Effects Of High Glucose And Free Fatty Acid On Insulin Signaling Proteins And Expression Of OB-R In Rat Skeletal Muscle Cells

ObjectiveDiabetes mellitus is a complex disorder that arises from various causes and dramatically rapid increase worldwide. The development of insulin resistance may be a major risk factor characterized by impaired stimulation of glucose disposal into muscle. In the present study, deterioration of the insulin receptor -signaling pathway at different levels accounts for the evolution from an insulin -resistant state to type2 diabetes.In recently published statements, PI3 - K/PKB pathway has played a very important role in insulin - signaling transduction. The effects of insulin on glucose uptake are mediated via the insulin receptor which, following insulin binding, undergoes autophosphorylation on tyrosine residues activating its tyrosine kinase activity. The activated IR then phosphorylates IRS - 1 and other substrates. Tyrosine phosphorylated IRS - 1 then serves as a docking protein for PI3 - kinase, which is activated by this interaction. The serine phosphorylation cascade initiated by PI 3 - kinase involves activation of PI3K - dependent serine/ tyrosine kinases, and, in turn, Akt and results in the translocation of intracellu-lar GLUT4 to the cell surface. It is the increased amount of GLUT4 on the cell plasma membrane that results in an increased rate of glucose transport into the cell.Glucose toxicity caused by postprandial hyperglycaemia has become a hot topic recently. In particular, the same HbAlc values may have different frequency of fluctuation in blood glucose levels. It is important to distinguish between the effect of glucose fluctuations and sustained high glucose. Experimentaland clinical evidence indicates that which in turn causes endothelial dysfunction and eventually atherosclerosis.Obesity is a major risk factor for the development of insulin resistance, characterized by impaired stimulation of glucose disposal into muscle. The adi-pocyte - derived hormone, leptin, regulates food intake and systemic fuel metabolism ; Leptin receptors are expressed most abundantly in the brain but are also present in several peripheral tissues. The role of leptin in controlling energy homeostasis has thus far focused on brain receptors and neuroendocrine pathways that regulate feeding behaviour and sympathetic nervous system activity. Now we focus on mounting evidence that leptin s effects on energy balance are also mediated by direct peripheral actions on key metabolic organs such as skeletal muscle. Strong evidence indicates that peripheral leptin receptors regulate triacylg-lycerol storage. Inducible leptin expression in rat skeletal muscle cell may be an important biochemical link between increased availability of nutrients and leptin expression. However, its precise role in controlling glucose - regulatory pathways remains uncertain and requires further investigation. The close association between obesity and insulin resistance, and their progression to type2 diabetes is a serious health problem.In summary, although the exact mechanisms underlying insulin resistances are not yet known, available evidence indicates that both postprandial hypergly-caemia and hypertriglyceridaemia may contribute to microvascular and macrovas-cular complications in diabetic patients. To gain insight high glucose and high lipid on skeletal muscle, here we examine the direct effect of on insulin - stimulated glucose uptake in muscle cells and evaluate the changes in insulin signaling pathways after the exposure of muscle cells to high glucose, and we investigate effect of free fatty acid on gene and protein expression of leptin receptor in rat skeletal muscle ceEs. In particular, we studied glucose fluctuation produce adverse effects on muscle cells and glucose toxicity and lipotoxicity in diabetes and a series of underlying molecular mechanisms.1. Primary culture of rat skeletal muscle cells and identification Skeletal muscle cells were isolated from new bora Sprague - Dawley rats.Trypsin digestion and tissue sections culture methods were adopted. A density gradient was used in the isolation and purification procedure of the rat skeletal muscle cell. Detection of surface antigen of cultured muscle cells was measured with immunocytochemistry method.2. Effects of high glucose on glucose transport activity, and expression of insulin signaling proteins in muscle cellsIsolated rat muscle cells were cultured at different glucose concentrations and fluctuant frequency. Then 3H - DG glucose uptake was measured by scintillation counting method, nonspecific and general 3H - DG uptake were measured in the presence and absence of cytochalasin B; The phosphorylation of protein ki-nase B as well as the protein expression of p85 subunit of phosphatidylinositol 3 - kinase and PKB were measured.3. Construction of expression vector for eukaryotic cells(1) Large scale preparation of a shuttle vector; (1) Large scale purification of plasmid vector after culture in LB medium contained 100u,g/Amp and extraction plasmid DNA. (2)Added 1 - 2ul of lOmg/ml EtBr for original DNA solution, d) after ultracentifugation, collected of closed circular DNA from CsCl -Gradients Ethidium Bromide using a long needle. ? Added an eaqual volume of CsCl saturated isopropanol in order to remove EtBr. ? Dialysis and dissolved in TE. (2) Digestion with EcoRI/XhoI and dephosphorylated by alkaline phospha-tase( vector pcDNA4 EcoRI/XhoI/CIP). (3) Cloning of DNA in a shuttle vector (l)Extraction plasmid DNA harboring OB - R cDNA. (D Digestion of plasmid DNA with restriction enzymes EcoRI/XhoI, reaction with Klenow enzyme and purification of DNA along the gel using SUPREC? "01(D Iigation of plasmid vector of pcDNA4 EcoRI/XhoI/CIP and OB - R DNA fragment/Klenow treated (without insert DNA as control). (4) Transformation .picking up the correct colony , and minipreparation of DNA, and the recombinant plasmid was identifiedby endonuclease.4. Transfection and characterization of expression vector in eukaryotic cells Eukaryotic expression vector was constructed, which contained leptin receptor gene; Cell was cultured in different groups ( recombinant transfection, empty plasmid transfection, and non - transfection). The recombinant with OB- R cDNA or empty plasmid was transfected into primary skeletal muscle cells of the rats mediated by lipofection when the cultured cells were 80% confluen-cy;The recombinant plasmid was identified by endonuclease, and expression level of OB — R mRNA in transfected cells was assayed by RT - PCR.5. Transfection procedure for Iipofectamine 2000 Reagent(l)Cells were plated in a 24 — well plate in growth medium containing serum one day prior to transfection. @Cells were trypsinized, counted, and 5x10 cells per well resulting in cultures that were 80 - 90% confluent on the day of transfection. (|￡) Combine Diluted Iipofectamine 2000 Reagent and 0. 8|xg of plasmid DNA, mix and incubate 20 min. (J) Add complexes to cells in OPT! - MEMI medium (no serum). (|5) Cells were changed medium 6 hours later after the addition of Iipofectamine 2000 and DNA complexes.6. FFA act on gene and protein expression of OB - R in rat skeletal muscle cellsAfter muscle cells were incubated by palmitate ( PA 0.25mmol/L) or ole-ate ( OA 0.125mmol/L) with different time respectively, Western blot was used to assess the protein abundance of OB - R in rat skeletal muscle cells. Total RNA was extracted from the FFA - treated muscle cells and control groups. Semi - quantitative RT - PCR was used to detect the mRNA expression level of OB - R with the |3 - actin as intrinsic control.Results1. Skeletal muscle cells were isolated from new born Sprague - Dawley rats. Cultured cells stably expressed the surface antigen of actin.2. Muscle cell treated with different high glucose showed the impairment of the basal and insulin - induced increase in glucose uptake. The exposure to...