels of miR-200a increased the levels of Rheb mRNA and protein by 50%and 33%,respectively.luciferase reporter gene experiment showed that miR-200a and Rheb mRNA interact directly and inhibit the expression of Rheb.5.miR-200a decreases downstream of Rheb and upregulates the IRS 1-dependent insulin signaling.Western blotting analysis showed that mTOR phosphorylation were less abundant in cells overexpressing miR-200a than in controls,thus causing high tyrosine phosphorylation of IRS 1/2 and serine phosphorylation of AKT.In contrast,miR-200a inhibitor transfection caused high mTOR phosphorylation and restrained the tyrosine phosphorylation of IRS 1/2 and serine phosphorylation of AKT.The serine phosphorilation of IRS2 remain unchanged when subjected to miR-200a mimic and inhibitor compared with each negative control.6.miR-200a inhibited Rheb expression and caused a series of change in glucose metabolism in post-DJB rats.After lentivirus injection and DJB,hepatic miR-200a expression reduced by 90%in DJB +miR-200a inhibitor group.DJB rats injected with miR-200a inhibitor displayed an obviously higher glucose level at all time points in the OGTT result.Insulin-stimulated glucose clearance test indicated that worse glucose clearance over the initial 30 min in the lentivirus-injected group compared to in the controls.7.In the DJB+miR-200a inhibitor group,the Rheb/mTOR cassette was upregulated,and IRS 1/2 tyrosine and AKT serine phosphorylation was reduced.The serine phosphorylation of IRS2 remained unchanged.IHC staining confirmed that Rheb was obviously higher in the DJB+miR-200a inhibitor group than in the DJB+vector group.PEPCK and G6Pase were significantly elevated in DJB+miR-200a inhibitor group.8.DM rats injected of miR-200a mimic showed lower blood glucose level both in OGTT and ITT.In the DM+miR-200a mimic group,the Rheb/mTOR cassette was downregulated,and IRS 1/2 tyrosine and AKT serine phosphorylation was increased.The serine phosphorylation of IRS2 remained unchanged.IHC staining confirmed that Rheb was obviously lower in the DM++miR-200a mimic group.PEPCK and G6Pase were significantly reduced in DM+miR-200a mimic group.9.qPCR analysis showed miR-200a expression remained unchanged between SG and sham group,whereas miR-200a expression is higher in DJB than SG group.Conclusions1.Upregulation of hepatic miR-200a inhibit the expression of Rheb,thus activating the insulin signaling and ameliorating insulin resistance postoperatively.2.Insuliin resistance of DM rats improved after DJB,but inhibition of hepatic miR-200a expression by lentivirus transfection can lead to recurrence of diabetes.3.Exogenous overexpression of liver miR-200a can improve insulin sensitivity in diabetic rats and relieve diabetes.Part ? miR-320 mediates diabetes amelioration after duodenal-jejunal bypass via targeting adipoRl.BackgroundsType 2 diabetes mellitus(T2DM)is a prevalent disease worldwide.Approximately 7%of the global population suffers from T2DM according to the International Diabetes Federation.High production of glucose contributes to fed and fasted hyperglycaemia,which is a major symptom of T2DM and can lead to damage to multiple organs.Bariatric surgery,such as duodenal jejunal bypass(DJB),is the most effective treatment for obesity.Often,DJB results in type 2 diabetes resolution,and possible prevention,of T2DM.Accumulating evidence has implicated reduction of circulating levels of adiponectin and deficiency of adiponectin signalling in the liver as contributors to disorders in gluconeogenesis,which have an essential role in the onset and development of T2DM.Adiponectin is an anti-diabetic adipokine expressed exclusively in adipose tissue.In patients with T2DM,obesity and the metabolic syndrome,serum levels of adiponectin are reduced significantly.The adiponectin receptors,AdipoRl and AdipoR2,are key membrane proteins that exert effects in opposition to the metabolic syndrome.Adiponectin accomplishes its biological effects by binding to AdipoRl and AdipoR2.MicroRNAs(miRNAs)are endogenous small non-coding RNAs of length ? 19-22 nucleotides.miRNAs are essential mediators in the development and progression of T2DM because they target pertinent genes.The aim of this study was to elucidate the essential role of miRNAs in regulation of adiponectin signaling by targeting AdipoRl in DJB and the underlying mechanisms.ObejectivesDJB and SHAM procedures were performed in a type 2 diabetes Wistar rat model induced by high-fat diet(HFD)/streptozotocin(STZ).The main objectives included:(1)How does the miRNA expression profile change after DJB?(2)Whether DJB could regulates adipoRl mediated hepatic adiponectin pathway activity or not?(3)Whether miR-320 can directly bind to the mRNA of adipoRl leads to its degradation and the expression of its protein level?(4)Whether miR-320 can induce the down-regulation of adipoRl expression in rat hepatocytes,which can increase the activity of hepatic adiponectin pathway?(5)Whether the T2DM rats after DJB induced by lentiviral vector transfected with miR-200a can influence effect of bariatric surgery?MethodsHFD/STZ induced T2DM rats were allocated to undergo DJB and SHAM procedures,and each group includes 5 rats.Each group undergoes ad libitum blood glucose levels,body weight,calorie intake,oral glucose tolerance test,insulin tolerance test.The liver tissue of rats were sacrificed and the key molecules of insulin pathway were examined by Western blotting and immunohistochemistry at 2 weeks,4 weeks and 8 weeks postoperatively.The expression of miRNA in liver of rats after DJB operation was analyzed by high throughput miRNA expression array.The effect of miR-320 on the adiponectin pathway in rat hepatocytes was studied by using the rat liver cell line and liposome-mediated siRNA transfection.Transfection of miR-320 using lentiviral expression system was used to study the