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        GLP-1受體激動(dòng)劑艾塞那肽緩解ob/ob小鼠骨骼肌脂質(zhì)沉積的作用不依賴于體重的下降*

        2020-12-03 14:21:04郭婉蓉陳宗蘭曹歡易鄧洪容蔡夢(mèng)茵梁華徐芬
        中國(guó)病理生理雜志 2020年11期
        關(guān)鍵詞:小鼠

        郭婉蓉, 陳宗蘭, 曹歡易, 鄧洪容, 蔡夢(mèng)茵, 梁華, 徐芬

        ·論著·

        GLP-1受體激動(dòng)劑艾塞那肽緩解/小鼠骨骼肌脂質(zhì)沉積的作用不依賴于體重的下降*

        郭婉蓉, 陳宗蘭, 曹歡易, 鄧洪容, 蔡夢(mèng)茵, 梁華, 徐芬△

        (中山大學(xué)附屬第三醫(yī)院,廣東省糖尿病防治重點(diǎn)實(shí)驗(yàn)室,廣東 廣州 510630)

        探討降糖藥物胰高血糖素樣肽1 (GLP-1)受體激動(dòng)劑艾塞那肽(Exe)對(duì)肥胖的/小鼠骨骼肌脂質(zhì)沉積的作用及相關(guān)機(jī)制。將8周齡雄性/小鼠隨機(jī)分為生理鹽水對(duì)照組(/組)和給藥組(/+Exe組),另以野生型(WT)小鼠為WT對(duì)照組(WT組)。對(duì)/+Exe組小鼠腹腔注射Exe (24 nmol·kg-1·d-1) 4周,而對(duì)WT組及/組小鼠腹腔注射等體積生理鹽水4周,定期監(jiān)測(cè)干預(yù)后小鼠的體重、空腹血糖(FBG)及體脂含量。用油紅O染色觀察骨骼肌脂質(zhì)沉積情況;用ELISA進(jìn)行血清中甘油三酯(TG)、游離脂肪酸(FFA)和總膽固醇及骨骼肌TG定量檢測(cè); Western blot檢測(cè)腺苷酸活化蛋白激酶(AMPK)及脂代謝相關(guān)蛋白的表達(dá)情況。采用小鼠C2C12成肌細(xì)胞進(jìn)行體外細(xì)胞實(shí)驗(yàn),棕櫚酸鈉(300 μmol/L)或Exe (20 nmol/L)干預(yù)24 h后,通過(guò)油紅O染色及細(xì)胞內(nèi)TG定量觀察細(xì)胞內(nèi)脂質(zhì)沉積情況; Western blot法觀察AMPK及脂代謝相關(guān)蛋白的表達(dá)情況;通過(guò)葡萄糖攝取實(shí)驗(yàn)觀察細(xì)胞的葡萄糖攝取能力。與/組相比, Exe治療對(duì)小鼠的體重、FBG、體脂含量及能量攝入無(wú)顯著影響(>0.05),但可顯著降低血清中FFA含量(<0.05)。油紅O染色及TG定量結(jié)果顯示, Exe明顯緩解/小鼠骨骼肌中的脂質(zhì)沉積(<0.05)。Western blot結(jié)果顯示, Exe可降低/小鼠骨骼肌中脂質(zhì)合成相關(guān)蛋白的表達(dá)水平,增加磷酸化AMPK (p-AMPK)和脂質(zhì)分解相關(guān)蛋白的水平(<0.05)。Exe顯著緩解棕櫚酸鈉誘導(dǎo)的C2C12細(xì)胞脂質(zhì)沉積(<0.05),顯著升高p-AMPK、脂質(zhì)分解相關(guān)蛋白和葡萄糖轉(zhuǎn)運(yùn)蛋白4的水平,降低脂質(zhì)合成相關(guān)蛋白的水平(<0.05),并且增強(qiáng)細(xì)胞的葡萄糖攝取能力(<0.05)。GLP-1受體激動(dòng)劑Exe可以緩解/小鼠骨骼肌中的脂質(zhì)沉積,其作用可能與抑制脂質(zhì)合成和促進(jìn)脂質(zhì)分解相關(guān),且該作用不依賴于體重的下降。

