Effects And Mechanisms Of Human Resistin Gene Transfection In Vitro On Glucose Uptake

For a long time, adipose tissue has been known as an inert storage organ. Only in the hunger needs of the body, fat tissue becomes active and release energy. However, in 1994, Friedman cloned the gene - leptin (Leptin) and found that it could inhibit appetite and reduced weight. Since then, people started to study the endocrine function of adipose tissue. With the foundings of other important hormones and cytokines from adipose tissue, including adiponectin, IL-8 and so on, adipose tissue is considered as the endocrine organ.Resistin, a small cysteinerich protein, belongs to a gene family found in inflammatory zone (FIZZ) or found in resistin-like molecule (RELM). It was found by Steppan in 2001 in white adipose tissue in mice and is considered as a link between obesity and insulin resistance. Its founding provides a new thinking to study obesity and insulin resistance.Studies in vitro and in vivo demonstrated that high concentration of resistin impaired sugar tolerance, decreased insulin sensitivity, and increased liver sugar export, which caused insulin resistance in rodents. Serum resistin levels were elevated in obesity genetic models (ob/ob and db/db) as well as in a diet-induced model of diabetes and obesity. In the contrast, reduction of resistin improved glucose homeostasis and insulin sensitivity. The antibody against resistin improved insulin sensitivity of diet-induced diabetes and obesity in a mouse model. These studies suggest that resistin is a potential link between obesity and insulin resistance in rodents. However, it is controversial in the studies evaluating resistin expression related with obesity and type 2 diabetes in humans. Both many existing clinical evidence and experimental results in vitro and in vivo support that resistin plays an important role to induce insulin resistance. However, many studies have reported that resistin has no correlation to obesity and type 2 diabetes. And the current understanding of the resistin is more based on the research of mice than that of human. It is necessary to investigate the effects of human resistin (hR) on target cells of insulin action.The role of resistin on the three main target cells of insulin action- fat cells, muscle cells and liver cells were studied here. Resistin impacts glycometabolism by endocrine, autocrine and paracrine ways. However, some reports considered that the activity of recombinant human resistin may be lower than that of the endogenous one, so we constructed human resistin eukaryotic expression vector to explore the function of it. There are five parts in our investigation. 1. The construction of human resistin expression vectors. 2. The expression vectors were transfected in 3T3-L1 and COS7 cells. (1) To induce differentiation of 3T3-L1 cells transfected with PcDNA-3.1(+) or PcDNA-hR and perform glucose uptake assays using [~3H] 2-deoxy-D-glucose. The mRNA levels of IR and GLUT4 were evaluated by semi-quantitative RT-PCR. The cell proliferation, cell cycle and differentiation in the absence of presence of Metformin were examined. (2) Incubated 3T3-L1 adipocytes and human adipocytes using the conditioned medium of COS7 cells and perform glucose uptake assays . 3. The expression vectors were transfected in C2C12 and COS7 cells. (1) To induce differentiation of C2C12 transfected with PcDNA-3.1(+) or PcDNA-hR and perform glucose uptake assays using [~3H] 2-deoxy-D-glucose. The mRNA levels of IR and GLUT4 were evaluated by semi-quantitative RT-PCR. The cell proliferation, cell cycle and expression of differentiation Marker including Desmin and Myoglobin were examined. ( 2 ) Incubated C2C12 myotubes using the conditioned medium of COS7 cells and perform glucose uptake assays. 4. To examine the expression of resistin in human hepatic tissue and hepatocytes. The recombinant plasmids were stably transfected into HepG2 cells to make human resistin overexpression. The uptake of [~3H] 2-deoxy-D-glucose was used to observe glucose transport in the absence or presence of rosiglitazone or Metformin. The effect of human resistin on glycogen synthesis was observed by Periodic acid-Schiff (PAS) staining. The mRNA levels of genes related insulin signal transduction pathway were evaluated by semi-quantitative RT-PCR. 5. The cells transfected with human resistin were treated with Metformin to investigate the effect of Metformin on action and expression of resistin. The results are as follows.1. The resistin gene eukaryotic expression vectors were constructed using genetic engineering recombination technique. It was determined by DNA sequencing analysis that resistin gene cDNA has been inserted into pcDNA3.1 (+).The direction and sequence is entirely correct. This established the experimental basis for future research on function of resistin.2. We transfected resistin in four