【作者】 刘倩；

【导师】 陈晓光； 叶菲；

【作者基本信息】 北京协和医学院 ， 药理学， 2009， 博士

【摘要】 己糖胺通路(Hexosamine biosymthesis pathway, HBP)是葡萄糖代谢的途径之一,其关键酶是谷氨酰胺：6-磷酸果糖酰胺转移酶(Glutamine:fructose-6-phosphate amidotransferase, GFAT),终产物是二磷酸尿嘧啶-N-乙酰葡萄糖胺(UDP-GlcNAc)。近年来研究发现该通路作为机体的能量感受器,在胰岛素抵抗、脂代谢紊乱、糖尿病及其血管并发症等的发生发展过程中发挥着重要作用。己糖胺通路的流量增加及O-GlcNAc的水平升高,与胰岛素抵抗及其相关代谢性疾病的代谢紊乱密切相关。对己糖胺通路的研究,将为探讨代谢综合征的发病机制及治疗途径提供新的线索。本论文分三个部分展开。一、人谷氨酰胺-6-磷酸果糖酰胺转移酶(hGFAT)抑制剂的筛选从人肝组织的cDNA中扩增出hGFAT1基因全长片段,构建了大肠杆菌表达质粒pET32b-GFAT,在大肠杆菌Origami (DE3)中表达了hGFAT蛋白。通过镍离子螯合柱(Ni-NTA)亲和层析柱,获得了纯度较高的hGFAT蛋白。使用GDH法检测了重组hGFAT蛋白的催化活性,并初步探讨了GFAT的酶动力学特性。利用重组hGFAT蛋白,建立了GFAT抑制剂体外筛选方法。并筛选天然产物和化学合成样品300多样次,发现大黄酸(Rhein)对GFAT的抑制作用。通过生物信息学分析hGFAT的抗原性和属间同源性,选择人和小鼠相同的基因区域；扩增小鼠肝脏cDNA中GFAT基因片段,构建了大肠杆菌表达质粒pET28b-GFAT。在大肠杆菌BL21(DE3)中表达了Ab-hGFAT蛋白,用Ni-NTA纯化了重组Ab-hGFAT,免疫BALB/c小鼠,得到特异性较高的抗hGFAT多克隆抗体。并观察了Ⅰ型糖尿病小鼠模型和胰岛素抵抗小鼠模型各组织GFAT的表达情况。Rhein可降低胰岛素抵抗小鼠骨骼肌组织中GFAT的表达。成功将hGFAT基因转入胰岛素受体多表达的HIRc细胞,建立了hGFAT高表达的hGFAT-HIRc细胞模型。与HIRc细胞比较,hGFAT-HIRc细胞GFAT的蛋白水平mRNA水平及活性均有所升高；其形态无明显变化,细胞周期改变不明显；MTT实验结果显示,转入基因的细胞对GFAT抑制剂的敏感性略有增加。胰岛素诱导的细胞葡萄糖摄取能力明显降低；胰岛素刺激下的细胞内O-糖基化水平升高；胰岛素刺激下Akt的磷酸化水平降低,Akt的O-糖基化水平升高。PRhein对hGFAT-HIRc的细胞周期无明显影响,但可降低GFAT的活性,抑制细胞总体的O-糖基化水平。Rhein还可改善hGFAT-HIRc细胞的胰岛素诱导的葡萄糖摄取能力,升高Akt的Ser473位磷酸化水平,并降低Akt的O-糖基化水平。二、Rhein改善胰岛素抵抗的药理作用及机制研究Rhein具有明显的GFAT抑制作用,降低高脂饲料诱导的胰岛素抵抗小鼠(HFD)升高的GFAT活性及蛋白表达,降低其肌肉中O-糖基化水平。GFAT印制剂Rhein可呈剂量依赖性地提高机体的胰岛素敏感性,降低HFD小鼠血清中胰岛素水平和胰岛素抵抗指数,改善葡萄糖耐受能力和胰岛素耐受能力,增加正糖钳实验中机体整体的胰岛素依赖的葡萄糖代谢能力和肝脏、骨骼肌、脂肪等胰岛素靶组织的胰岛素依赖的葡萄糖摄取能力。