        胰高血糖素樣肽1;艾塞那肽;骨骼??;脂質(zhì);體重

        胰高血糖素樣肽1 (glucagon-like peptide-1, GLP-1)主要由腸道L細(xì)胞分泌,作用于胰腺上的GLP-1受體(GLP-1 receptor, GLP-1R),促進(jìn)胰島素分泌并且減少胰高血糖素的分泌,從而達(dá)到降低血糖效果,其作用具有葡萄糖依賴性[1]。GLP-1R激動(dòng)劑(GLP-1R agonist, GLP-1RA)是一類已被廣泛應(yīng)用于臨床的降糖藥物[2],除了具有降糖作用以外,近年來(lái)的臨床研究還顯示了其顯著的減重作用[3-4]。

        本課題組前期研究表明,作為最早批準(zhǔn)應(yīng)用于臨床治療的GLP-1RA,艾塞那肽(exenatide, Exe;又稱exendin-4)可以減輕高脂飲食所致小鼠的肥胖、糖耐量受損、胰島素抵抗、肝臟脂質(zhì)沉積及白色脂肪增生、肥大[5-7]。除了肝臟及白色脂肪,骨骼肌也是胰島素作用的重要靶器官之一,在機(jī)體糖脂代謝穩(wěn)態(tài)中發(fā)揮著重要作用。骨骼肌脂質(zhì)沉積與胰島素抵抗以及糖代謝異常有著密切聯(lián)系,骨骼肌脂質(zhì)沉積的改善可以提高胰島素敏感性[8-10]。雖然Exe減重作用確定,而本課題組前期的研究也證實(shí)肝臟和白色脂肪為Exe減輕高脂飲食所致小鼠肥胖的靶器官,但Exe對(duì)骨骼肌脂質(zhì)沉積的作用及其與體重減輕的關(guān)系仍未明確。因此,本研究在瘦素(leptin)基因突變的/肥胖小鼠模型和C2C12小鼠成肌細(xì)胞模型上觀察Exe對(duì)骨骼肌脂質(zhì)沉積的作用及其與體重的關(guān)系,并探討可能的分子機(jī)制。

        材料和方法

        1 實(shí)驗(yàn)動(dòng)物和材料

        7周齡雄性ob/ob小鼠12只及其野生型(wild-type, WT)小鼠6只(南京大學(xué)模式動(dòng)物研究所,動(dòng)物合格證號(hào)為1107272011000847)。

        2 方法

        2.1動(dòng)物實(shí)驗(yàn)實(shí)驗(yàn)獲中山大學(xué)動(dòng)物實(shí)驗(yàn)倫理委員會(huì)批準(zhǔn)。小鼠適應(yīng)性喂養(yǎng)1周后隨機(jī)分為WT對(duì)照組(WT組)、/小鼠生理鹽水對(duì)照組(/組)及/小鼠給藥組(/+Exe組),每組6只。每組小鼠給予普通飼料(質(zhì)量百分比碳水化合物占58%、蛋白質(zhì)占18%、脂肪占4%),自由飲水。隨機(jī)分組后,/+Exe組每天腹腔注射Exe (24 nmol/kg), WT組和ob/ob組給予等體積生理鹽水,持續(xù)4周。每周監(jiān)測(cè)小鼠體重、空腹血糖及能量攝入,并在結(jié)束前進(jìn)行體脂含量測(cè)定。實(shí)驗(yàn)結(jié)束后,用異氟烷麻醉小鼠后摘除眼球取血,并頸椎脫臼處死小鼠。取小鼠腓腸肌標(biāo)本兩份,一份在-80℃冰箱保存用于Western blot實(shí)驗(yàn),另一份保存用4%多聚甲醛固定用于形態(tài)學(xué)實(shí)驗(yàn)。

        2.2C2C12細(xì)胞誘導(dǎo)分化及干預(yù)用含10%胎牛血清的高糖(4.5 g/L) DMEM培養(yǎng)基進(jìn)行細(xì)胞培養(yǎng),待細(xì)胞長(zhǎng)至80%時(shí),換為含2%馬血清的高糖DMEM培養(yǎng)基進(jìn)行誘導(dǎo)分化,分化成功后進(jìn)行后續(xù)實(shí)驗(yàn)。用無(wú)血清高糖DMEM培養(yǎng)基進(jìn)行饑餓4~6 h后分為3組,分別用棕櫚酸鈉(300 μmol/L)、棕櫚酸鈉聯(lián)合Exe (20 nmol/L)及牛血清白蛋白(bovine serum albumin, BSA;作為實(shí)驗(yàn)對(duì)照)干預(yù)24 h。

