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Effect of Leucine on Glucose Uptake in Primary Cultured Skeletal Muscle Cells and the Underlied Mechanisms Mediated by mTOR

Author: LiuRui
Tutor: YangXueFeng
School: Huazhong University of Science and Technology
Course: Nutrition and Food Hygiene
Keywords: leucine glucose uptake Wortmannin Rapamycin AKT mTOR
CLC: R151
Type: Master's thesis
Year: 2011
Downloads: 40
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Abstract

The incidence of type 2 diabetes mellitus and metabolic syndrome is increasing rapidly worldwide. Insulin resistance plays an important pathophysiological role and is a key feature of above diseases. Dietary intervention has been considered as an effective measure in prevention and treatment of insulin resistance.Accumulating evidence shows that an essential amino acid-leucine plays a key regulatory role in numerous metabolic processes including protein synthesis, energy homeostasis and insulin-signaling pathway. The role of leucine in insulin resistance has been greatly paid attention. Several reports have suggested that leucine have an important effect on decreasing high fat-induced hyperglycemia and improving insulin sensitivity in adipocytes. Clinical studies in obesity and type 2 diabetes revealed that consumption of leucine reduced postprandial insulin and blood glucose level, had benefical effects on glucose homeostasis and insulin resistance. However, the contribution of leucine on insulin resistance and the underlying mechanisms are still not well documented. Some results remain controversial..Skeletal muscle is one of the major targets responsible for insulin physiological function. After a meal, approximately two third of postprandial glucose is taken up by skeletal muscle. Disregulation of insulin-stimulated glucose disposal in skeletal muscle plays a crucial role in insulin resistance. Leucine is the most potent activator of the mammalian target of rapamycin (mTOR), which can exerts multiple roles at different levels of insulin-signaling pathway. Protein kinase B (PKB/AKT) is the downstream effecter of insulin signaling. Our study aimed to determine the effect of leucine on glucose uptake of primary cultured skeletal muscle cells. At the same time , we investigated the protein expression and phosphorylation level of AKT, mTOR to explore the underlying mechanisms . The results were summarized as follows:PartⅠEffect of leucine on glucose uptake in primary cultured skeletal muscle cells of ratsObjective: To examine the effects of leucine on glucose uptake in primary cultured skeletal muscle cells of rats, and determine the optimal concentration and stimulation time.Method: Exposure to different doses of leucine (0, 0.4, 2, 4, 8 mmol/l) with or without insulin(100 nmol/l) for 80 min or 45 min, 2-deoxyglucose uptake of skeletal muscle cells was determined by a nonradioisotope enzymatic microplate assay.Results: Under insulin-free conditions, leucine showed little or no effect on glucose uptake. The effect of leucine on glucose uptake was time- and does- dependent. In the presence of insulin, the increase in 2-deoxyglucose uptake was significant in primary skeletal muscle cells when incubated at 2 mmol leucine for 45 min, But the effects of leucine decreased with prolong of exposure time.Conclusion: Leucine can time- and does- dependently improve insulin sensitivity in primary cultured skeletal muscle cells.PartⅡThe mechanisms of leucine promoting glucose uptake in primary cultured skeletal muscle cellsObjective: To examine the effects of leucine on glucose uptake in Wortmannin and/or Rapamycin treated primary cultured skeletal muscle cells of rats. Then we further detect the protein expression and phosphorylation level of AKT, mTOR to explore the possible molecular mechanisms underlying its effect. Methods: 1. Cells pretreated with Wortmannin or /and Rapamycin for 30 min were incubated with 2 mmol/L leucine or/and insulin for 45min, then 2-deoxyglucose uptake of skeletal muscle cells was determined.2. Western blot was used to detect AKT , phosphoAKT (Ser473) , mTOR and phospho mTOR (Ser2448) expression after cells were treated as above described.Results: 1. Either Wortmannin or Rapamycin significantly reduced insulin-induced glucose uptake (P<0.05). Leucine diminished the inhibitory effect of Wortmannin and Rapamycin on insulin-induced glucose uptake, insulin-induced glucose transport was increased by 61% and 71% respectively when incubated with leucine. Leucine showed little effect on glucose uptake when cells were pretreated with Wortmannin and Rapamycin,it was only increased by 21%.2. In the presence of insulin, leucine had no significant effect on AKT when PI-3K was blocked. Wortmannin inhibits insulin-induced AKT phosphorylation on ser473 sites (P<0.05), this inhibitory effect of Wortmannin was partially reversed in the presence of leucine. (P<0.05). Rapamycin has little effect on insulin-induced pSer473AKT even in the presence of insulin or/and leucine. However, Rapamycin significantly prevented AKT phosphorylation stimulated by insulin and leucine with Wortmannin. (P<0.05). 3. Wortmannin inhibits insulin-induced mTOR and pSer2448 mTOR expression (P<0.05). Leucine can rescue their expression despite the presence of Wortmannin(P<0.05), while Rapamycin prevents this positive effect of leucine on mTOR when insulin signaling is inhibited by Wortmannin.Conclusion: Leucine can improve insulin sensitivity of skeletal muscle cells. The underlying mechanism is partly independent of PI-3K insulin signaling. It is associated with the phosphorlation of AKT and is dependent of mTORC1 signaling pathway.

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