本文選題：睪酮 + C2C12細(xì)胞��； 參考：《復(fù)旦大學(xué)》2007年博士論文

【摘要】： 第一部分睪酮對C2C12骨骼肌細(xì)胞胰島素敏感性的影響 目的：觀察不同濃度的睪酮短時間和長時間處理對C2C12骨骼肌細(xì)胞胰島素刺激的葡萄糖攝取能力的影響，探討睪酮對C2C12骨骼肌細(xì)胞胰島素敏感性的影響及與濃度和時間的關(guān)系。 方法：誘導(dǎo)C2C12成肌細(xì)胞系分化為成熟的C2C12骨骼肌細(xì)胞，用高于生理濃度(10~(-5)、10~(-6)M)、近于生理濃度(10~(-9)、10~(-8)M)和低于生理濃度(10~(-11)M)的睪酮分別處理細(xì)胞30分鐘和24小時，然后利用[~3H]-2-脫氧葡萄糖摻人法，觀察上述各處理方式對C2C12骨骼肌細(xì)胞基礎(chǔ)和胰島素刺激的葡萄糖攝取能力的影響。 結(jié)果：(1)短時間處理：用高于生理濃度(10~(-5)M)、近于生理濃度(10~(-9)M)的睪酮處理細(xì)胞30分鐘，對于C2C12骨骼肌細(xì)胞基礎(chǔ)的葡萄糖攝取(未加胰島素刺激)沒有影響；近于生理濃度(10~(-9)、10~(-8)M)和低于生理濃度(10~(-11) M)的睪酮和胰島素共同作用于細(xì)胞30分鐘，睪酮有增加胰島素刺激的葡萄糖攝取的趨勢，但差別沒有統(tǒng)計學(xué)意義，皆P＞0.05；10~(-6)M(高濃度)的睪酮對胰島素刺激的葡萄糖攝取的影響沒有統(tǒng)計學(xué)意義，P＞0.05；但10~(-5)M(高濃度)的睪酮明顯抑制胰島素刺激的葡萄糖攝取，P=0.002。(2)長時間處理：生理濃度(10~(-9)、10~(-8)M)和低于生理濃度(10~(-11)M)的睪酮預(yù)處理C2C12骨骼肌細(xì)胞24小時，，對胰島素刺激的葡萄糖攝取沒有影響，P＞0.05；高于生理濃度(10~(-5)、10~(-6)M)的睪酮明顯抑制胰島素刺激的葡萄糖攝取，P分別為0.011，0.006。 結(jié)論：睪酮可以影響C2C12骨骼肌細(xì)胞的胰島素敏感性，高濃度的睪酮短時間和長時間處理細(xì)胞均能明顯降低C2C12骨骼肌細(xì)胞的胰島素敏感性，導(dǎo)致細(xì)胞胰島素抵抗的發(fā)生。 第二部分睪酮激活的ERK1／2導(dǎo)致C2C12骨骼肌細(xì)胞胰島素抵抗的形成 目的：睪酮引起細(xì)胞胰島素抵抗的機制尚不清楚。本部分在第一部分研究的基礎(chǔ)上觀察睪酮短時間和長時間刺激對C2C12骨骼肌細(xì)胞ERK1／2的活性、IRS-1蛋白水平、IRS-1酪氨酸及絲氨酸磷酸化變化的影響，并探討應(yīng)用MEK抑制劑PD98059抑制ERK1／2磷酸化后上述蛋白表達和活性的變化；進一步運用葡萄糖攝取實驗觀察PD98059預(yù)處理對睪酮引起的C2C12骨骼肌細(xì)胞胰島素敏感性下降的影響，探討睪酮導(dǎo)致C2C12骨骼肌細(xì)胞胰島素抵抗的可能機制。 方法：(1)先用不同濃度的睪酮(10~(-11)、10~(-9)、10~(-8)、10~(-6)、10~(-5)M)作用于誘導(dǎo)分化成熟的C2C12骨骼肌細(xì)胞30分鐘，然后選定用高濃度睪酮(10~(-5)M)處理細(xì)胞0、15、30、60分鐘、2小時、24小時，應(yīng)用western blotting方法檢測細(xì)胞ERK1／2、p-ERK1／2的蛋白表達，了解睪酮對ERK1／2磷酸化的影響及與濃度和時間的關(guān)系。