        2.3血脂測(cè)定血清中甘油三酯(triglyceride, TG)、游離脂肪酸(free fatty acids, FFA)及總膽固醇(total cholesterol, TC)水平的測(cè)定均嚴(yán)格按照BioVision相應(yīng)試劑盒進(jìn)行測(cè)定。

        2.4骨骼肌及細(xì)胞TG的測(cè)定(1)骨骼肌TG的萃?。杭羧?0 mg腓腸肌標(biāo)本并加入200 μL 5% NP-40溶液,進(jìn)行勻漿;勻漿完全后,沸水加熱5 min后冷卻至室溫再重復(fù)加熱1次;以14 000 r/min室溫離心2 min后取上清,即骨骼肌中的TG。(2) C2C12細(xì)胞中的TG通過(guò)5% NP-40進(jìn)行裂解獲得。后續(xù)嚴(yán)格按照BioVision相關(guān)試劑盒說(shuō)明書(shū)進(jìn)行實(shí)驗(yàn)。

        2.5油紅O染色(1)骨骼肌:小鼠腓腸肌于4%多聚甲醛中浸泡過(guò)夜,分別用20%及30%蔗糖溶液進(jìn)行過(guò)夜脫水,脫水后經(jīng)OCT膠包埋進(jìn)行切片,切片厚度10 μm,得骨骼肌冰凍切片;稱取0.15 g油紅O粉末,溶于30 mL異丙醇中,混勻得油紅儲(chǔ)存液,避光長(zhǎng)期4℃保存,以油紅儲(chǔ)存液∶雙蒸水=3∶2配制油紅工作液,現(xiàn)配現(xiàn)用;骨骼肌冰凍切片于油紅工作液中避光染色75 min,雙蒸水輕柔沖洗20 min; 20%蘇木精染細(xì)胞核1 min后雙蒸水漂洗返藍(lán), 1%鹽酸乙醇分色5 s后在此雙蒸水漂洗;最后以10%甘油封片;封片后用倒置顯微鏡進(jìn)行拍照。(2) C2C12細(xì)胞:干預(yù)結(jié)束后,用1× PBS洗2次,加入4%多聚甲醛進(jìn)行室溫固定15 min;再次用1× PBS洗2次后, 60%異丙醇預(yù)處理20 s;加入油紅工作液室溫避光染色30 min后, 1× PBS洗2次,并用60%異丙醇分色5 s,再次1× PBS洗2次;最后用倒置顯微鏡進(jìn)行拍照,整個(gè)過(guò)程不超過(guò)2 h。

        2.6Western blot實(shí)驗(yàn)取小鼠腓腸肌30 mg至150 μL裂解液中,勻漿后14 000 r/min、4℃離心20 min,取中間清液層得總蛋白。C2C12細(xì)胞總蛋白提取同樣使用上述裂解液,裂解結(jié)束后利用超聲破碎儀進(jìn)行細(xì)胞破碎,離心步驟同上。利用BCA試劑盒進(jìn)行蛋白濃度并配平后取40 μg總蛋白進(jìn)行電泳,恒壓90 V電泳30 min后再恒壓110 V電泳60 min,再以恒流300 mA轉(zhuǎn)膜90 min。室溫下5%脫脂奶粉封閉1 h后,TBST洗膜3次。根據(jù)目標(biāo)蛋白分子量進(jìn)行切膜,并分別加入相應(yīng)的Ⅰ抗稀釋液進(jìn)行過(guò)夜4℃孵育。孵育結(jié)束后,回收Ⅰ抗稀釋液并再次進(jìn)行洗膜,并以相應(yīng)的Ⅱ抗稀釋液(LI-COR)進(jìn)行室溫避光孵育1 h,隨后再次進(jìn)行洗膜,用Odyssey紅外熒光成像系統(tǒng)掃描,保存圖片后利用Image-Pro Plus軟件進(jìn)行灰度分析??瓜佘账峄罨鞍准っ福ˋMP-activated protein kinase, AMPK)、磷酸化AMPK (phosphorylated AMPK, p-AMPK)、固醇調(diào)節(jié)元件結(jié)合蛋白1c(sterol regulatory element binding protein 1c, SREBP1c)、磷酸化SREBP1c (phosphorylated SREBP1c, p-SREBP1c)、脂肪甘油三酯脂肪酶(adipose triglyceride lipase, ATGL)及葡萄糖轉(zhuǎn)運(yùn)蛋白4(glucose transporter 4, GLUT4)抗體均購(gòu)自CST;抗脂蛋白脂肪酶(lipoprotein lipase, LPL)抗體購(gòu)自Santa Cruz。每組實(shí)驗(yàn)重復(fù)次數(shù)≥3。

