| Hexosamine biosynthesis pathway (HBP) is one of the glucose metabolic pathways with the rate-limiting enzyme glutamine:fructose 6-phosphate amidotransferase (GFAT) and the end product UDP-N-acetylglucosamine (UDP-GlcNAc). HBP is regarded as a cellular nutrient sensor for its variational flux which is depended on the levels of extracellular glucose and plays an important role in the development of insulin resistance and vascular complications of diabetes. It is noteworthy that most of the metabolic abnormalities associated with insulin resistance and diabetes are all related with HBP over flux and increased O-GlcNAc levels. Understanding the diverse roles of HBP will facilitate to investigate the pathogenesy and therapy of insulin resistance and diabetes.Part 1:The screening of human glutamine:fructose 6-phosphate amidotransferase (GFAT) inhibitorHuman GFAT1 gene (cDNA) was obtained from human liver tissue and full length of hGFAT 1 gene was cloned to the pET-32b plasmid. Then the recombinant plasmid hGFAT-pET32b was transformed into Escherichia coli Origami (DE3). The expressed recombinant protein was purified by His-Bind column. The catalytic activity of recombinant hGFAT protein and the affinity to two substrates were determined by GDH method, and the kinetic analysis of hGFAT was assayed. By screening over 300 samples of natural products, Chinese medicine compounds and chemical compounds, the effect of NP07008 (Rhein) on GFAT inhibition has been found.The antigenic of GFAT was analyzed and the half length of GFAT gene was cloned to the pET-28b plasmid. Then the recombinant plasmid was transformed into Escherichia coli BL21(DE3)and the expressed recombinant protein was purified by His-Bind column. The purified recombinant protein was injected to generate the polyclonal antibody. The Western blot results showed that the antibody could recognize GFAT from mouse tissues specifically. The expressions of GFAT in muscle and liver tissue of alloxan-induced hyperglycaemia mice and high fat diet-induced insulin resistance mice were analyzed. Rhein could down-regulate the expression of GFAT in the skeletal muscle of insulin resistance mice.The HIRc cell was stably transfected with an expression vector pcDNA 3.1(+) harboring the cDNA for the full length of hGFAT. The expression levels of hGFAT mRNA and protein were increased in the clones harboring the hGFAT-containing expression vector, and the activity of hGFAT was upgraded, too. Compared with that in HIRc cells, after insulin stimulating, the O-glycosylation of protein was increased in hGFAT-HIRc cells. After transfected with hGFAT1, morphology of HIRc was not changed, and there were no significant differences in cell-cycle between HIRc cells and hGFAT-HIRc cells. hGFAT-HIRc cells were more sensitive to Azaserine than HIRc cells. The insulin stimulated glucose uptake rate declined in the hGFAT-HIRc cells. The phosphorylation of Akt was decreased; otherwise the O-glycosylation of Akt was increased in the hGFAT-HIRc cells after insulin stimulating. Rhein could decrease the activity of GFAT and inhibit the level of O-glycosylation in hGFAT-HIRc cells. Rhein could improve the glucose uptake rate, up-regulate the degree of Akt serine (ser 473) phosphorylation and reduce the degree of Akt O-glycosylation in the hGFAT-HIRc cells.Part 2:Studies on the effects of Rhein on insulin resistance and its mechanismsRhein could decrease the activity and expression of GFAT and O-glycosylation of total protein in the skeletal muscle and the liver of high-fat diet induced mice.Rhein dose-dependently improved hyperinsulinemia, glucose tolerance and insulin resistance. During the hyperinsulinemic-euglycemic clamp tests, the value of GIR was increased in Rhein (60 mg/kg) treatment mice compared with that in HFD mice, and the glucose uptake of liver, skeletal muscle and white adipocytes in RH-M mice were improved by Rhein (60 mg/kg) treatment. Immunoblotting displayed the tyrosine phosphorylation of IR?and IRS-1, the serine phosphorylation of Akt and GSK3?were down-regulated in HFD mice, and were reversed by Rhein treatment. Furthermore, Rhein treatment improved the defective insulin action on the translocation of GLUT 4 in insulin-resistant skeletal muscle.Rhein dose- and time-dependently increased the glucose consumption on 3T3-L1 adipocytes and C2C12 myotubes. High concentration of glucose and insulin could induce insulin resistant cell models. Rhein promotes JH-glucose uptake in insulin resistant 3T3-L1 adipocytes and C2C12 myotubes, benefits insulin signal transduction by up-regulating the serine phosphorylation of Akt and translocation of GLUT 4 to cell membrane. Part 3:Studies on the effects of Rhein on lipid metabolism and its mechanismsRhein inhibited the differentiation of 3T3-L1 preadipocytes induced by differentiation medium (DM) in a time-and dose-dependent manner. Rhein down-regulates the expression of adipogenesis-related transcription factors PPAR?and C/EBPa and their upstream regulator, C/EBP?. In addition, Rhein also reversed high fat diet-induced body weight gain and adiposity in mice, down-regulated the mRNA levels of PPARy and C/EBPa, and their downstream target genes.Rhein could improve non-alcoholic steatohepatitis (NASH) by decreasing liver index, serum ALT and TC levels, hepatic triglyceride contents of the model mice. In the further study, Rhein displayed beneficial effects on lipid metabolic disorders via inhibiting the up-regulation expressions of PPARy and PGC-1a induced by high fat diet, and the expressions of several genes such as FAS, UCP2 and ap2 involved in fatty acid and TG metabolism in liver in high-fat-diet-induced mice.Rhein could decrease triglyceride contents of skeletal muscle of high-fat-diet induced mice. In the further study, Rhein displayed beneficial effects on lipid metabolic disorders via inhibiting the up-regulation expressions of CD36 and FATP1 induced by high fat diet, and increasing the down-regulation expressions of CPT1 and the serine phosphorylation of AMPK induced by high fat diet, which leds to decline fatty acid transportation and disturb fatty acid?-oxidation.In conclusion, recombinant human GFAT protein was obtained by prokaryotic expression system and screening system of GFAT inhibitor in vitro was established. The inhibition of Rhein on GFAT was found and was conformed in GFAT overexpression cell model, type 1 diabetic mice model and insulin resistance mice model. Then, the pharmacological effects and mechanisms of Rhein improving insulin resistance was observed in high fat-diet-induced mice modeland high concentration of glucose and insulin induced insulin resistant cell models. At last, Rhein inhibited adipocyte differentiation and adipogenesis in 3T3-L1 adipocytes and in rodent models of obesity, improved non-alcoholic steatohepatitis (NASH) by inhibiting the expressions of several genes involved in fatty acid and TG metabolism in liver, improved sarcous ectopic lipid accumulation by declining fatty acid transportation and disturbing fatty acid P-oxidation in high-fat-diet induced insulin resistance mice. In this paper, GFAT was found as a new target of insulin sensitizer. From the search for highly selective and efficient inhibitor of GFAT, a new avenue was opened to the combined treatment of insulin resistance and lipid metabolism disorder. |
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