| 【Background】In the last decades, global T2DM has been increasing dramatically. Althoughscientists and organization have known the prevalence and risk of, and thetreatment of T2DM has been made a great progress, T2DM is still increasing outof control and the transition from “treatment” to “prevention” is slow. The keyproblem lies in that the cause of T2DM is unclear and the mechanism on theregulation of insulin signaling and glucose and lipid metabolism is still needed tobe clarified. Our previous results showed that in IR-sensitive animals, theexpression of Nrf2and TCF2were significantly down-regulated. In response tovarious stimuli, the transcription factors Nrf2and TCF2could, in turn, regulate aseries of molecules and processes. Several questions are imperiously needed to beanswered. Could the up-regulation of Nrf2and TCF2decrease blood glucose andimprove IR and β cell dysfunction? Are there interactions between Nrf2and TCF2? Are there chemicals that can target Nrf2and TCF2and treat T2DM?Answers to these questions are important for the study of the pathogenesis ofT2DM, and important for the exploration of potential drugs and the control ofT2DM prevalence.【Aims】(1) To study role and mechanism of disorder of Nrf2-regulated redox balance inhigh glucose-induced oxidative injury, IR and β cell dysfunction.(2) To clarify role of Nrf2-antioxidant enzyme chain in the decrease of bloodglucose, the improvement of IR and β cell dysfunction and the regulation ofOA.(3) To study insulin-exerted regulation on Nrf2-redox balance.(4) To study the function of TCF2, role of TCF2in IR and β cell dysfunction andthe regulation of RA.(5) To study the interaction between Nrf2and TCF2and role of the interaction inthe pathogenesis of T2DM.【Methods】(1) Role of Nrf2-antioxidant enzyme chain in high glucose-induced oxidativeinjury. Hepatocytes were cultured in medium with high level of glucose and RSG,and high glucose toxicity and the protective effect of RSG were observed. MTTwas used to detect cell viability. DCFH-DA was used to detect ROS generation.Western blot and immunofluorescence were used to determine related proteinexpression.(2) Role of Nrf2-antioxidant enzyme chain in ROS-induced IR. Based on traditional high fat diet-induced IR animal model, injection of oxidant tBHP wasintroduced to induce IR model. In addition, GOX was injected to establish instantIR model. FBG, IPGTT and IPITT were assayed and FFAs was determined.Transmission electron microscope was used to observe the changes of liverstructure. RT-PCR was used to measure mRNA expression of related factors.Na+/K+-ATPase and Ca2+-ATPase were detected and the activities ofmitochondrial respiratory chain enzyme complex. Western blot was used to detectrelated protein expression and insulin-stimulated signal.(3) Role of Nrf2-antioxidant enzyme chain in ROS-induced β cell dysfunction.Immunohistochemistry and immunofluorescence were used to detect thedistribution of Nrf2in rat pancreatic islet and the sub-distribution of Nrf2inINS-1cell. INS-1cells were treated with FFAs. MTT was used to detect cellviability. Hoechst was used to measure apoptosis. DCFH-DA was used to detectROS generation. Western blot was conducted to detect Nrf2and relatedantioxidant enzymes expression. ELISA was conducted to measure GSIS.RT-PCR was used to detect mRNA expression of related factors. siRNA targetingNrf2was synthesized and adenovirus over-expressing Nrf2was construct toobserve the effect of Nrf2on cell viability and FFAs on cell toxicity. Short termand long term of high fat diet was introduced to observe the changes of Nrf2expression and insulin secretion. Several inhibitors were used to clarify thesignaling pathway responsible for the effect of FFAs on Nrf2. GSIS was detectedto observe the alteration of ROS generation and Nrf2expression.(4) Regulation of insulin on Nrf2-redox balance.100nM insulin was added in themedium of BRL-3A hepatocyte to observe the effect of insulin on Nrf2-redoxbalance in vitro.10IU/kg insulin was injected to observe the effect of insulin on Nrf2-redox balance in liver in vivo. DCFH-DA was used to detect ROSgeneration and dynamic ROS production during1.5h was recorded. Western blotwas conducted to detect the expression of Nrf2and insulin-stimulated signalingpathways. Several inhibitors were used to examine the signaling pathwaysresponsible for Nrf2-redox balance.