        2.7葡萄糖攝取實(shí)驗(yàn)C2C12細(xì)胞干預(yù)結(jié)束后再次饑餓2 h,隨后換為含胰島素(100 nmol/L)的PBS干預(yù)30 min。胰島素干預(yù)結(jié)束后,更換為含同位素的緩沖液(140 mmol/L NaCl、20 mmol/L HEPES-Na、5 mmol/L KCl、0.5 mCi/L 2-deoxy-D-[3H]glucose、2.5 mmol/L MgSO4和1.0 mmol/L CaCl2, pH 7.4)室溫孵育5 min,用預(yù)冷的PBS中止反應(yīng)。加入NaOH(1 mol/L)裂解細(xì)胞并收集至含閃爍液的管子中,使用液體閃爍計(jì)數(shù)儀進(jìn)行檢測(cè),并用蛋白濃度進(jìn)行校正。

        3 統(tǒng)計(jì)學(xué)處理

        利用SPSS 20.0軟件進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)誤(mean±SEM)表示,多組計(jì)量資料用單因素方差分析并用Bonferroni校正的檢驗(yàn)進(jìn)行兩兩比較。以<0.05為差異有統(tǒng)計(jì)學(xué)意義。

        結(jié)果

        1 Exe對(duì)ob/ob小鼠體重、空腹血糖和能量攝入的影響

        /小鼠的體重、空腹血糖、能量攝入及體脂含量均顯著高于WT小鼠(<0.05);而Exe干預(yù)4周后,/小鼠的體重、空腹血糖、能量攝入及體脂含量均無(wú)明顯下降(>0.05),見(jiàn)表1。

        表1 艾塞那肽對(duì)ob/ob小鼠表型的影響

        ?<0.05WT group.

        2 Exe對(duì)ob/ob小鼠血脂的影響

        /小鼠血清中的TG、FFA及TC均顯著高于WT小鼠;而Exe干預(yù)4周后,/小鼠血清中的FFA顯著下降(<0.05),見(jiàn)表2。

        3 Exe對(duì)ob/ob小鼠骨骼肌形態(tài)學(xué)的影響

        油紅O染色結(jié)果顯示,/小鼠較WT小鼠有更為明顯的骨骼肌脂質(zhì)沉積;而Exe干預(yù)4周后,/小鼠骨骼肌的脂質(zhì)沉積明顯減少,見(jiàn)圖1。骨骼肌甘油三酯定量結(jié)果同樣證明Exe具有減少/小鼠骨骼肌中脂質(zhì)沉積的作用(<0.05),見(jiàn)表2。

        Figure 1. The oil red O staining of skeletal muscle in ob/ob mice after exenatide (Exe) treatment (×200).

        表2 艾塞那肽對(duì)ob/ob小鼠血脂譜及骨骼肌中甘油三酯含量的影響

        ?<0.05WT group;?<0.05/group.