effect of miR-320 on adiponectin sensitivity of T2DM rats after DJB.Results1.A minimum of blood glucose levels in rodents fed ab libitum in the DJB group(6.42±1.35mmol/L)was observed at week 8 and the difference between the DJB and sham group was increasingly obvious from week 4 to week 8.There was no significant alteration in the OGTT,ITT,serum level of insulin,serum level of glucagon-like peptide-1(GLP-1),the homeostatic model assessment of insulin resistance(HOMA-IR)and the Matsuda-DeFronzo insulin sensitivity index(CISI)during OGTT 2 weeks after DJB,However 4 weeks after DJB,the postprandial blood glucose level at all measured time points after glucose challenge was significantly lower in the DJB group than in the sham group.Insulin sensitivity(assessed using ITT),HOMA-IR and CISI indicated an improvement in systemic insulin sensitivity in the DJB groups at week 4.During the OGTT,no alteration in plasma levels of insulin was observed in the DJB group at week 4;in contrast,plasma levels of GLP-1 were increased slightly in the DJB group.2.We found five miRNAs(miR-503-5p,miR-320-3p,miR-133a-3p,miR-542-3p and miR-133b-3p)to have significantly reduced expression(P<0.05 two-tailed;Student's t-test)in post-DJB diabetic rats compared with sham-operated rats.Typically these DJB-altered microRNAs exhibited a 29-81%reduction compared with sham-operated rats.miR-320-3p was among the five most down-regulated miRNAs in livers,and was the one predicted to target AdipoR1.The serum level of adiponectin remained comparable between DJB and sham groups of rats postoperatively in the fed state.However,a clear difference in hepatic levels of miR-320 between DJB and sham groups of rats was noted postoperatively.This difference became increasingly distinct from week 2 to week 8.3.We found that AdipoRl expression was increased in the rat liver 2,4 and 8 weeks after DJB at mRNA and protein levels,but a significant difference with AdipoR2 expression was not observed.AMPK is a downstream mediator of AdipoRl,and was activated in the rat liver at the protein level all three time points measured after DJB compared with the sham group.There was no significant difference in hepatic protein expression of downstream mediator of AdipoR2,PPAR-?,after DJB compared with sham rats.DJB significantly reduced expression of gluconeogenesis-associated genes(glucose-6-phosphatase(G6pc),phosphoenolpyruvate carboxykinasel(Pck1),Ppargcla),inflammation markers(C-C motif ligand 2(CCL2),tumor necrosis factor-alpha(TNF-?)),an oxidative-stress marker such as thiobarbituric acid reactive substances(TARS)and hepatic triglyceride content 2,4 and 8 weeks after DJB compared with the sham group.4.A mimic of miR-320 increased miR-320 levels by 200 fold,and an inhibitor of miR-320 reduced miR-320 levels by 70%.miR-320 over-expression in Buffalo rat liver(BRL)cells resulted in a 60%reduction of AdipoRl mRNA and protein levels;in contrast,low expression of miR-320 increased levels of AdipoRl mRNA and protein by 70%and 90%;respectively.The luciferase activity of cells that had been transfected with miR-320 and p-Luc-3'UTR AdipoRl was decreased by 50%compared with cells that had been co-transfected with control and p-Luc-3'UTR AdipoR1.Western blotting showed that phosphorylated-AMPK was less abundant in cells overexpressing miR-320,and that inhibitor transfection caused high expression phosphorylated-AMPK protein.Also,the low expression of miR-320 resulted in more potent enhancement of AMPK phosphorylation in response to stimulation of arctiin(AdipoRl agonist)whereas transfection with a miR-320 mimic did not change the phosphorylation level of AMPK significantly.5.Seven days after lentivirus injection,hepatic expression of miR-320 increased by almost four fold in the DJB+miR-320 mimic group.Blood glucose levels in rodents fed ad libitum began to show a significant difference 4 weeks after DJB.A difference of change in body weight or calorie intake postoperatively was not observed.DJB rats injected with a miR-320 mimic displayed obviously higher glucose levels at all time points in the OGTT 8 weeks postoperatively.An insulin-stimulated glucose clearance test suggested that worse glucose clearance over the initial 30 min in the lentivirus-injected group compared with that in controls.DJB rats injected with a miR-320 mimic showed higher HOMA-IR and lower CISI compared with that of control.Reduced expression of AdipoRl and lower signalling of activated AMPK were observed in DJB rats injected with an miR-320-mimic lentivirus,confirming that miR-320 targets adipoRl in vivo.As predicted by those earlier studies,we found expression of G6pc,Pckl and Ppargcla to be increased significantly in DJB rats injected with a miR-320-mimic lentivirus.Expression of inflammatory cytokines such as TNF-a and CCL2 increased,as did that of oxidative-stress markers such as TBARS and hepatic Triglyceride content in DJB rats injected with a miR-320-mimic.6.To ascertain which part of the intestine may be responsible for the alteration in miR-320 expression,we measured alteration of hepatic levels of miR-320 level in DJB(n=5),jejunectomy(n=5),ileectomy(n=5),sleeve gastrectomy(n=5)and sham operation(n=5).Data from quantitative polymerase chain reactions showed that miR-320 expression remained unchanged between sleeve gastrectomy(n=5),ileectomy(n=5)and sham groups(n=5),whereas miR-320 expression was reduced in DJB and jejunectomy groups,by 60%and 40%3 respectively,compared to sham group.Conclusions1.After DJB,the expression of miR-320 decreased,the expression of adipoRl was increased,and the activation of AMPK mediated adipoRl pathway activity in the patients with type 2 diabetes was improved.2.The insulin sensitivity of diabetic rats was improved after DJB,but the increase of the expression of miR-320 in the liver was induced by lentivirus can lead to recurrence of T2DM.