【摘要】：研究目的:葡萄糖代謝異常是引發(fā)代謝相關(guān)疾病的重要因素之一。眾所周知,規(guī)律的有氧運(yùn)動(dòng)是防治機(jī)體肥胖、胰島素抵抗(IR)和2型糖尿病(T2DM)的有效手段。機(jī)體運(yùn)動(dòng)主要是通過(guò)骨骼肌收縮和舒張來(lái)完成,人體內(nèi)約80%由胰島素介導(dǎo)的葡萄糖轉(zhuǎn)運(yùn)和利用是由骨骼肌組織完成,因此,促進(jìn)骨骼肌細(xì)胞葡萄糖代謝能夠增強(qiáng)機(jī)體組織對(duì)胰島素的敏感性,改善IR、預(yù)防T2DM的發(fā)生。Ⅱ型組蛋白去乙�；�(HDACs)作為轉(zhuǎn)錄抑制因子對(duì)組織細(xì)胞葡萄糖代謝起著重要的調(diào)節(jié)作用,Ⅱ型組蛋白去乙�；钢械腍DAC4和HDAC5是運(yùn)動(dòng)敏感性的組蛋白去乙�；浮Ｓ醒踹\(yùn)動(dòng)作為預(yù)防和治療肥胖、IR和多種代謝性疾病的有效手段,其作用機(jī)理是否與骨骼肌組織Ⅱ型HDACs的調(diào)控有關(guān)目前尚不完全明了。本研究以Ⅱ型HDACs調(diào)控組織細(xì)胞葡萄糖代謝為線索、以在體動(dòng)物模型和離體細(xì)胞模型為對(duì)象,研究HDAC4和HDAC5在有氧運(yùn)動(dòng)改善骨骼肌細(xì)胞葡萄糖攝取及利用的作用機(jī)制。通過(guò)在體動(dòng)物實(shí)驗(yàn)和離體細(xì)胞培養(yǎng)相結(jié)合的方式,研究有氧運(yùn)動(dòng)對(duì)C57BL/6小鼠骨骼肌細(xì)胞HDAC4和HDAC5調(diào)控葡萄糖轉(zhuǎn)運(yùn)體4(GLUT4)基因表達(dá)相互作用關(guān)系的影響;并在離體狀態(tài)下探討微小RNA-206(mi R-206)調(diào)節(jié)氧化型骨骼肌細(xì)胞HDAC4基因表達(dá)的具體機(jī)制,同時(shí)本研究還采用一磷酸腺苷活化蛋白激酶α2(AMPKα2)基因敲除小鼠,研究在AMPKα2缺失的情況下骨骼肌細(xì)胞HDAC4和HDAC5調(diào)控細(xì)胞葡萄糖代謝的變化,為進(jìn)一步揭示HDAC4和HDAC5在骨骼肌細(xì)胞葡萄糖代謝中的作用機(jī)制提供理論依據(jù)。研究方法:1.動(dòng)物模型的建立5周齡、雄性C57BL/6小鼠隨機(jī)分為正常安靜組(NC)和正常運(yùn)動(dòng)組(NE)。5周齡AMPKα2基因敲除小鼠共有三種基因型,即野生型(WT)、雜合子型(AMPKα2+/-)和純合子型(AMPKα2-/-)。分別將各基因型小鼠隨機(jī)分為安靜對(duì)照組(C)和有氧運(yùn)動(dòng)組(E)。2.腺病毒的構(gòu)建利用Gateway克隆技術(shù)構(gòu)建HDAC4,HDAC5,MEF2A和AMPKα2過(guò)表達(dá)重組腺病毒質(zhì)粒,采用Pac I內(nèi)切酶線性化后轉(zhuǎn)染293A細(xì)胞進(jìn)行腺病毒包裝。AMPKα2顯性負(fù)突變腺病毒是通過(guò)Gateway克隆技術(shù)構(gòu)建過(guò)表達(dá)質(zhì)粒后進(jìn)行點(diǎn)突變,再進(jìn)行腺病毒載體的重組及包裝。3.細(xì)胞培養(yǎng)及干預(yù)小鼠肌原細(xì)胞系C2C12經(jīng)2%馬血清培養(yǎng)誘導(dǎo)分化為肌管細(xì)胞。觀察HDAC4,MEF2A和HDAC5過(guò)表達(dá)腺病毒以及HDAC4和HDAC5 si RNA干預(yù)肌管細(xì)胞對(duì)葡萄糖代謝的影響;檢測(cè)AMPKα2過(guò)表達(dá)腺病毒及AMPKα2顯性負(fù)突變腺病毒對(duì)肌管細(xì)胞HDAC4的影響;使用mi R-206 mimic和mi R-206 inhibitor轉(zhuǎn)染肌管細(xì)胞,觀察葡萄糖代謝的變化。4.生化指標(biāo)檢測(cè)采用Real-time PCR檢測(cè)基因表達(dá);采用Western Blot方法檢測(cè)蛋白表達(dá);使用2-脫氧葡萄糖熒光類似物(2-NBDG)檢測(cè)C2C12肌管細(xì)胞葡萄糖攝取能力;采用免疫共沉淀(Co-IP)檢測(cè)蛋白間的相互結(jié)合;采用免疫熒光檢測(cè)蛋白表達(dá)及細(xì)胞內(nèi)定位。研究結(jié)果:1.HDAC4/5在有氧運(yùn)動(dòng)改善骨骼肌糖代謝中的作用6周有氧運(yùn)動(dòng)可以顯著增加小鼠骨骼肌組織GLUT4 m RNA和蛋白表達(dá)、HDAC4 Ser246位點(diǎn)磷酸化,同時(shí)促進(jìn)HDAC4從細(xì)胞核轉(zhuǎn)移至胞漿。通過(guò)離體細(xì)胞實(shí)驗(yàn)發(fā)現(xiàn)肌管細(xì)胞HDAC4表達(dá)的增加可抑制細(xì)胞GLUT4轉(zhuǎn)錄及其對(duì)葡萄糖攝取能力,當(dāng)增加MEF2A的表達(dá)水平可逆轉(zhuǎn)HDAC4的抑制作用。HDAC4 si RNA顯著增加肌管細(xì)胞GLUT4基因轉(zhuǎn)錄水平,在此基礎(chǔ)上給予HDAC5 si RNA,GLUT4基因轉(zhuǎn)錄水平被大幅度提高。與之相反,同時(shí)過(guò)表達(dá)HDAC4和HDAC5對(duì)于C2C12肌管細(xì)胞GLUT4的轉(zhuǎn)錄抑制效率顯著高于單獨(dú)過(guò)表達(dá)HDAC4或HDAC5。由此推測(cè),骨骼肌細(xì)胞HDAC4與HDAC5可能在GLUT4的轉(zhuǎn)錄調(diào)控過(guò)程中發(fā)揮著協(xié)同作用。有氧運(yùn)動(dòng)顯著降低氧化型肌纖維HDAC4和HDAC5蛋白表達(dá),然而在酵解型肌纖維中卻無(wú)顯著變化。此外,有氧運(yùn)動(dòng)也顯著降低骨骼肌細(xì)胞HDAC4和HDAC5的相互結(jié)合(圖1.8)。由此提示,有氧運(yùn)動(dòng)對(duì)HDAC4和HDAC5蛋白表達(dá)調(diào)控具有肌纖維類型的特異性,對(duì)其下游靶基因的抑制作用可能與二者的相互結(jié)合有關(guān)。