        4 Exe對(duì)ob/ob小鼠骨骼肌中脂代謝相關(guān)蛋白的影響

        與WT小鼠相比,/小鼠骨骼肌中p-AMPK及脂質(zhì)分解相關(guān)蛋白ATGL和LPL水平均顯著降低(<0.05),脂質(zhì)合成相關(guān)蛋白p-SREBP1c和FAS水平顯著升高(<0.05);在Exe干預(yù)4周后, p-AMPK、ATGL和LPL蛋白水平顯著升高(<0.05),而p-SREBP1c和FAS蛋白水平顯著降低(<0.05),見(jiàn)圖2。

        Figure 2. The effects of exenatide (Exe) on the expression levels of AMPK and lipid metabolism-related proteins in the skeletal muscle of ob/ob mice. Mean±SEM. n=3. ?P<0.05 vs WT group; ?P<0.05 vsob/ob group.

        5 Exe對(duì)棕櫚酸鈉誘導(dǎo)的C2C12細(xì)胞脂質(zhì)沉積的影響

        油紅O染色結(jié)果顯示, Exe干預(yù)后,棕櫚酸鈉所致的C2C12細(xì)胞脂質(zhì)沉積顯著減輕(圖3A),并且TG定量結(jié)果同樣顯示C2C12細(xì)胞內(nèi)的TG含量有所降低(圖3B)。Western blot結(jié)果顯示, Exe可以顯著增加p-AMPK及脂質(zhì)分解相關(guān)蛋白ATGL和LPL的水平,同時(shí)顯著減少脂質(zhì)合成相關(guān)蛋白p-SREBP1c及FAS的水平(<0.05),見(jiàn)圖3C。此外,葡萄糖攝取實(shí)驗(yàn)結(jié)果顯示, Exe顯著增強(qiáng)C2C12細(xì)胞的葡萄糖攝取能力,見(jiàn)圖3D; Western blot結(jié)果也同樣顯示, Exe顯著上調(diào)C2C12細(xì)胞GLUT4蛋白的表達(dá)(<0.05),見(jiàn)圖3C。

        Figure 3. The effects of exenatide (Exe) on the lipid accumulation, the ability of glucose uptake, the expression levels of AMPK and lipid metabolism-related proteins in the C2C12 cells induced by sodium palmate, a sodium salt of palmitic acid (PA). A: oil red O staining was used to detected the lipid accumulation (×200); B: TG content in the C2C12 cells was detected; C: Western blot was used to detected the protein levels; D: glucose uptake was detected. Mean±SEM. n=3. ?P<0.05 vs BSA group; ?P<0.05 vs PA group.

        討論

        GLP-1RA是治療糖尿病的常用藥物之一,除了其降糖作用以外,近年來(lái)越來(lái)越多臨床研究發(fā)現(xiàn)GLP-1RA在減重上的顯著效果[3-4, 11-12]。有研究表明,在飲食和生活方式干預(yù)的基礎(chǔ)上給予肥胖患者皮下注射GLP-1RA治療56周后,與安慰劑組相比,GLP-1RA治療組有更加顯著的降低體重、空腹血糖、糖化血紅蛋白、空腹胰島素水平以及減少血脂(TC、TG及FFA)的作用[4]。Exe作為經(jīng)典的GLP-1RA,早期多項(xiàng)研究表明其可以減少肝臟脂質(zhì)沉積[6-7, 12-14],抑制白色脂肪增生和肥大[5, 15]。骨骼肌作為機(jī)體胰島素作用重要的靶器官之一,同時(shí)是體內(nèi)葡萄糖利用的重要場(chǎng)所之一,其脂質(zhì)沉積對(duì)機(jī)體胰島素敏感性及葡萄糖代謝均有負(fù)面影響[8-10]。本課題組前期研究的結(jié)果表明, Exe可以降低體重并減少高脂飲食所致C57BL/6小鼠的骨骼肌脂質(zhì)沉積,其作用可能與促進(jìn)骨骼肌中脂解作用及抑制脂質(zhì)合成有關(guān)[16]。