(5) OA improves IR and β cell dysfunction through activating Nrf2-antioxidnatenzyme chain. db/db diabetic mice were injected with OA for2weeks. FBG、IPGTT and IPITT were assayed. Animal weight and visceral fat was observed.Liver was collected and insulin signaling was measured. Frozen section ofpancreas was made and TUNEL was conducted to detect apoptosis. INS-1cellswere cultured with medium in the presence of FFAs and OA, and MTT and GSISwas conducted. Annexin Ⅴ-FITC/PI was used to detect cell apoptosis. Theexpression of Nrf2and antioxidant enzymes and the phosphorylation of relatedkinases were measured by Western blot. siNrf2was used to observe role of Nrf2in protecting β cell function. Inhibitors of Akt, ERK and PPARγ were used tostudy the signaling pathways responsible for the effect of OA on Nrf2.(6) Key role of TCF2and the regulation of RA. Immunofluorescence wasconducted to observe the sub-distribution of TCF2in BRL-3A. Rat pancreas wascollected and immunohistochemistry and immunofluorescence were conducted todetect the distribution of TCF2in pancreatic islets. In INS-1cells, thesub-distribution of TCF2was assayed by immunofluorescence. siTCF2andlentivirus over-expressing TCF2were used to treat BRL-3A cell and insulinsignaling was determined. Rats were injected with lentivirus over-expressingTCF2, and FBG, IPGTT, IPITT and liver insulin signaling were measured.GOX-treated rats were injected with lentivirus over-expressing TCF2, and FBG, IPGTT, IPITT and liver insulin signaling were measured. siTCF2and lentivirusover-expressing TCF2were used to treat INS-1cell and GSIS was assayed.BRL-3A cell was treated with RA, and the expression of TCF2and insulinsignaling was detected. INS-1cells were treated with FFAs and RA, and theexpression of TCF2and insulin secretion-related factors and GSIS weredetermined.(7) Nrf2-TCF2interaction and mechanism on the application of OA-RAcombination. BRL-3A cell was transfected with adenovirus over-expressing Nrf2and lentivirus over-expressing TCF2, respectively. Western blot was conducted toevaluate the expression of Nrf2, TCF2, antioxidant enzymes and glucosemetabolism related factors. DHE was used to detect ROS generation in BRL-3Acell and TCF2-BRL-3A cell. BRL-3A cell and TCF2-BRL-3A cell were exposedto different concentrations of GOX. Cell viability and cell appearance wereobserved. Western blot was used to evaluate the effect of GOX on Nrf2and TCF2expression. STZ was used to induce diabetic animals and OA, RA, and OA+RA(half dose) were given to animals. In addition, STZ was used to treat INS-1cellsand ROS and GSIS was detected. TCF2-INS-1cell was transfected withadenovirus over-expressing Nrf2, and the effect of STZ on GSIS was determined.【Results】(1) High level of glucose decreased cell viability and induced evident cytotoxicity.Along with the increase of glucose concentration, ROS production increasedsignificantly. Low level of glucose activates Nrf2. In the presence of high level ofglucose, the expression of Nrf2and a series of antioxidant enzymes were reduced.In addition, high level of glucose decreased PPARγ and COX-2expression, promoted the phosphorylation of PKC, and suppressed the phosphorylation ofAkt and ERK. RSG notably reduced the cytotoxicity induced by RSG, suppressedROS generation, and up-regulate PPARγ and Nrf2expression. Inhibitor of PPARγnotably down-regulated the effect of RSG on Nrf2and COX-2expression andAkt and ERK phosphorylation, but had no effect on PKC phosphorylation.(2) tBHP aggravated HF diet-induced disorder of lipid metabolism, IR andimpairment of glucose tolerance, promoted HF diet-induced disturbance ofinsulin signaling, mitochondrial injury, endocytoplasmic reticulum stress andimbalance of Nrf2-redox system. GOX treatment resulted in impairment ofglucose and insulin tolerance and disorder of insulin signaling, affected thephosphorylation of JNK, ERK and p38, induced endocytoplasmic reticulumstress and mitochondrial dysfunction, and decreased Nrf2-controlled antioxidantenzyme chain. Antioxidant NAC could notably suppress GOX-induced IR.