睾酮对C2C12骨骼肌细胞胰岛素敏感性的影响及分子机制研究

发布时间：2018-05-04 11:54

本文选题：睾酮 + C2C12细胞　；参考：《复旦大学》2007年博士论文

【摘要】： 第一部分睾酮对C2C12骨骼肌细胞胰岛素敏感性的影响目的：观察不同浓度的睾酮短时间和长时间处理对C2C12骨骼肌细胞胰岛素刺激的葡萄糖摄取能力的影响，探讨睾酮对C2C12骨骼肌细胞胰岛素敏感性的影响及与浓度和时间的关系。方法：诱导C2C12成肌细胞系分化为成熟的C2C12骨骼肌细胞，用高于生理浓度(10~(-5)、10~(-6)M)、近于生理浓度(10~(-9)、10~(-8)M)和低于生理浓度(10~(-11)M)的睾酮分别处理细胞30分钟和24小时，然后利用[~3H]-2-脱氧葡萄糖掺人法，观察上述各处理方式对C2C12骨骼肌细胞基础和胰岛素刺激的葡萄糖摄取能力的影响。结果：(1)短时间处理：用高于生理浓度(10~(-5)M)、近于生理浓度(10~(-9)M)的睾酮处理细胞30分钟，对于C2C12骨骼肌细胞基础的葡萄糖摄取(未加胰岛素刺激)没有影响；近于生理浓度(10~(-9)、10~(-8)M)和低于生理浓度(10~(-11) M)的睾酮和胰岛素共同作用于细胞30分钟，睾酮有增加胰岛素刺激的葡萄糖摄取的趋势，但差别没有统计学意义，皆P＞0.05；10~(-6)M(高浓度)的睾酮对胰岛素刺激的葡萄糖摄取的影响没有统计学意义，P＞0.05；但10~(-5)M(高浓度)的睾酮明显抑制胰岛素刺激的葡萄糖摄取，P=0.002。(2)长时间处理：生理浓度(10~(-9)、10~(-8)M)和低于生理浓度(10~(-11)M)的睾酮预处理C2C12骨骼肌细胞24小时，，对胰岛素刺激的葡萄糖摄取没有影响，P＞0.05；高于生理浓度(10~(-5)、10~(-6)M)的睾酮明显抑制胰岛素刺激的葡萄糖摄取，P分别为0.011，0.006。结论：睾酮可以影响C2C12骨骼肌细胞的胰岛素敏感性，高浓度的睾酮短时间和长时间处理细胞均能明显降低C2C12骨骼肌细胞的胰岛素敏感性，导致细胞胰岛素抵抗的发生。第二部分睾酮激活的ERK1／2导致C2C12骨骼肌细胞胰岛素抵抗的形成目的：睾酮引起细胞胰岛素抵抗的机制尚不清楚。本部分在第一部分研究的基础上观察睾酮短时间和长时间刺激对C2C12骨骼肌细胞ERK1／2的活性、IRS-1蛋白水平、IRS-1酪氨酸及丝氨酸磷酸化变化的影响，并探讨应用MEK抑制剂PD98059抑制ERK1／2磷酸化后上述蛋白表达和活性的变化；进一步运用葡萄糖摄取实验观察PD98059预处理对睾酮引起的C2C12骨骼肌细胞胰岛素敏感性下降的影响，探讨睾酮导致C2C12骨骼肌细胞胰岛素抵抗的可能机制。方法：(1)先用不同浓度的睾酮(10~(-11)、10~(-9)、10~(-8)、10~(-6)、10~(-5)M)作用于诱导分化成熟的C2C12骨骼肌细胞30分钟，然后选定用高浓度睾酮(10~(-5)M)处理细胞0、15、30、60分钟、2小时、24小时，应用western blotting方法检测细胞ERK1／2、p-ERK1／2的蛋白表达，了解睾酮对ERK1／2磷酸化的影响及与浓度和时间的关系。(2)用高浓度睾酮(10~(-5)M)分别作用于诱导分化成熟的C2C12骨骼肌细胞30分钟和24小时后用或不用胰岛素刺激，应用western blotting方法检测细胞IRS-1、IRS-1丝氨酸307位磷酸化、酪氨酸941位磷酸化蛋白的表达，观察睾酮处理细胞后对上述蛋白表达的影响。(3)检测应用MEK抑制剂PD98059预处理后睾酮对上述蛋白的表达的影响。(4)PD98059预处理C2C12骨骼肌细胞，进一步应用葡萄糖摄取实验研究睾酮(10~(-5)M)引起的C2C12骨骼肌细胞胰岛素刺激的葡萄糖摄取降低的影响。结果：(1)睾酮可以以浓度依赖的方式促进ERK1／2的磷酸化，10~(-8)M睾酮可以明显促进ERK1／2的磷酸化；10~(-5)M睾酮作用5分钟即可以使ERK1／2的磷酸化明显增强，作用2小时ERK1／2的磷酸化开始下降，作用24小时ERK1／2的磷酸化下降更明显，但仍高于对照组水平。(2)①空白对照组细胞基础状态下IRS-1的Ser~(307)和Tyr~(941)磷酸化水平均极低，胰岛素刺激使IRS-1 Tyr~(941)磷酸化水平明显升高，P＜0.05。②高浓度睾酮(10~(-5)M)作用30分钟可使IRS-1的Ser~(307)磷酸化增加，P＜0.05；胰岛素刺激的IRS-1的Tyr~(941)磷酸化水平明显低于对照组，P＜0.05；总IRS-1水平在睾酮处理30分钟后没有改变，P＞0.05；②睾酮(10~(-5)M)作用24小时可见IRS-1蛋白水平降低，P＜0.05；此时IRS-1的Ser~(307)磷酸化增加不明显，胰岛素可以诱导IRS-1的Tyr~(941)磷酸化水平，P＜0.05。(3)PD98059预处理可以抑制睾酮诱导的ERK1／2的磷酸化，短时间睾酮刺激的IRS-1的Ser~(307)磷酸化、睾酮对胰岛素刺激的IRS-1的Tyr~(941)的抑制作用可以被PD98059逆转，P＜0.05；PD98059对睾酮长时间处理IRS-1水平减少没有作用，P＞0.05。(4)睾酮对胰岛素刺激的葡萄糖摄取的抑制作用可被PD98059部分阻断，P＜0.05；但仍低于正常对照组水平，P＜0.05。结论：高浓度睾酮短时间处理C2C12骨骼肌细胞可能通过促进ERK1／2的磷酸化增强，因而与胰岛素促代谢通路交叉对话，直接或间接磷酸化IRS-1的Ser~(307)位点，使其磷酸化过度，进而导致胰岛素刺激的IRS-1的Tyr~(941)磷酸化水平下降，引起胞内信号传导障碍而导致细胞胰岛素抵抗的发生，短时间睾酮作用于C2C12骨骼肌细胞对胰岛素信号的影响是影响磷酸化水平而不是改变蛋白水平；睾酮长时间处理C2C12骨骼肌细胞主要通过IRS-1蛋白水平的降低从而导致胰岛素抵抗的发生；高浓度睾酮尚存在其它途径引起细胞胰岛素抵抗，关于其机制有待进一步探讨。