Rhein对HFD小鼠的胰岛素增敏作用,可能与增强胰岛素信号通路中胰岛素诱导的IR-β和IRS-1的酪氨酸磷酸化水平、提高Akt和GSK3β的磷酸化水平、以及增加肌肉中GLUT4向细胞膜上的转位等调节作用相关。Rhein可促进3T3-L1脂肪细胞和C2C12成肌细胞的葡萄糖消耗,且呈剂量依赖性和时间依赖性。对高糖高胰岛素诱导产生胰岛素抵抗的IR-3T3-L1细胞模型和IR-C2C12肌细胞模型,Rhein可明显增强其胰岛素诱导的葡萄糖摄取能力、提高胰岛素刺激下GLUT 4向细胞膜上的转位及p-Akt (Ser 473)的表达。三、Rhein改善脂质代谢的药理作用及机制研究GFAT抑制剂Rhein对3T3-L1脂肪细胞增殖和分化均具有抑制作用,与抑制3T3-L1细胞分化相关的PPARγ和C/EBPa蛋白及mRNA水平有关。Rhein还可降低高脂饲料诱导的肥胖小鼠的体重、白色脂肪重量和脂肪指数,并抑制其白色脂肪组织中与脂肪生成相关的基因表达。Rhein可改善HFD小鼠非酒精性脂肪肝病,降低肝指数、血清ALT水平和肝脂的堆积；降低血清TC水平。抑制肝脏中脂质合成的重要调节因子PPARγ及其转录激活因子PGC-1α、抑制脂肪酸从头合成的关键酶—FAS和SCD-1、抑制脂质生成相关基因ACCα、ap2和UCP2的(?)nRNA水平,可能是Rhein改善HFD小鼠的肝脏脂代谢紊乱的主要作用机制。Rhein可降低HFD小鼠骨骼肌中甘油三酯含量。降低骨骼肌中CD36和(?)FATP1的mRNA水平、增加CPT1的mRNA水平、增加AMPK的丝氨酸磷酸化水平,从而减少肌肉细胞脂肪酸吸收和促进脂肪酸在线粒体的氧化,可能是Rhein改善骨骼肌脂质异位堆积的主要作用机制。总之,本论文首先采用原核表达体系获得重组的人GFAT蛋白,并建立了GFAT抑制剂的体外筛选方法；在筛选中发现了Rhein对GFAT的抑制作用。分别通过体外高表达GFAT的细胞模型、1型糖尿病及胰岛素抵抗的动物模型,证明了Rhein对GFAT的抑制作用。其次,分别在高脂饲料诱导的胰岛素抵抗的动物模型和高糖高胰岛素诱导的胰岛素抵抗的细胞模型上,观察到Rhein改善胰岛素抵抗的药理作用并探讨了其作用机制。最后,在3T3-L1脂肪细胞模型上观察到Rhein(?)印制脂肪细胞分化的作用；并通过抑制白色脂肪组织中与脂肪生成相关的基因表达,减轻高脂饲料诱导的肥胖小鼠的体重；通过抑制肝脏中脂质合成相关因子,改善HFD小鼠非酒精性脂肪肝病；并通过减少肌肉细胞脂肪酸吸收和促进脂肪酸在线粒体的氧化,改善骨骼肌的脂质异位堆积。本论文以GFAT作为胰岛素增敏的新靶点,从寻找高效高选择性的GFAT抑制剂出发,为胰岛素抵抗合并脂代谢紊乱防治开辟了新的途径。更多还原

【Abstract】 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 IRβand IRS-1, the serine phosphorylation of Akt and GSK3βwere 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 PPARγand C/EBPa and their upstream regulator, C/EBPβ. 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 acidβ-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.更多还原