(2)用高濃度睪酮(10~(-5)M)分別作用于誘導(dǎo)分化成熟的C2C12骨骼肌細(xì)胞30分鐘和24小時后用或不用胰島素刺激，應(yīng)用western blotting方法檢測細(xì)胞IRS-1、IRS-1絲氨酸307位磷酸化、酪氨酸941位磷酸化蛋白的表達，觀察睪酮處理細(xì)胞后對上述蛋白表達的影響。(3)檢測應(yīng)用MEK抑制劑PD98059預(yù)處理后睪酮對上述蛋白的表達的影響。(4)PD98059預(yù)處理C2C12骨骼肌細(xì)胞，進一步應(yīng)用葡萄糖攝取實驗研究睪酮(10~(-5)M)引起的C2C12骨骼肌細(xì)胞胰島素刺激的葡萄糖攝取降低的影響。 結(jié)果：(1)睪酮可以以濃度依賴的方式促進ERK1／2的磷酸化，10~(-8)M睪酮可以明顯促進ERK1／2的磷酸化；10~(-5)M睪酮作用5分鐘即可以使ERK1／2的磷酸化明顯增強，作用2小時ERK1／2的磷酸化開始下降，作用24小時ERK1／2的磷酸化下降更明顯，但仍高于對照組水平。(2)①空白對照組細(xì)胞基礎(chǔ)狀態(tài)下IRS-1的Ser~(307)和Tyr~(941)磷酸化水平均極低，胰島素刺激使IRS-1 Tyr~(941)磷酸化水平明顯升高，P＜0.05。②高濃度睪酮(10~(-5)M)作用30分鐘可使IRS-1的Ser~(307)磷酸化增加，P＜0.05；胰島素刺激的IRS-1的Tyr~(941)磷酸化水平明顯低于對照組，P＜0.05；總IRS-1水平在睪酮處理30分鐘后沒有改變，P＞0.05；②睪酮(10~(-5)M)作用24小時可見IRS-1蛋白水平降低，P＜0.05；此時IRS-1的Ser~(307)磷酸化增加不明顯，胰島素可以誘導(dǎo)IRS-1的Tyr~(941)磷酸化水平，P＜0.05。(3)PD98059預(yù)處理可以抑制睪酮誘導(dǎo)的ERK1／2的磷酸化，短時間睪酮刺激的IRS-1的Ser~(307)磷酸化、睪酮對胰島素刺激的IRS-1的Tyr~(941)的抑制作用可以被PD98059逆轉(zhuǎn)，P＜0.05；PD98059對睪酮長時間處理IRS-1水平減少沒有作用，P＞0.05。(4)睪酮對胰島素刺激的葡萄糖攝取的抑制作用可被PD98059部分阻斷，P＜0.05；但仍低于正常對照組水平，P＜0.05。 結(jié)論：高濃度睪酮短時間處理C2C12骨骼肌細(xì)胞可能通過促進ERK1／2的磷酸化增強，因而與胰島素促代謝通路交叉對話，直接或間接磷酸化IRS-1的Ser~(307)位點，使其磷酸化過度，進而導(dǎo)致胰島素刺激的IRS-1的Tyr~(941)磷酸化水平下降，引起胞內(nèi)信號傳導(dǎo)障礙而導(dǎo)致細(xì)胞胰島素抵抗的發(fā)生，短時間睪酮作用于C2C12骨骼肌細(xì)胞對胰島素信號的影響是影響磷酸化水平而不是改變蛋白水平；睪酮長時間處理C2C12骨骼肌細(xì)胞主要通過IRS-1蛋白水平的降低從而導(dǎo)致胰島素抵抗的發(fā)生；高濃度睪酮尚存在其它途徑引起細(xì)胞胰島素抵抗，關(guān)于其機制有待進一步探討。
[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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R341

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