| Insulin resistance (IR) is a phenomenon that the requirement of insulin to maintain normoglycaemia is higher than normal concentrations of insulin, with lower sensibility or depressed responsibility of insulin's target tissue towards endogenous or exogenous insulin. Long-term insulin resistance can cause a series of closely coherent clinical abnormities, such as obesity (especially visceratonia obesity), glyco-accommodate damage, type 2 diabetes, adiposis hepatica, high blood pressure, coronary artery disease, lipid metabolism disorder and so on.The principal function of insulin is to maintain the glucose homeostasis in the body. Liver, fatty tissue and muscle tissue are the three target organs of insulin. When sensitivity of organism towards insulin degrades, more glucose will release from liver and synthesis of amylon will decline in liver. Thus, the uptake of glucose by the peripheral tissues will decrease. When sensitivity of periphery tissue towards insulin has degraded for a long time, fatty deposition can deposit translocationally in muscle tissue and liver. Skeletal muscle, a periphery target tissue, is also an important organism for glycometabolism and one of the main causes for insulin resistance. Because of the complexity of insulin resistance, there are ponderous disputes for the mechanism of insulin resistance.The relationship between free fatty acid and insulin resistance is very close. Fatty acid is primary substance of man's body fat and adipoid (such as phosphatide, glucolipid, steroid) which have very important physiologic function. Fatty acid can be divided into saturated fatty acid and unsaturated fatty acid, according to the existence of double bond between carbo-hydrogen bonds. FFA-induced insulin resistance often accompany with abnormity of many elements of signal transmission (such as the decrease in the capability for insulin to bind with its receptor; the decline in activity of insulin receptor and substrate of insulin receptor; the depression in expression of glucose transporter). Those above can all be regarded as possible mechanism of insulin resistance induced by FFA. However, the machanism for the change of insulin molecule signal or its upper mechanism is unknown. Recently, some researchers propose that PI3-K is not the only way that regulates GLUT4 translocation and glucose uptake. Along with the discovery of lipid rafts, researchers begin to pay close attention towards effect of Small G protein , TC10.Lipid rafts are microdomains which contain distinct, characteristic proteins involved in cellular membrane. The main compositions of fatty substance in lipid rafts are cholesterin, phosphosphingolipid and ganglioside. There is long saturated fatty acid chain in phosphosphingolipid, which can form a kind of lipid ordered phase when interacting with cholesterin. This structure (lipid ordered phase) can be more stable compared with area around. This structure can be called Liquid-ordered which is something between liquid crystal and fluid without order. Some researches show that lipid rafts participate in the intra-cellular transmembrane transport of fat acid.Skeletal muscle, a major tissue of energy metabolism in body, plays an important role in insulin resistance, because the metabolism of most of carbohydrate and fat in the body occurs in it.The object of this study is primarily cultured skeletal muscle cells. Through observing the effect of palmic acid (PA) and insulin on glucose transport in primary cultured skeletal muscle cells, we found a feasible condition to establish a cell model of insulin resistance in primary skeletal muscle cells. On the foundation of those work, we further our research on the different expression of signal protein GLUT4, TC10, cbl mRNA. We provided an investigation on the mechanism of insulin resistance in skeletal muscle. Furthermore, we also investigate the effect of lipid rafts in insulin signal conduction and provide a new point to investigate insulin resistance.Methods1. Cell culture: this study culture and identify primary skeletal muscle cells of new born SD rats.2. Establishment of insulin resistance model in primary skeletal muscle cells: according to the treating factors, skeletal muscle cells were divided into different groups: normal control group, insulin groups (5×10-7,1×10-7 M) and PA groups (0.25,0.4,0.6,1mM). The concentration of glucose were observed in culture medium at different time points (2h,6h,12h,24h) by Glucose Detection Kit(GOD-POD). Then, in each group, some cells were treated with insulin for 20 min, the others not. We investigated the different ability of glucose uptake in different groups with or without insulin and estimated the efficacy of insulin. The concentration of glucose was assayed by Glucose Detection Kit (GOD-POD).3. Detection of the abundance of cbl,TC10,GLUT4 mRNA in cell model of insulin resistant skeletal muscle cells which are induced by PA and insulin: according to the different culturing condition, skeletal muscle cells were divided into normal control group, insulin groups (5×10-7,1×10-7 M) , groups with different PA density(0.25,0.4,0.6mM,t=12), groups of the same PA concentration but different time(0.6mM, 6h,12h,24h). Using qPCR, we detected the relative abundance of cbl,TC10,GLUT4mRNA in those groups.4. Influence of lipid rafts towards the conduction of insulin signal: lipid rafts were covered withβ-CD and the transmission electron microscope was used to investigate the change of lipid rafts. The normal cell group and the covered group were stimulated with insulin for 20 min. The relative abundance of cbl,TC10,GLUT4 mRNA were detected in the two groups.Results1. When primary skeletal muscle cells were successfully cultured, the beating of myotube could be seen under microscope, the dyeing result in positive.2. When the cells were incubated in cultured medium with Palmic Acid (PA,≥0.6 mM) for 12 h, or with 5×10-7 M insulin for 24 h, the glucose transport of cultured medium was significantly lower than that in control group. The degrading of physiological effect indicates that hexadecanoic acid inducing groups became tolerant towards insulin.3. The results of observation by qPCR are as follows: low-insulin density group had higher relative abundance of cbl,TC10,GLUT4 mRNA than the normal group. The group induced by 5×10-7 M insulin had a significant lower relative abundance of cbl,TC10,GLUT4 mRNA. Different density or different time of PA inducing have a lower relative abundance of cbl, TC10,GLUT4 mRNA and have time-dose dependence.4.β-CD covered skeletal muscle cells showed blocked lipid rafts on membrance under electron microscope. Using qPCR, we can see that lipid raft block group have a lower relative abundance of cbl,TC10,GLUT4 mRNA than normal group.ConclusionsInsulin resistance model in primary skeletal muscle cells can be established by culturing primary skeletal muscle cells in high concentration of PA or insulin. GLUT4 as rate limiting protein for skeletal muscle cells'glucose uptake is not only impacted by PI3K access, but also influenced by cbl,TC10 signal. PA inducing group have a dose- and time-dependent manner. When lipid drat are covered, relative abundance of cbl,TC10,GLUT4 mRNA can be apparently depressed.
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