| ObjectiveInsulin resistance is a major characteristic of Type 2 diabetes, and is also commonly associated with obesity, hypertension and cardiovascular disease. It can be defined as a failure of target tissue to increase whole body glucose disposal in response to insulin. The elevated levels of plasma free fatty acids ( FFA) noted in patients with Type 2 diabetes and obesity may lead to insulin resistance.The latest developments in insulin signaling cascade show that insulin stimulated - glucose metabolism is mainly involved in phosphatidylinositol - 3 ki-nase (PI-3K) pathway. A fundamental event , following the binding of insulin to insulin receptor(IR) , is the activation of the intrinsic tyrosine kinase, leading to autophosphorylation. This in turn enables a number of docking proteins, including insulin receptor substrate - 1 (IRS - 1) , - 2, to be recruited. Activation of PI-3K through binding of its p85 adaptor subunit to IRS -1 raises the levels of several second messengers and hence activated protein kinase B (PKB). PKB has been implicated in the translocation of glucose transporter 4 (GLUT4) to the plasma membrane, which underlies insulin - stimulated glucose uptake. Therefore, any factor that alters the insulin signalling pathway and the translocation of GLUT4 may lead to the attenuation of insulin signal resulting in insulin resistance.Elevated FFA levels may exert multiple negative effects, depending on the differentenl target tissue. Therefore, the ungerlying intracellular signaling mechanisms that are affected by elevated FFA levels are best deciphered in vitro.3T3-L1 cells represent an useful model for studies of insulin action, particularly glucose metabolism, since these cells can differentiate from fibroblasts to adipocytes that are sensitive to insulin under appropriate culture conditions.Mesenchymal stem cells (MSC) is separated from bone marrow and contributes to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, adipose, etc.In this study, two kinds of adipocytes differentiated from 3T3-L1 cells and rat MSCs were cultured, stained and compared. After 3T3-L1 adipocytes were treated by palmitic acid (PA) or oleic acid(OA) , glucose uptake were measured to examine the FFA - induced cell model of insulin resistance. The protein expression of PI-3K, PKB, phosphated - PKB ( p - PKB) and GLUT4 were further detected; as well as the mRNA expression and the translocation of GLUT4.Method1. 3T3-L1 cells were differentiated into adipocytes in DMEM supplemented with dexmethason, IBMX, insulin andlO%FBS.2. A density gradient was used in the isolation and purification procedure of the rat MSCs. Detection of surface antigen of cultured MSCs were conveyed with immunocytochemistry method. MSCs were differentiated into adipocytes in DMEM supplemented with dexmethason, IBMX, insulin andlO%FBS.3. Oil Red 0 stain was performed to examine the differentiated adipocytes.4. 3T3-L1 adipocytes were incubated with PA or OA of different concentration for different duration.5. 3H - DG glucose uptake was measured by scintillation counting method, nonspecific 3H - DG uptake were measured in the presence of cytochalasin B.6. Total cell protein was extracted from the FFA - treated 313-LI adipocytes and controls.7. Western blot analysis was employed to detect the protein expression of PI-3K p85a, PKB -a, p -PKB and GLUT4.8. Total RNA was extracted from the FFA - treated 3T3-L1 adipocytes and controls. Semi - quantitative RT-PCR was used to detect the mRNA expression of GLUT4 with the house - keeping gene p - actin as intrinsic control.9. Indirect immunofluorescence was processed to observe the translocation of GLUT4 of the FFA - treated 3T3-L1 adipocytes and controls.10. Immunocytochemistry method was processed to quantitatively analysis the translation of GLUT4 of the FFA - treated 313-LI adipocytes and controls.Result1. 3T3-L1 cells were differentiated into adipocytes and approved by Oil Red 0 stain at a rate rang...
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