2.AMPKα2在有氧運(yùn)動(dòng)調(diào)控骨骼肌組織HDAC4/5中的作用離體狀態(tài)下敲低AMPKα2可導(dǎo)致HDAC4 Ser246/632位點(diǎn)的磷酸化水平顯著降低,促使HDAC4從細(xì)胞核轉(zhuǎn)移至胞漿。提示骨骼肌細(xì)胞AMPKα2是HDAC4的上游激酶。單次有氧跑臺(tái)運(yùn)動(dòng)可以激活野生型小鼠骨骼肌AMPK-HDAC4信號(hào)通路;然而有氧運(yùn)動(dòng)未能增加AMPKα2缺失小鼠骨骼肌HDAC4 Ser246/632磷酸化水平,提示運(yùn)動(dòng)對(duì)于骨骼肌組織HDAC4的激活依賴于AMPKα2。3.mi R-206介導(dǎo)HDAC4在有氧運(yùn)動(dòng)提高骨骼肌葡萄糖代謝中的作用機(jī)制6周的有氧運(yùn)動(dòng)顯著增加氧化型肌纖維mi R-206的表達(dá)水平、抑制HDAC4蛋白表達(dá)、顯著增加GLUT4 m RNA及蛋白表達(dá)。由此推測(cè)有氧運(yùn)動(dòng)可能通過(guò)mi R-206降低HDAC4的蛋白表達(dá),進(jìn)而增加骨骼肌細(xì)胞GLUT4的轉(zhuǎn)錄。離體研究發(fā)現(xiàn),mi R-206水平的增高可顯著降低HDAC4的蛋白表達(dá)、增加骨骼肌細(xì)胞GLUT4基因的轉(zhuǎn)錄、提高了骨骼肌細(xì)胞對(duì)葡萄糖的攝取。因此提示mi R-206可能通過(guò)抑制HDAC4促進(jìn)骨骼肌細(xì)胞GLUT4的轉(zhuǎn)錄,進(jìn)而增加了對(duì)葡萄糖的攝取能力。采用mi R-206 mimic和HDAC4 si RNA共轉(zhuǎn)染肌管細(xì)胞,發(fā)現(xiàn)mi R-206和GLUT4的轉(zhuǎn)錄水平顯著低于單獨(dú)使用mi R-206 mimic干預(yù)的肌管細(xì)胞。隨后檢測(cè)發(fā)現(xiàn),采用HDAC4 si RNA可顯著降低C2C12肌管細(xì)胞mi R-206的表達(dá)水平,甚至與mi R-206 inhibitor干預(yù)后結(jié)果無(wú)顯著差異。由此說(shuō)明,HDAC4si RNA反饋性的抑制骨骼肌細(xì)胞mi R-206的表達(dá)。此外,以不同濃度梯度mi R-206 mimic孵育肌管細(xì)胞,GLUT4的基因表達(dá)隨著濃度梯度增加而增加,之后轉(zhuǎn)染肌管細(xì)胞過(guò)表達(dá)HDAC4腺病毒,GLUT4基因表達(dá)雖顯著高于對(duì)照組,但顯著低于單獨(dú)使用mi R-206 mimic干預(yù)組,提示mi R-206通過(guò)HDAC4調(diào)控肌管細(xì)胞GLUT4的基因表達(dá)。研究結(jié)論:1.HDAC4通過(guò)MEF2A調(diào)控骨骼肌細(xì)胞GLUT4的基因轉(zhuǎn)錄,且在轉(zhuǎn)錄調(diào)控的過(guò)程中HDAC4/5可能發(fā)揮著協(xié)同作用。有氧運(yùn)動(dòng)對(duì)于HDAC4/5的影響具有肌纖維類型的特異性,唯有在氧化型肌纖維中的表達(dá)水平顯著降低,可能是有氧運(yùn)動(dòng)促進(jìn)骨骼肌有氧氧化,提高機(jī)體對(duì)糖脂代謝的重要機(jī)制。2.AMPK能夠調(diào)控骨骼肌細(xì)胞HDAC4活性。單次有氧運(yùn)動(dòng)通過(guò)AMPKα2所依賴的方式激活骨骼肌細(xì)胞HDAC4。3.mi R-206 mimic顯著抑制骨骼肌細(xì)胞HDAC4的蛋白表達(dá),同時(shí)增加GLUT4轉(zhuǎn)錄、促進(jìn)骨骼肌對(duì)葡萄糖的攝取。此外,有氧運(yùn)動(dòng)可能通過(guò)mi R-206調(diào)控氧化型肌纖維HDAC4的蛋白表達(dá)、促進(jìn)機(jī)體的葡萄糖代謝。
[Abstract]:Objective: abnormal glucose metabolism is one of the important factors that cause metabolic related diseases. It is well known that regular aerobic exercise is an effective means to prevent body obesity, insulin resistance (IR) and type 2 diabetes (T2DM). Body exercise is accomplished mainly through skeletal muscle contraction and relaxation, and about 80% of the human body is mediated by insulin. Glucose transport and utilization are accomplished by skeletal muscle tissue. Therefore, promoting glucose metabolism in skeletal muscle cells can enhance the sensitivity of body tissue to insulin, improve IR and prevent the occurrence of T2DM. Type II histone deacetylase (HDACs) plays an important role in regulating glucose metabolism in tissue cells as a transcriptional inhibitor, type II egg HDAC4 and HDAC5 in white deacetylase are motor sensitive histone deacetylases. Aerobic exercise is an effective means to prevent and