        從本研究的結(jié)果可見(jiàn), 4周的Exe干預(yù)可以緩解瘦素基因突變所致肥胖小鼠骨骼肌中的脂質(zhì)沉積,說(shuō)明Exe對(duì)骨骼肌脂質(zhì)沉積的緩解作用并不完全依賴于瘦素的作用。但與本課題組前期在高脂飲食所致的肥胖C57BL/6小鼠模型上的研究[5, 7, 16]相比, Exe干預(yù)對(duì)/小鼠減重和改善糖代謝的作用顯著削弱。本課題組前期的研究表明,給予高脂飲食誘導(dǎo)的肥胖C57BL/6小鼠4周Exe干預(yù)后,小鼠的體重顯著下降[5, 7, 16]。在本研究中,給予/小鼠4周等劑量的Exe干預(yù)后,未能觀察到明顯的體重下降。攝食量結(jié)果顯示, 4周Exe干預(yù)并不減少/小鼠的攝食量,但能明顯減少C57BL/6小鼠的攝食量。Williams等[17]的研究表明, GLP-1RA并不能降低瘦素受體缺乏的Koletsky大鼠的攝食量和體重,與我們的觀察結(jié)果一致。因此, 4周的Exe干預(yù)在不同模型上對(duì)攝食量及體重產(chǎn)生不一樣的效果可能與瘦素相關(guān)。此外, Ding等[13]的研究表明,給予/小鼠60 d的Exe干預(yù)可以減輕小鼠的體重。本研究中,給予更短時(shí)間(4周)的Exe干預(yù)并未顯著降低/小鼠的體重,但能明顯減輕小鼠骨骼肌脂質(zhì)沉積,除了干預(yù)時(shí)間不同可能會(huì)導(dǎo)致以上差異外,也提示Exe減輕骨骼肌脂質(zhì)沉積是直接作用而并不單純是減重的伴隨結(jié)果,其對(duì)骨骼肌的直接作用可能參與到減重的機(jī)制當(dāng)中。在C2C12細(xì)胞模型中,我們同樣觀察到Exe可以緩解棕櫚酸鈉所致的細(xì)胞脂質(zhì)沉積,與Choung等[18]的研究結(jié)果一致,說(shuō)明Exe對(duì)骨骼肌的確存在直接的作用,而不依賴于瘦素的作用。

        動(dòng)物及體外細(xì)胞研究表明,Exe可以上調(diào)肝臟中p-AMPK的蛋白水平[6, 19-20]。AMPK作為機(jī)體的能量感受器,在維持機(jī)體能量平衡中具有重要的作用。p-AMPK為AMPK的激活狀態(tài),其上調(diào)可以抑制脂質(zhì)合成關(guān)鍵酶的表達(dá)[21]。Li等[22]的研究表明,激活小鼠肝臟AMPK可以明顯抑制核中脂質(zhì)合成關(guān)鍵蛋白SREBP1和SREBP2的表達(dá),從而抑制脂質(zhì)合成并達(dá)到減輕肝臟脂質(zhì)沉積的效果。ATGL是脂肪組織中重要的脂質(zhì)分解相關(guān)蛋白。脂肪組織特異性敲除的小鼠表現(xiàn)為棕色脂肪白色化和白色脂肪增生肥大, AMPK可以促進(jìn)脂肪組織中ATGL的磷酸化并增強(qiáng)ATGL的脂質(zhì)分解能力,從而促進(jìn)脂代謝的平衡[23-24]。LPL是一種水解酶,同樣在脂質(zhì)的分解中起著重要的作用[25-26],亦有報(bào)道指出LPL的活性可能也受到AMPK的調(diào)節(jié)[27]。本研究發(fā)現(xiàn), Exe能上調(diào)/小鼠骨骼肌中能量感受器p-AMPK蛋白水平,并且下調(diào)脂質(zhì)合成蛋白p-SREBP1c和FAS蛋白水平和上調(diào)脂質(zhì)分解相關(guān)蛋白ATGL和LPL的表達(dá)。Exe緩解/小鼠骨骼肌的脂質(zhì)沉積可能與其直接上調(diào)骨骼肌中p-AMPK的表達(dá),從而抑制脂質(zhì)合成和促進(jìn)脂質(zhì)分解有關(guān)。在C2C12細(xì)胞模型中,我們觀察到Exe對(duì)上述蛋白的調(diào)控作用與動(dòng)物實(shí)驗(yàn)一致。