(3) Under normal condition, low level of Nrf2is present in pancreatic β cell andin the cytoplasm. FFAs could induce evident cell apoptosis and decreased themRNA expression of insulin synthesis and secretion-related factors and GSIS.Low level of FFAs could promote the expression of Nrf2and antioxidantenzymes, and enhance cell viability. High level of FFAs decreased the expressionof Nrf2and antioxidant enzymes, and decrease cell viability. siNrf2notablyinhibited the up-regulation of cell viability and antioxidant enzyme expression.Over-expression of Nrf2could significantly inhibit the impairment of GSIS andcell viability induced by high level of FFAs. Short term of HF diet promotedinsulin secretion and pancreatic Nrf2expression. However, long term of HF dietinhibited insulin secretion and pancreatic Nrf2expression. FFAs promoted ROSgeneration in mitochondria and NADPH oxidase. Low level of FFAs promoted Akt and ERK phosphorylation. Inhibition of ROS generation and Akt and ERKphosphorylation could notably inhibit FFAs-induced Nrf2expression. GSIS inINS-1cells was coupled with transient increase of Nrf2.(4) Under the treatment of insulin, the expression of Nrf2and antioxidantenzymes increased transiently and then reduced to basic level. siNrf2couldsignificantly inhibit insulin-stimulated antioxidant enzyme expression. Insulinpromoted ROS generation in BRL-3A cell and30min after the treatment showedthat most evident production of ROS. Antioxidant, DPI and3NP couldsignificantly inhibit insulin-exerted ROS production. In the presence of insulin,the changes of Akt and ERK phosphorylation was similar with Nrf2. Inhibition ofAkt and ERK could significantly suppressed insulin-stimulated Nrf2expression.Pre-treatment of cells with insulin could prevent against tBHP-induced decease ofcell viability and apoptosis. Inhibition of Nrf2could notably reduce the protectiveeffect of insulin.(5) In db/db diabetic mice, OA could decrease blood glucose, improve IR andprotect β cell. OA could reduce body weight and visceral fat content, and promotelipolysis. In db/db diabetic mice, OA inhibited pancreatic cell apoptosis throughactivating Nrf2-antioxidant enzyme chain. FFAs-induced decrease of cell viability,GSIS and apoptosis was significantly inhibited by OA. siNrf2significantlyinhibited the protective effect of OA in INS-1cells. Inhibition of Akt, ERK andPPARγ could suppress OA-activated Nrf2.(6) Under normal condition, in BRL-3A cells, TCF2is mainly expressed incytoplasm. siTCF2could significantly inhibit insulin signaling in BRL-3A cells.In TCF2-BRL-3A cells, GLUT2and GCK expression was increased, PEPCK expression was reduced, and insulin signaling was enhanced. In rats injected withTCF2lentivirus, insulin sensitivity was enhanced and GOX-induced IR wasinhibited. In rat pancreas, TCF2is mainly in β cells. In INS-1cells, TCF2ismainly present in cytoplasm. siTCF2could significantly suppress GSIS. InTCF2-INS-1cells，GLUT2and GCK expression were increased, the expression ofPDX-1and GSIS were decreased. In BRL-3A cells treated with RA, TCF2expression and insulin signaling were increased, and GOX-induced insulinsignaling disturbance was notably inhibited. In INS-1cells treated with RA, theexpression of TCF2, GLUT2, GCK and PDX-1and GSIS were enhanced. RAcould significantly inhibit FFAs-induced lipotoxicity.(7) Compared with BRL-3A cell, in Nrf2-BRL-3A cell, the expression ofantioxidant enzymes were significantly up-regulated. However, the expression ofTCF2and PPARγ were not significantly altered. When TCF2was over-expressed,the expression of SOD1was significantly decreased, while GCLm and GPx1expression was notably up-regulated. Moreover, up-regulation of TCF2increasedPPARγ, without significant effect on Nrf2and antioxidant enzyme expression. InTCF2-BRL-3A cell, ROS production was significantly increased. In BRL-3A cell,in the presence of GOX, the expression of Nrf2and TCF2increased transientlyand then decreased. Compared with BRL-3A cell, TCF2-BRL-3A was moreresistant to GOX toxicity. In STZ-induced diabetic rats, OA and RA alone coulddecrease blood glucose. Moreover, the combination of OA and RA could alsodecrease blood glucose, which was more potent. In INS-1cell, STZ resulted indecreased cell viability and GSIS. Compared with INS-1cell, TCF2-INS-1cellwas more resistant to STZ toxicity. In TCF2-INS-1cell, over-expression of Nrf2could further protect cells against STZ toxicity. 