The Study On The Role And Mechanism Of Antioxidant Reversing Insulin Resistance-repairing Glycometabolism Disorder

BackgroundType2Diabete Mellitus(T2DM) is one of the ordinary endocrine diseaseswith the development of economy and the richness of people’s living, theincidence of T2DM increased quickly, and becomes a serious public healthconcern. Now，Insulin resistance(IR) and hypofunction of β cell of islet wereknown as the two key points of T2DM. But the etiology and pathogenesis ofT2DM has not been fully elucidated. In recent years, many evidences sugestobesity is accompanied by cellular oxidative stress and inflammatory signalingpathway activation. Some theories such as inflammatory etiology theory,oxidative stress and the others have been put up, but the origin of cellular stressis not clear. Preliminary studies have showed oxidative stress is closely relatedwith IR and hypofunction of β cell of islet and fatty toxicity induced by the abnormal lipid metabolism.Although IR was the key point of T2DM had been accepted generally, butoxidative stress may be play a very important role in development of IR; it isdefinitive that IR induced glycometabolism disorder, but it is not clear that IR whether haddirect associativity with glycometabolism disorder. Studies suggested G-6-Pase may bepart of a switch that controls glycometabolism, however, there were three paths aboutglycometabolism and blood sugar.①The main path is tricarboxylic acid cycle ofaerobic metabolism;②The secondary path iszymolysis of phosphoric acid pentaose that isthe main form of blood sugar metabolism after sport. The potency ratio is lower than TCAcycle without oxygen molecule.③The shunt pathway is glyconeogenesis with deficientglucose supply. PEPKC and G6Pase were the key enzyme in glyconeogenesis, but wealways pay close attention to the regulation relationship between oxidative stress and IR.We ignored influence of oxidative stress to the source and regulation of glucose and themolecular mechanism of the contribution of oxidative stress in advance of blood sugarlevel.Many studies show that IR is closely related with glyconeogenesis, however, whatis the “IR resulted in glyconeogenesis” or “glyconeogenesis induced IR”? There has beenno conclusion. Oxidative stress associated with development of IR, but isglyconeogenesis affected by Oxidative stress or not? What is the relationship ofthe three of them? It is not reported up to now.TZDs are the highly selective agonist of PPARγ and the effective drugs ofinsulin resistance against target tissues, can activation the gene up-regulationexpression of PPARγ, improvement of islet K+, Ca2+channel, promote increasedinsulin secretion, accelerate the metabolism of fat, thus has the reverse theeffects of IR. But, we also cannot explain the key problem of activation ofPPARγ how to improve insulin sensitivity. In recent years, a fat cell sources of polypeptide hormones with action of resistance to insulin has been found, wascalled resistin.The plasma level of Resistin can be inhibited by rosiglitazone or other TZDsdrugs, and is thought to be directly regulated by PPARγ. Meanwhile, somestudies show that AMP-activated protein kinase(AMPK) as an important Serine/threonine protein kinase have the potential of improving IR. AMPK also joins in theregulation of insulin sensitivity.ObjectivesTo observe the change of glycolipid metabolism and the key point targetmolecule such as PPARγ、Resistin、AMPK indexs by establishment of the newIR model of rats induced by high fat high glucose and tBHP. To study theregulation relationship between ROS and IR and to investigate whetherglyconeogenesis join in the mobilization and regulation and the mechanism ofROS regulating glyconeogenesis-related key enzyme by determination ofG6Pase、 PEPCK. To study the interaction of “oxidativestress-IR-glycometabolism dropout” in DM, T2DM specially. To elucidate themolecule mechanism of pathopoiesis chain: oxidative stress is the reason, IR isthe key point, and glycometabolism dropout is the presentate. To try to block uppathopoiesis chain on the three links by using antioxidant. To provideexperiment basis for looking for a new therapia and prevention.Methods1. Study on IR model of rats induced by ROS and correlated regulationmechanismTo prove oxidative stress is the main cause of IR by the following research①To induce rats IR using low dose tBHP injection based on high fat and high glucose feeding.②Oxidative stress-related and glucolipid metabolism-related indexes of serum,liver and fat were measured to testify whether the oxidative injury wereexisted continuously and stably and aggravate IR.③The expression and activites of antioxidative regulation enzymogram, such asPPARγ、SOD1、Gpx1、CAT、GCLC and GR mRNA and protein weredetermined.④Observation of liver morphology and structure changes stimulated by ROSand high fat and glucose using Liver histopathologic.2. Study on the molecular regulation mechanism of glycometabolismcorrelation factors and IR based on oxdative stressThe further study on the interaction of PPARγ-Resistin-AMPK and regulativemechanism to IR on the base of oxidative stress. To prove IR is the core of DMand glycometabolism dropout is the presentate because of IR induce the increasing of bloodsugar.①Human normal hepatocyte cell line(QZG) was used to study thedose-effect relation of different consentrations of tBHP to the signal ofinsulin.②The composite antioxidants were added in cell culture medium taking TZDsas positive control group to observe the effect of oxidative stress on celldamage and the protection of antioxidant on cell injury in rats.③To build IR model of rats of experiment one, AMPK inhibitors, Resistinneutralizing antibody were injected intraperitoneal through the TZDs ascontrol drug therapy. Oxidative stress and glucose metabolism-relatedindexes and changes of liver tissue in IR associated factor in changes of protein expression were determined.