        此外,我們?cè)贑2C12細(xì)胞模型中觀察到Exe可以顯著增強(qiáng)細(xì)胞的葡萄糖攝取能力。一方面,骨骼肌的脂質(zhì)沉積可以損傷骨骼肌的葡萄糖攝取能力[10],臨床研究也表明骨骼肌的脂質(zhì)沉積可以導(dǎo)致機(jī)體糖耐量受損[9];另一方面,有研究表明激活骨骼肌中的AMPK可以促進(jìn)骨骼肌的葡萄糖攝取能力[28]。本研究也同樣觀察到Exe顯著增加C2C12細(xì)胞中p-AMPK的蛋白水平。因此, Exe通過(guò)緩解C2C12細(xì)胞的脂質(zhì)沉積及激活A(yù)MPK而增強(qiáng)細(xì)胞的葡萄糖攝取能力,提示骨骼肌是改善糖代謝的靶器官之一。

        綜上所述, GLP-1RA艾塞那肽可以減輕瘦素基因突變(/)所致肥胖小鼠骨骼肌的脂質(zhì)沉積,其機(jī)制可能與促進(jìn)脂質(zhì)分解和抑制脂質(zhì)合成有關(guān),并且不依賴于體重的下降。

        [1] Holst JJ. The physiology of glucagon-like peptide 1[J]. Physiol Rev, 2007, 87(4):1409-1439.

        [2] Buse JB, Wexler DJ, Tsapas A, et al. 2019 Update to management of hyperglycemia in type 2 diabetes, 2018. A consensus report by the ADA and the EASD[J]. Diabetes Care, 2020, 2(43):487-493.

        [3] Abreu M, Tumyan A, Elhassan A, et al. A randomized trial comparing the efficacy and safety of treating patients with type 2 diabetes and highly elevated HbA1c levels with basal‐bolus insulin or a glucagon‐like peptide‐1 receptor agonist plus basal insulin: the SIMPLE study[J]. Diabetes Obes Metab, 2019, 21(9):2133-2141.

        [4] Pi-Sunyer X, Astrup A, Fujioka K, et al. A randomized, controlled trial of 3.0 mg of liraglutide in weight management[J]. N Engl J Med, 2015, 373(1):11-22.

        [5] Xu F, Lin B, Zheng X, et al. GLP-1 receptor agonist promotes brown remodelling in mouse white adipose tissue through SIRT1[J]. Diabetologia, 2016, 59(5):1059-1069.

        [6] Xu F, Li Z, Zheng X, et al. SIRT1 mediates the effect of GLP-1 receptor agonist exenatide on ameliorating hepatic steatosis[J]. Diabetes, 2014, 11(63):3637-3646.

        [7] Zheng X, Xu F, Liang H, et al. SIRT1/HSF1/HSP pathway is essential for exenatide-alleviated, lipid-induced hepatic endoplasmic reticulum stress[J]. Hepatology, 2017, 66(3):809-824.

        [8] Anderwald C, Bernroider E, Krssák M, et al. Effects of insulin treatment in type 2 diabetic patients on intracellular lipid content in liver and skeletal muscle[J]. Diabetes, 2002, 51(10):3025-3032.

        [9] Kautzky-Willer A, Krssak M, Winzer C, et al. Increased intramyocellular lipid concentration identifies impaired glucose metabolism in women with previous gestational diabetes[J]. Diabetes, 2003, 52(2):244-251.

        [10] Szendroedi J, Roden M. Ectopic lipids and organ function[J]. Curr Opin Lipidol, 2009, 20(1):50-56.

        [11] Yan J, Yao B, Kuang H, et al. Liraglutide, sitagliptin, and insulin glargine added to metformin: the effect on body weight and intrahepatic lipid in patients with type 2 diabetes mellitus and nonalcoholic fatty liver disease[J]. Hepatology, 2019, 6(69):2414-2426.

        [12] Dutour A, Abdesselam I, Ancel P, et al. Exenatide decreases liver fat content and epicardial adipose tissue in patients with obesity and type 2 diabetes: a prospective randomized clinical trial using magnetic resonance imaging and spectroscopy[J]. Diabetes Obes Metab, 2016, 18(9):882-891.

        [13] Ding X, Saxena NK, Lin S, et al. Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, reverses hepatic steatosis in/mice[J]. Hepatology, 2006, 43(1):173-181.

        [14] Gupta NA, Mells J, Dunham RM, et al. Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway[J]. Hepatology, 2010, 51(5):1584-1592.