【Conclusions】(1) In hepatocytes, glucose activates PKC and thus promotes the generation ofROS. Low level of glucose generates low level of ROS, which in turn activatesNrf2and antioxidant enzymes and enhance antioxidant defense. High level ofglucose generates high concentration of ROS, down-regulates Nrf2andantioxidant enzymes, leading to oxidative stress. The antioxidant enzymescontrolled by Nrf2may constituent a potential “antioxidant enzyme chain”,enhancing the cell defense system. Once ROS is excessive, Nrf2-antioxidantenzyme chain could be injured and oxidative stress may be aggravated. Theinjury of Nrf2-antioxidant enzyme chain is pivotal for high glucose-inducedoxidative injury. In one hand, RSG inhibits PKC and thus ROS production in aPPARγ-independent manner, in the other hand, RSG activates Nrf2anddownstream antioxidant enzymes and thus promotes the elimination of ROS,which is PPARγ-dependent.(2) tBHP induces oxidative stress, aggravates HF diet-induced IR. Either in vivoor in vitro, GOX could generate ROS and down-regulate Nrf2-antioxidantenzyme chain, and influence various factors, including down-regulation ofPPARγ, the alteration of MAPKs, endocytoplasmic reticulum stress andmitochondrial dysfunction, leading to disturbance of insulin signaling andelevation of blood glucose. ROS may be the causative factor of IR, andNrf2-antioxidant enzyme chain plays a crucial role in the defense system. In thepresent experiment, we established an animal model of IR, using GOX, paveways for the study of the pathogenesis and the treatment of IR.(3) ROS plays a double-edged sword in normal β cell function and in thepathogenesis of β cell dysfunction. Chronic exposure to FFAs, ROS was generated and β cell function could be influenced. Low level of ROS generatedby low level of FFAs and in the process of GSIS could activate ERK and Aktsignaling and thus activates Nrf2-antioxidant enzyme chain. The activation ofNrf2reflects the responsive and protective effect in a body. Nrf2-antioxidantenzyme chain plays a key role in determining the effect of ROS direction and inprotecting β cell against potential oxidative injury.(4) Insulin regulates antioxidant system through activating Nrf2. Insulin-exertedROS may derive from mitochondrial respiratory chain and NADPH oxidase.ROS is responsible for insulin-stimulated regulation on Nrf2. Insulin regulatesredox balance via ROS-ERK-Akt-Nrf2, which prompts us to re-recognize theessence of insulin effect.(5) Oxidative stress plays a key role in the lipotoxicity in β cells, andNrf2-antioxidant enzyme chain plays a protective role. OA could decrease ROScontent, and inhibit the influence of PA on Nrf2-antioxidant enzyme chain. Theactivation of Nrf2-antioxidant enzyme chain is responsible for the protectiveeffect of OA. And ERK/Akt/PPARγ signaling is responsible for OA-activatedNrf2.(6) Under normal condition, TCF2is mainly present in cytoplasm. Uponsimulation, TCF2may be translocated into nucleus. In hepatocytes, TCF2promotes insulin signaling, regulates glucose metabolism. In β cell, TCF2regulates insulin synthesis and secretion positively and negatively, which meansthat the homeostasis of TCF2is critical for β cell function.(7) In the one hand, Nrf2is the key regulator of redox balance, and in the otherhand, Nrf2could also regulates lipid metabolism. For TCF2, it can regulate glucose metabolism and influence redox balance in the mean time. In addition,TCF2may influence Nrf2indirectly through PPARγ. Moreover, Nrf2mayinfluence TCF2via regulating ROS. There may be complicated interactionbetween Nrf2and TCF2, in which ROS is the center. The homeostasis of theinteraction between Nrf2and TCF2is pivotal for the maintenance of normalfunction in a body and for the protection against IR and β cell dysfunction. |
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