kinds of cell lines including 3T3-L1, C2C12, HepG2 and COS7 cells. It was confirmed that human resistin was expressed or overexpressed in the four kinds of cells' endochylema and homologus supernatant by ELISA, semiquantitative RT-PCR analysis, Western blot and immunocytochemistry staining methods. This supplied experimental evidence for the further essay to study the effect of resistin on target cells.3. After the 3T3-L1 cells were transfected with human resistin, cell proliferation was dramatically stimulated, S phase cells were increased whereas G0/G1 phase cells were decreased. It also decreased mature adipocytes and suppressed the expression of adipogenic markers including C/EBP-αand PPAR-γgenes. Simultaneously, the glucose uptake and expression of IR and GLUT4 were decreased in 3T3-L1 adipocytes transfected with human resistin and insulin sensitivity was impaired. However, human resistin condition medium from COS7 cells did not affect glucose uptake and insulin sensitivity of 3T3-L1 adipocytes and human adipocytes. The results indicated that human resistin maybe impaired insulin sensitivity of adipocytes through promoting preadipocytes proliferation and suppressing adipogenesis.4. After the C2C12 cells were transfected with human resistin, cell proliferation was dramatically stimulated, S phase cells were increased whereas G0/G1 phase cells were decreased. It also decreased diameter of myotubes and suppressed the expression of myogenic markers including Desmin and Myoglomin genes. Simultaneously, the glucose uptake and expression of IR and GLUT4 were decreased in C2C12 myotubes transfected with human resistin and insulin sensitivity was impaired. However, human resistin condition medium from COS7 cells did not affect glucose uptake and insulin sensitivity of C2C12 myotubes. The results indicated that human resistin maybe impaired insulin sensitivity of myocytes through promoting myoblasts proliferation and suppressing myogenesis.5. It was confirmed by semiquantitative RT-PCR analysis and Western blot analysis that human resistin is expressed in HepG2 cells. After the HepG2 cells were transfected with human resistin, the glucose uptake and glycogen synthesis was decreased and insulin sensitivity was impaired. That the decrease of the expression of insulin receptor substrate 2 (IRS-2) and c-cbl associated protein (CAP) and the increase of the expression of glycogen synthetase kinase 3β(GSK-3β) contributed to the results, which suggested that human resistin blocked the two insulin signal transduction pathways of PI-3K/Akt and of CAP/c-cbl in hepatocytes.6. The adipogenesis of the 3T3-L1 cells transfected with human resistin was suppressed. However, after treated with Metformin, the adipocytes population was increased, as well as the expression of C/EBP-αand PPAR-γgenes, which suggested Metformin reversed the suppression of human resistin on adipogenesis. Moreover, normal 3T3-L1 cells were treated with Metformin and the results indicated that Metformin promoted adipogenesis of 3T3-L1 cells and the effect was dose-dependent. The insulin sensitivity of HepG2 cells transfected with human resistin decreased. However, after treated with Metformin, the insulin-stimulated glucose uptake increased and expression of insulin signaling pathway relevant genes including IRS-2, GSK-3βand CAP were accommodated. The results suggested that Metformin againsted the action of human resistin inducing insulin resistance. Further investigation indicated that Metformin downregulated expression of human resistin in hepatocytes.In a word, our results indicated that three insulin target cells are all targets of human resistin and human resistin maybe induced periphery insulin resistance and hepatic insulin resistance by different mechanisms. That hR maybe had no direct effect on insulin sensitivity of adipocytes and myocytes. However, it suppressed adipogenesis and myogenesis, and stimulated proliferation of preadipocytes and myoblasts, so that the population of mature adipocytes and myocytes, as well as expression of IR and GLUT4 cut down, which led to impairment of insulin sensitivity. As to hepatocytes, human resistin impaired directly insulin sensitivity through downregulating the two insulin signaling pathway PI/3K and CAP/cbl-1. In addition, the results indicated for the first time that Metformin reversed inhibition of resistin on adipogenesis, downregulated expression of resistin in hepatocytes and improved insulin resistance induced by resistin. On the whole, our results revealed relationship between human resistin and insulin resistance, which demonstrated human resistin is close correlated to obesity and insulin resistance. Moreover, the results indicated effect of human resistin on insulin signal transduction pathway on cell level and molecular level. This establishs groundwork for further study medicine targeting on resistin curing T2DM and obesity.