| IntroductionType 2 diabetes accurate pathogenesis at present is not extremely clear, but the nearly all 2 diabetes patients all have the insulin to resist (IR), even in normal physiological situation, also can have IR, like pregnancy and so on. Many studies have shown that skeletal muscle insulin resistance and type 2 diabetes is linked to one of the important shortcomings. Insulin combination with the insulin receptor play its physiological effects, and post-receptor abnormalities or abnormalities of insulin signal transduction is an important reason leading to insulin resistance. Protein kinase B (protein kinase B, PKB) is regulated by a number of post-receptor insulin signal transduction factor is one of the main function of the insulin signaling pathway, one of phosphatidylinositol 3-kinase (phosphatidylinositol 3-kinase, PI3K) downstream of important ways serine/threonine kinase. PKB can affect insulin signal to the actual adjustment glucose ingestion of raw sugar synthetic, sugar, insulin to resolve the metabolic response [1]. Rosiglitazone, as insulin-sensitizing agent can reduce the IR, increasing glucose uptake, but the role of specific steps are not very clear. LY294002 is a commonly used PI3K pathway inhibitor, can be transparent cells, specifically inhibit the PI3K, inhibited PI3K/PKB signaling pathways. In this study, palmitic acid-induced SD rat primary cultured skeletal muscle cells to form limb model of insulin resistance, testing the model in rat skeletal muscle cells in the rosiglitazone and LY294002, respectively, after the role of insulin-induced protein expression of PKB and phosphorylation, and explore whether Rosiglitazone improves insulin sensitivity through the pathway.MethodsIn this study, we used collagenase and trypsin digestion method of dual-SD rat primary cultured skeletal muscle cells in fetal mice, using differential Purification of adherent cells, using morphological analysis and immunofluorescence techniques to the cells double identification. With different concentrations of palmitic acid acting on the cells after induction compared with a glucose test kit (glucose oxidase) determination of glucose in the culture medium content analysis to ensure a stable model of insulin resistance. Establishment of a normal cell group (NG), insulin resistance group cells (IR), "the methadone intervention group (RO), and" LY 294,002 methadone in intervention group (RL).Groups were given rosiglitazone by 20μmol/L address 24h, 10μmol/L concentration of LY294002 treatment factors, such as pre-24h,, after incubation of 10-7 insulin, using western blotting to detect the protein expression of PKB and phosphorylation.Results1.10 days in vitro by mononuclear cells into the muscle long spindle cells gradually merge into a single multi-core tubular muscular tube, with the growth of sexual arrangement, or even contraction of muscle fibers may be;Immunofluorescence identification of cells, a-Sarcomeric Actin-specific antibody staining results showed that about 96% of the cells showed positive cytoplasm, nucleus and cytoplasm of peripheral red fluorescence,showed that cells cultured skeletal muscle cells.2.0.6mM palmitic acid in normal culture conditions, the role of primary cultured rat skeletal muscle cells in 12-24h,significantly inhibited insulin-stimulated skeletal muscle glucose transport under the condition that this model has physiological dose of insulin tolerance, and this inhibition was dose-time-dependent.3.PKB protein expression of RO group was lower than NG group, higher than the IR group, RL group was lower than RO group, but were not statistically significant. But the RO group PKB473 Ser phosphorylation level was significantly higher than the IR group (P＜0.05), RL Group PKB473 Ser phosphorylation level significantly lower than the RO group (P＜0.05).Conclusions1.Collagenase and trypsin using a double digestion and differential adhesion method can be purified in good condition and the high purity of the primary culture of skeletal muscle cells. 2.0.6mM palmitic acid in normal culture conditions, the role of primary cultured rat skeletal muscle cells in 12-24h, can form a stable model of insulin resistance in skeletal muscle cells.3.Palmitic acid-induced insulin resistance in skeletal muscle cell model, there is impaired activation of PKB. Rosiglitazone increases insulin resistance in skeletal muscle cells have the role of the sensitivity of the involvement of PKB and increases skeletal muscle cells by insulin signal transduction pathway of PI3K PKB 473 level of serine phosphorylation mediated. |
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