treat obesity, IR and multiple metabolic diseases. The mechanism of its action is not completely clear to the regulation of skeletal muscle type II HDACs. This study uses type II HDACs to regulate tissue cell Portuguese. Glucose metabolism was used as a clue to study the effect mechanism of HDAC4 and HDAC5 on improving glucose uptake and utilization of skeletal muscle cells by aerobic exercise in vivo animal model and isolated cell model. The HDAC4 and HDAC5 modulation of skeletal muscle cells in C57BL/6 mice was studied by combining in vivo and in vitro culture. The interaction relationship between the gene expression of glucose transporter 4 (GLUT4) was controlled, and the specific mechanism of the HDAC4 gene expression of the oxidized skeletal muscle cells regulated by tiny RNA-206 (MI R-206) was investigated in the isolated state, and the deletion of AMPK alpha 2 was also studied in this study by the gene knockout mice of adenosine activated protein kinase alpha 2 (AMPK alpha 2). The changes in skeletal muscle cells HDAC4 and HDAC5 regulate cell glucose metabolism and provide a theoretical basis for further revealing the mechanism of HDAC4 and HDAC5 in the glucose metabolism of skeletal muscle cells. The research methods: 1. animal models were established at 5 weeks of age, and male C57BL/6 mice were randomly divided into normal and quiet group (NC) and normal exercise group (NE).5 weeks AMP AMP There were three genotypes in K alpha 2 gene knockout mice, namely, wild type (WT), heterozygote type (AMPK alpha 2+/-) and homozygote type (AMPK alpha 2-/-). The mice were randomly divided into the quiet control group (C) and the aerobic exercise group (E).2. adenovirus construction using Gateway cloning technology to construct HDAC4, HDAC5, 2+/- and alpha 2 overexpressed the recombinant adenosine toxicity. After linearization of Pac I endonuclease, 293A cells were transfected into 293A cells to carry on the adenovirus packaging.AMPK alpha dominant negative mutation, and the overexpression plasmid was constructed by Gateway cloning technology to make the point mutation, then the recombinant adenovirus vector was reorganized and the.3. cell culture was packaged and the mouse myogenic cell line C2C12 was induced by 2% horse sera culture to induce differentiation. The effects of HDAC4, MEF2A and HDAC5 overexpressed adenoviruses and HDAC4 and HDAC5 Si RNA on glucose metabolism were observed, and the effects of AMPK a 2 overexpressed adenovirus and AMPK alpha 2 dominant negative mutant adenovirus on myotubular HDAC4 were detected. The changes of glucose metabolism.4. biochemical indexes were detected by Real-time PCR, Western Blot was used to detect protein expression, and 2- deoxyglucose fluorescence analogue (2-NBDG) was used to detect the glucose uptake ability of C2C12 myotube cells; immunofluorescence (Co-IP) was used to detect the interaction of protein, and the immunofluorescence was used to detect eggs. White expression and intracellular localization. Results: the effect of 1.HDAC4/5 on aerobic exercise improvement of skeletal muscle glucose metabolism 6 weeks of aerobic exercise can significantly increase the expression of GLUT4 m RNA and protein in skeletal muscle tissue of mice, phosphorylation of HDAC4 Ser246 site, and promote the transfer of HDAC4 from the nucleus to the cytoplasm. The myotube cell H is found through the cell experiment in vitro. The increase in expression of DAC4 inhibits the transcription of GLUT4 and its ability to absorb glucose, while increasing the expression level of MEF2A reverses the inhibition of HDAC4,.HDAC4 Si RNA significantly increases the transcriptional level of GLUT4 gene in myotube cells, on the basis of which HDAC5 Si RNA is given, and the transcriptional level of the GLUT4 gene is greatly increased. The transcriptional inhibition efficiency of HDAC4 and HDAC5 on C2C12 myotubular GLUT4 was significantly higher than that of HDAC4 or HDAC5. alone. The skeletal muscle cells HDAC4 and HDAC5 may play a synergistic role in the transcriptional regulation of GLUT4. Aerobic exercise significantly reduces the expression of HDAC4 and HDAC5 protein in the oxidized muscle fibers, however, in the glycolytic muscle fibers In addition, the aerobic exercise also significantly reduced the interaction of HDAC4 and HDAC5 in skeletal muscle cells (Figure 1.8). It suggests that the regulation of the expression of HDAC4 and HDAC5 proteins by aerobic exercise is specific to the type of muscle fiber, and the inhibition of the target gene of the downstream target may be associated with the interaction of the two persons with.2.AMPK alpha 2 in the aerobic exercise. The regulation of HDAC4/5 in skeletal muscle tissue in vitro knockout low AMPK alpha 2 can lead to a significant decrease in the phosphorylation level of the HDAC4 Ser246/632 site, prompting the transfer of HDAC4 from the nucleus to the cytoplasm. It suggests that the skeletal muscle cells AMPK alpha 2 is the upstream kinase of the HDAC4. The single aerobic table exercise can activate the AMPK-HDAC4 signal of the skeletal muscle of the wild type mice. However, aerobic exercise failed to increase the level of HDAC4 Ser246/632 phosphorylation in the skeletal muscle of AMPK alpha 2 deficient mice, suggesting that the activation of HDAC4 in skeletal muscle tissue depends on the mechanism of AMPK alpha 2.3.mi R-206 mediated HDAC4 in aerobic exercise to increase skeletal muscle glucose metabolism and the 6 week aerobic exercise