[Abstract]:Part one effect of testosterone on insulin sensitivity in C2C12 skeletal muscle cells
Objective: To observe the effect of different concentrations of testosterone on glucose uptake ability of insulin stimulation in C2C12 skeletal muscle cells for a short and long time treatment, and to explore the relationship between testosterone on insulin sensitivity of C2C12 skeletal muscle cells and its concentration and time.
Methods: the C2C12 myoblast line was induced to differentiate into mature C2C12 skeletal muscle cells, and the cells were treated with the physiological concentration (10~ (-5), 10~ (-6) M), the physiological concentration (10~ (-9), 10~ (-8) M) and the testosterone below the physiological concentration of 30 minutes and 24 hours respectively. Effects of formula on basal muscle cell and glucose uptake capacity of C2C12 stimulated insulin.
Results: (1) short time treatment: testosterone treated cells with higher physiological concentration (10~ (-5) M), near physiological concentration (10~ (-9) M), had no effect on glucose uptake (without insulin stimulation) on the basis of C2C12 skeletal muscle cells, near the physiological concentration (-9), 10~ (-8) M) and testosterone and insulin below the physiological concentration. For 30 minutes, testosterone had a tendency to increase glucose uptake by insulin stimulation, but the difference was not statistically significant, all P > 0.05; 10~ (-6) M (Gao Nongdu) testosterone had no significant effect on glucose uptake by insulin stimulation, P > 0.05; but 10~ (-5) M (Gao Nongdu) testosterone significantly inhibited insulin stimulation Glucose uptake, P=0.002. (2) long time treatment: physiological concentration (10~ (-9), 10~ (-8) M) and testosterone preconditioning for C2C12 skeletal muscle cells below the physiological concentration (10~ (-11) M) for 24 hours, there was no effect on glucose uptake by insulin stimulation, P > 0.05; testosterone that was higher than the physiological concentration inhibited insulin stimulated grapes. Sugar intake, P is 0.011,0.006., respectively
Conclusion: testosterone can affect insulin sensitivity of C2C12 skeletal muscle cells. High concentration of testosterone in short time and long time treated cells can significantly reduce the insulin sensitivity of C2C12 skeletal muscle cells and lead to cell insulin resistance.
