Effect Of Glucose Tolerance Factor On Glucose Metabolism In Hep-G2 Cells

Chromium is an essential trace element, which has an important role in the metabolism of glucose and lipid metabolism in human body. Diabetic patients and the elderly people through supplement the right amount of chromium ( Cr3+ ) can significantly restore the impaired glucose tolerance status and maintain the balance of glucose metabolism. The active center of the glucose tolerance factor (GTF) is Cr3+, which extracted and purified from yeast. Therefore, in this paper, we extract the GTF from yeast and apply the GTF into Hep-G2 cells model of insulin resistant to study the structure of GTF and the effect of the GTF on the metabolism of glucose and lipid metabolism. The full text aims to establish an excellent and efficient method of GTF purification, and to get high-yield and high purity of GTF substance. Meanwhile, the study aim to lay the theoretical foundation for the research of GTF in the hypoglycemic function.First of all, this experiment screen out the resistant chromium yeast strain, which can grow up in the high concentration of chromium (1000 ?g/mL) , then inoculate it into YPD medium, and gradually increase the concentration of chromium ion in the medium. The research shows that it can up to 1818 ?g/g, which is the optimal organic chromium content,when the chromium ion concentration reached 500 ?g/mL, and the content of organic chromium can reach to 90.34%. At the same time, the study use the yeast strain for fermenting in different sizes to measure the content of organic chromium. The fermentation scale is expanded from shake flask fermentation to 500 L, and the content of organic chromium decreased gradually from 2160 ?g/mL to 765 ?g/mL. The percentage of total chromium content in organic chromium is from 87.17% to 61.55%. However the biomass dry biomass has increased from 0.89 g/100 mL to 1.01 g/100 mL. Overall, with the expansion of fermentation, the content of organic chromium and total chromium in yeast is decreased.Secondly, the GTF yeast powder extract with ammonia, then precipitate with ethanol(30%) at 0?for 20 min, centrifuge and take the liquid supernatant, measure the ratio of chromium content and protein content is 5.02. This method is compared with the extraction only with ammonia (0.818), GTF extraction rate has increased by more than six times. The crude extract from ethanol precipitation has isolated by SuperdexTM75 and Sephadex G25 chromatography column in turn,and purified GTF substance has purified and analyzed by high performance liquid chromatography (HPLC) . The results has shown that the purified GTF can be obtained after the extraction of ammonia water, ethanol precipitation and two stages of column chromatography purification.Meanwhile, this study analyzes the structure of the GTF with the method of HPLC and nuclear magnetic resonance (NMR) , the result has shown that glutamic acid and aspartic acid is the main amino acids of GTF components. The chromium valence state of GTF has analyzed by the technology of HPLC and inductively coupled plasma mass spectrometry(ICP-MS ) . The result shows that there is only Cr3+ existing in the GTF structure.Finally, to establish insulin resistance cell model, Hep-G2 cells is incubated with insulin (10-6 mol/L) at 37? for 48 h in this study. This cell model of insulin resistance can maintain for 48 h. After successful modeling, this study apply the GTF and other chromium complexes into the Hep-G2 cells model of insulin resistance by measuring the content of glucose metabolism, triglyceride and non-esterified fatty acid, to evaluate the effect of GTF on the glucose and lipid metabolism. The glucose consumption can reach to 18.07 mmol/L,when the chromium content of GTF arrive at 1.0?g/mL. Compared with the control group increase by 5%, this result is significantly promoted the metabolism of glucose. In this paper, the same chromium content (1.0 ?g/mL) of GTF and other chromium complexes act on the insulin resistance cells, the results show that the chromium complexes can promote the metabolism of glucose in the cells. GTF has the most significantly effect on glucose metabolism in cells (P < 0.01) .In summary, this study screen for the yeast strain (YSI-3.7 ) with high chromium resistance by domestication of chromium. And we can obtain the purified GTF after extracte with ammonia, precipitate with 30% ethanol at 0?for 20 min and separate from two stages of chromatography column. Through the method of amino acid composition analysis and the test of NMR, we can know the main amino acid in GTF are aspartic acid and glatllnic acid; There is only one kind of chromium valence of (Cr3+) in GTF, which analyzed by the method of HPLC-ICP-MS. We also use high concentration of insulin to induce the Hep-G2 cells model of insulin resistance. Apply the GTF and other chromium complexes into the cell model to analyze the effect on glucose and lipid metabolism.