④Western blot and RT-PCR were used to evaluate the phosphorylation ofAkt、AMPK and the expression of glucose metabolism and oxidativestress-related indexes and the effect of different sensitizer and inhibiter to IRand glyconeogenesis-related enzyme.3. To study the molecular mechanism of composite antioxidants blocking upglycometabolism disorder and reverses insulin resistance.To investigate the depressant effect of antioxidants to the three important points on thebase of proving oxidative stress is the reason, IR is the key point, and glycometabolismdropout is the presentate in order to prove the molecular mechanism of antioxidantsreversing IR.①To observe the effect of classic antidiabetic drugs and compoundantioxidants on oxidative stress induced hepatic gluconeogenesi-relatedfactors and and insulin signal transduction through liver microsomeperoxidation model to screen appropriate doses of compound antioxidantsand with QZG cells as research object.②To observe the difference effect of classic antidiabetic drugs and compoundantioxidants and the protection of liver, pancreas injury in rats on STZinduced diabetic rat.③To observe the effect of inhibiting ROS、IR and glycometabolism correlationfactors and to study the mechanism of antioxidative therapy in the development of DM.Results1. We made them in a state of oxidative stress by giving rats with high-fatfeed and tBHP injection after a month, the generation of ROS was increased， antioxidant capacity was diminished and lipid peroxidation was increased.Compared with the control group, HGF group and HGF+tBHP group weightchanged obviously, fasting glucose and fasting insulin level were markedlyelevated, serum cholesterol, serum triglyceride and serum free fatty acid contentincreased, decreased insulin sensitivity. Our experiment proved that IR modelcan be established rapidly and stablely by high fat and glucose diet plus tBHP, atthe same time oxidative ROS damage in liver cells pathology subjected liverdamage has a great effect on glucose and lipid metabolism, have protection ofIR.2. We found ROS have significant effects of Akt and AMPKphosphorylation stimulated by insulin through the study of different doses oftBHP effects in normal liver cell QZG. While at low doses when ROS promotedAkt, AMPK expression, but at high doses showed obvious inhibition effect.tBHP also had significant effect of glucolipid-related factors stimulated byinsulin. Low concentration of tBHP promoted the phosphorylation of IRS1,GLUT-4and PPARγ, but high concentration of tBHP inhibited theirphosphorylation. The expression of Resistin was opposite. At the same time, weused PPARγ sensitizer RSG, AMPK inhibitors and Resisitin neutralizingantibodies, and found that the levels of AMPK phosphorylation wassignificantly increased. We also studied the effect of intervention of sensitizerand inhibitors to the mRNA expression of glycometabolism-related enzyme in100μM tBHP on insulin stimulation. The result suggested that tBHPsignificantly increased the mRNA expresstion of gluconeogenesis enzymesPEPCK and G6Pase, but inhibited the mRNA level of GLUT-4. RSG group andResistin inhibits significantly changed this situation, the mRNA level ofGLUT-4were significantly elevated. Instead of PEPCK, G6Pase the expression were inhibited.On the base of the model established successfully in experiment1, weobserved serum insulin levels in AMPK inhibitor group was significantlyelevated and insulin sensitivity index decreased significantly by given IR ratsintraperitoneal injection of resistin neutralizing antibody and AMPK inhibitors,with RSG as positive control group. Insulin sensitivity can be improved. Thelevels of Serum cholesterol, triglyceride and free fatty acid in the resistinantibody group were also significantly lower, while in AMPK group weresignificantly elevated by inhibitor. The changes of oxidative damage index inSerum and liver tissue suggested that oxidative stress was the essential factorinduce IR, oxidative stress in Resistin antibody group was reduced apparentlythrough the intervention. It also prompted the glycolipid metabolism disorderwas improved by increasing the activity of antioxidant enzymes in amelioratingoxidative stress injury.3. The two drugs combined composite antioxidant significantly reduced bloodglucose and the level of oxidative stress were also significantly reduced withrosiglitazone and metformin administered only. At the same time, we weresurprised to find that the treatment group combined with antioxidants not onlyreduce the level of blood sugar but was more stable and persistent compared tothe group of administered alone. The vitro experiment showed that combinedgroup had more apparente hypoglycemic effect than administered only, at the sametime by stimulating upregulation the expression of PPARγ in liver, it inhibitedthe level of Resistin to increased the expression of AMPK and inhibited theactivity of gluconeogenesis enzyme, improved glucose metabolism, reversed thedevelopment of diabetes. The pathological section of Liver and pancreas revealedalthough hypoglycemic effect of rosiglitazone is obvious, but there was a certain side effects of liver injury as well as Metformin Hydrochloride. But it hadimproved liver injury with composite antioxidant, and did not affect the stabilityof blood sugar. The pancreas had the same results, combined treatment groupwas more significant protection of the pancreatic injury and improve thefunction of pancreatic islets.ConclusionOur study observed the important role of ROS in the development of IR byestablishing the IR model of rats induced by high fat and glucose with low doseof oxidant, and investigated the regulation relationship of PPARγ-Resisitin-AMPK ofinsulin signal pathway in liver cell which was stimulated by ROS. At the sametime, we found that the combination group showed more hypoglycemic effectremarkably and stably than the single group which also significantly reduced theoxidative damage of organs.This is the first study demonstrating that oxidative stress is the reason, IR is the keypoint and glycometabolism dropout is the presentate in the development of DM andantioxidants can block up the “morbigenous chain”. Our results will provideimportant experimental evidence for studying the pathogenic mechanism onT2DM. It is also very significant for prevention and treatment on T2DM.