The second part of testosterone activated ERK1 / 2 resulted in the formation of insulin resistance in C2C12 skeletal muscle cells.
Objective: the mechanism of testosterone induced insulin resistance is not clear. On the basis of the first part of this study, the activity of ERK1 / 2 in C2C12 skeletal muscle cells, the level of IRS-1 protein, the changes of IRS-1 tyrosine and serine phosphorylation were observed on the basis of the first part of the first part of the study, and the application of MEK inhibitor PD98059 to the inhibition of ERK1 was also discussed. The changes in the expression and activity of these proteins after 2 phosphorylation, and the effect of PD98059 pretreatment on the decrease of insulin sensitivity in C2C12 skeletal muscle cells induced by testosterone, and the possible mechanism of testosterone induced insulin resistance in C2C12 skeletal muscle cells were further investigated by glucose uptake.
Methods: (1) first use different concentrations of testosterone (10~ (-11), 10~ (-9), 10~ (-8), 10~ (-6), 10~ (-5) M) to induce the differentiation of mature C2C12 skeletal muscle cells for 30 minutes, and then selected high concentration testosterone to treat cells for minutes, 2 hours, and 24 hours. To understand the effect of testosterone on ERK1 / 2 phosphorylation and the relationship between testosterone and concentration and time. (2) using high concentration of testosterone (10~ (-5) M) to induce differentiation of mature C2C12 skeletal muscle cells respectively for 30 minutes and 24 hours with or without insulin stimulation, Western blotting square method was used to detect cell IRS-1, IRS-1 serine 307 phosphorylation, tyrosine 9 The expression of 41 phosphorylated proteins and the effect of testosterone treated cells on the expression of the above protein. (3) the effect of testosterone on the expression of the above protein was detected by MEK inhibitor PD98059 pretreatment. (4) PD98059 pretreated C2C12 skeletal muscle cells and further applied glucose uptake to study the skeletal muscle fine of C2C12 (10~ (-5) M). Impaired glucose uptake stimulated by insulin.
Results: (1) testosterone can promote phosphorylation of ERK1 / 2 in a concentration dependent manner. 10~ (-8) M testosterone can obviously promote the phosphorylation of ERK1 / 2; 10~ (-5) M testosterone can increase the phosphorylation of ERK1 / 2 obviously, and the phosphorylation of ERK1 / 2 in 2 hours begins to decrease, and the phosphorylation of ERK1 / 2 in 24 hours is more obvious. But it was still higher than that of the control group. (2) the level of Ser~ (307) and Tyr~ (941) phosphorylation of IRS-1 in the blank control group was very low, and the level of phosphorylation of IRS-1 Tyr~ (941) was significantly increased by insulin stimulation, and P < 0.05. (10~ (-5) M) could increase the IRS-1 Ser~ (307) phosphorylation in 30 minutes. The level of Tyr~ (941) phosphorylation of stimulated IRS-1 was significantly lower than that of the control group, P < 0.05; the total IRS-1 level did not change after 30 minutes of testosterone treatment, P > 0.05; and the effect of testosterone (10~ (-5) M) showed that the level of IRS-1 protein decreased, P < 0.05; IRS-1 Ser~ (307) phosphorylation was not significantly increased at this time, and insulin could induce the phosphorylation (941) of phosphorus. The level of acidification, P < 0.05. (3) PD98059 pretreatment could inhibit the phosphorylation of testosterone induced ERK1 / 2, Ser~ (307) phosphorylation of testosterone stimulated IRS-1, and the inhibitory effect of testosterone on IRS-1 Tyr~ (941) stimulated by insulin can be reversed by PD98059, P < 0.05; PD98059 has no effect on the reduction of IRS-1 level by testosterone for a long time. 0 The inhibitory effect of.05. (4) testosterone on insulin stimulated glucose uptake was partially blocked by PD98059, P < 0.05, but it was still lower than that of normal control group, P < 0.05.
Conclusion: high concentration of testosterone in short time treatment of C2C12 skeletal muscle cells may be enhanced by promoting the phosphorylation of ERK1 / 2, thus cross dialogue with insulin metabolism pathway, directly or indirectly phosphorylate the Ser~ (307) site of IRS-1, resulting in excessive phosphorylation, resulting in a decrease in the level of Tyr~ (941) phosphorylation of insulin stimulated IRS-1 and the cause of the cell. The effect of short time testosterone on insulin signaling in C2C12 skeletal muscle cells affects the level of phosphorylation rather than changes in protein levels; testosterone for long time treatment of C2C12 skeletal muscle cells is mainly caused by the decrease of the level of IRS-1 protein to lead to insulin resistance. There are still other ways to induce insulin resistance in high concentration testosterone.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341

【参考文献】