significantly increases the mi R- of the oxidized muscle fibers. The expression level of 206 inhibits the expression of HDAC4 protein and significantly increases the expression of GLUT4 m RNA and protein. Therefore, it is suggested that aerobic exercise may reduce the expression of HDAC4 protein through mi R-206, and then increase the transcription of GLUT4 in skeletal muscle cells. In vitro studies have found that the increase of MI R-206 level can significantly reduce the HDAC4 protein expression and increase skeletal muscle cell GLUT4. Gene transcription increases the uptake of glucose in skeletal muscle cells. Therefore, MI R-206 may promote the transcription of GLUT4 in skeletal muscle cells by inhibiting HDAC4 and then increase the uptake of glucose. Mi R-206 mimic and HDAC4 Si RNA are used to co transfect myotube cells, and MI R-206 and transcriptional levels are found to be significantly lower than that of individual cells. It was found that the expression level of MI R-206 in C2C12 myotubular cells was significantly reduced by using HDAC4 Si RNA, and there was no significant difference between the C2C12 myotubular mi R-206 and the prognosis of MI R-206 inhibitor. 06 mimic incubated myotube cells, the gene expression of GLUT4 increased with the increase of concentration gradient and then transfected with myotube cells to overexpress HDAC4 adenovirus. The expression of GLUT4 gene was significantly higher than that of the control group, but it was significantly lower than the MI R-206 mimic intervention group, suggesting mi R-206 through HDAC4 to regulate the GLUT4 gene expression of myotube cells. 1.HDAC4 regulates the gene transcription of GLUT4 in skeletal muscle cells by MEF2A, and HDAC4/5 may play a synergistic role in the process of transcription regulation. The effect of aerobic exercise on HDAC4/5 has the specificity of the type of muscle fiber, only the expression level in the oxidized muscle fibers is significantly reduced, and it may be aerobic exercise to promote the aerobic oxidation of skeletal muscle. .2.AMPK, an important mechanism for the metabolism of glycolipid, can regulate the activity of HDAC4 in skeletal muscle cells. The activation of HDAC4.3.mi R-206 mimic in skeletal muscle cells by AMPK alpha 2 on single aerobic exercise significantly inhibits the protein expression of HDAC4 in skeletal muscle cells, and increases the GLUT4 conversion, and promotes the uptake of glucose in skeletal muscle. In addition, Aerobic exercise may regulate the protein expression of oxidized muscle fiber HDAC4 through mi R-206, and promote glucose metabolism in the body.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R87

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本文編號(hào)：2167743