Protective Effect Of Vitamin C On TNF-? Mediated Insulin Resistance And Its Mechanism

The morbidities of metabolic syndrome associated diseases such as obesity,diabetes,cardiovascular diseases are increasing year by year,which is becoming a global health problem.Insulin resistance has been considered as the common pathophysiological basis of metabolic syndrome.Many people with obesity,diabetes and cardiovascular diseases have been proven to be in insulin resistant state.Currently,development in drugs to improve insulin resistance have always been an important field for the prevention and treatment of metabolic syndrome,while the insulin signal pathway IRS-PI3K-AKT has always been regarded as the most important target.At normal condition,insulin in circulation binds to its receptors located on the membrane of hepatocytes,adipocytes and muscle cells,which then activate the IRS-PI3K-AKT signal pathway.The activated signal pathway promotes glucose uptake,glucose utilization,glycogen synthesis?fat synthesis and storage,inhibits gluconeogenesis and glycogenolysis in those targeted cells,finally maintain glucose balance.Studies have shown that patients with obesity and diabetes are in a kind of chronic inflammatory state.Inflammatory cytokines in circulation are closely involved in the development of insulin resistance by impairing insulin signal transduction,among which,researches on insulin resistance induced by TNF-a have been highlighted.Vitamin C is a small molecule compound,as a soluble antioxidant and enzyme cofactors,participates in various biological function in vivo.Studies have shown that vitamin C can protect against diabetes and its associated complication.However,the direct effects of vitamin C on insulin resistance are unclear.Therefore,in this study,we explore the correlation between vitamin C deficiency and insulin resistance in Gulo gene knockout(Gulo-/-)mice,which can't synthesize vitamin C by itself,and further validate and explore the underlying molecular mechanism in HepG2 cells,which may provide a theoretical basis for the administration of vitamin C in prevention and treatment of metabolic syndrome.Palrt ?Correlation between vitamin C deficiency and insulin resistancePurpose:To explore the correlation between vitamin C deficiency and insulin resistance.Insulin resistant Gulo-/-~mice model have been induced by chronic TNF-a exposure,and the effect of supplement vitamin C to normal level on relevance indexes of insulin resistance has been evaluated.Method:Gulo-/-mice were treated with TNF-a exposure(8?g/kg·day)for 7 days to induce insulin resistance,supplemented with vitamin C(0.33 g/L and 3.3 g/L)in drinking water as the vitamin C deficiency group and vitamin C normal group,respectively.Intraperitoneal glucose tolerance test was performed.Fasting plasma insulin levels was measured by ELISA,hepatic triglyceride content,cholesterol content and glycogen content were measured by microplate assay.Hepatic hematoxylin and eosin(HE)staining and immunohistochemistry staining of NF-?cBp65 were performed.Protein level and phosphorylation of AKT and GSK-3? were measured by western blot,and mRNA level of IL-6,IL-la,PEPCK,G6P,ACC1 and FAS were measured by real-time PCR.Results:Vitamin C deficiency aggravated TNF-a-induced insulin resistance in Gulo-/-mice,impaired glucose tolerance,increased fasting serum insulin level and liver lipid accumulation,lead to higher mRNA level of gluconeogenesis-related genes and lipogenesis-related genes,decreased hepatic glycogen synthesis and impaired insulin signal pathway,manifesting as decreased phosphorylation of AKT and GSK3?.Vitamin C deficiency also increased TNF-a-induced activation of NF-?Bp65 and mRNA levels of IL-6 and IL-1? in liver tissues of Gulo-/-mice.Compared will-Tulo-/-mice in vitamin C deficiency group,mice in vitamin C normal group presented better glucose tolerance,lower fasting serum insulin level,less liver lipid accumulation,decreased hepatic mRNA levels of PEPCK,G6P,ACC1 and FAS,increased hepatic glycogen level and improved hepatic insulin signal pathway,manifesting as increased phosphorylation of AKT and GSK3?.Moreover,supplementation with vitamin C inhibited TNF-a-induced activation of NF-?Bp65 and decreased mRNA levels of IL-6 and IL-1? in liver tissues of Gulo-/-mice.Conclusions Vitamin C deficiency aggravated TNF-a-induced insulin resistance in Gulo-/-mice,while supplementation with vitamin C could improve TNF-?-induced insulin resistance,the mechanism may involve in its anti-inflammatory effect.Part ? Mechanism of vitamin C on the improvement of insulin resistancePurpose:To validate the effect of vitamin C on amelioration of insulin resistance and explore its underlying mechanism.Method:HepG2 cells were exposed to TNF-?(10 ng/ml)for 24 h with pretreatment of different concentration of vitamin C(0-200 ?M)for different time(4 h-48 h).Cell viabilities were measured by CCK-8 assay,hepatic glycogen contents were measured by microplate assay,glucose uptakes were measured by flow cytometry,nuclear translocation of p-NF-KBp65 was determined by immunofluorescence,mRNA level of gluconeogenesis-related genes(PEPCK and G6P),lipogenesis-related genes(ACC1 and FAS)were measured by real-time PCR,protein level and phosphorylation of IRS-1,AKT,GSK-3?,GLUT2,JNK,p38,ERK,IKK?,I?B? and NF-?Bp65 were measured by western blot.Results:1)Vitamin C had no suppressive effect on HepG2 cells growth at concentration of 100 ?M and below.2)Vitamin C improved TNF-?-induced reduction of glycogen level in a dose and time-dependent manner.3)Vitamin C improved TNF-a-induced reduction of glucose uptake.4)Vitamin C improved TNF-a-impaired insulin signal pathway,manifesting as reduced serine phosphorylation along with increased tyrosine phosphorylation of IRS-1,increased phosphorylation of AKT and GSK3?,increased protein level of GLUT2.5)Vitamin C inhibited mRNA levels of PEPCK,G6P,ACC1 and FAS in TNF-?-induced insulin resistant HepG2 cells.6)Vitamin C inhibited TNF-a-induced activation of JNK,p38 and IKK?/I?B?/NF-kB signal pathway,but without significant inhibitory effect on ERK activation.Conclusions:Vitamin C ameliorates TNF-a-induced insulin resistance in HepG2 cells through improving IRS1/AKT/GSK3? signal pathway and increasing GLUT2 protein expression,the mechanism may involve in inhibition of TNF-?-induced activation of JNK,p38 and IKK?/IkB?/NF-?B signal pathway.