        [15] Tanaka K. Exenatide improves hepatic steatosis by enhancing lipid use in adipose tissue in nondiabetic rats[J]. World J Gastroenterol, 2014, 20(10):2653-2663.

        [16] 曹歡易,徐芬,陳宗蘭,等. 艾塞那肽對(duì)高脂飲食誘導(dǎo)的肥胖小鼠骨骼肌脂質(zhì)沉積的作用及機(jī)制探討[J]. 中華醫(yī)學(xué)雜志, 2017, 97(2):131-136.

        Cao HY, Xu F, Chen ZL, et al. Effect of exendin-4 on lipid deposition in skeletal muscle of diet-induced obese mice and its underlying mechanism[J]. Natl Med J China, 2017, 97(2):131-136.

        [17] Williams DL, Baskin DG, Schwartz MW. Leptin regulation of the anorexic response to glucagon-like peptide-1 receptor stimulation[J]. Diabetes, 2006, 55(12):3387-3393.

        [18] Choung J, Lee Y, Jun H. Exendin-4 increases oxygen consumption and thermogenic gene expression in muscle cells[J]. J Mol Endocrinol, 2017, 2(58):79-90.

        [19] Svegliati-Baroni G, Saccomanno S, Rychlicki C, et al. Glucagon-like peptide-1 receptor activation stimulates hepatic lipid oxidationand restores hepatic signalling alteration induced by a high-fat diet in nonalcoholic steatohepatitis[J]. Liver Int, 2011, 9(31):1285-1297.

        [20] Jinmi L, Seok-Woo H, Wan CS, et al. Exenatide improves hepatic steatosis by enhancing lipid use in adipose tissue in nondiabetic rats[J]. PLoS One, 2012, 2(7):e31394.

        [21] Carling D, Mayer FV, Sanders MJ, et al. AMP-activated protein kinase: nature's energy sensor[J]. Nat Chem Biol, 2011, 7(8):512-518.

        [22] Li Y, Xu S, Mihaylova MM, et al. AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice[J]. Cell Metab, 2011, 13(4):376-388.

        [23] Ahmadian M, Abbott MJ, Tang T, et al. Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotype[J]. Cell Metab, 2011, 13(6):739-748.

        [24] Kim S, Tang T, Abbott M, et al. AMPK phosphorylates desnutrin/ATGL and hormone-sensitive lipase to regulate lipolysis and fatty acid oxidation within adipose tissue[J]. Mol Cell Biol, 2016, 36(14):1961-1976.

        [25] Jansen H, Breedveld B, Schoonderwoerd K. Role of lipoprotein lipases in postprandial lipid metabolism[J]. Atherosclerosis, 1998, 141:S31-S34.

        [26] Wang H, Astarita G, Taussig MD, et al. Deficiency of lipoprotein lipase in neurons modifies the regulation of energy balance and leads to obesity[J]. Cell Metab, 2011, 13(1):105-113..

        [27] An D, Pulinilkunnil T, Qi D, et al. The metabolic "switch" AMPK regulates cardiac heparin-releasable lipoprotein lipase[J]. Am J Physiol Endocrinol Metab, 2005,288(1):E246-E253.

        [28] Musi N, Hirshman MF, Nygren J, et al. Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes[J]. Diabetes, 2002, 51(7):2074-2081.

        Alleviating effect of exenatide on ectopic lipid accumulation in skeletal muscle of/mice is independent on reducing body weight

        GUO Wan-rong, CHEN Zong-lan, CAO Huan-yi, DENG Hong-rong, CAI Meng-yin, LIANG Hua, XU Fen

        (,,510630,)

        To investigate the alleviating effect of exenatide (Exe), a glucagon-like peptide-1 (GLP-1) receptor agonist, on the ectopic lipid accumulation in skeletal muscle of/mice and its mechanism.Eight-week-old male/mice and their wild-type (WT) littermates were randomly divided into 3 groups,/group,/+Exe group and WT group, and treated with Exe at 24 nmol/kg or the same volume of saline intraperitoneally once daily for 4 weeks. The body weight, fasting blood glucose (FBG) and fat content were measured after the 4-week treatment. The oil red O staining and the quantification of triglyceride (TG) were performed on the skeletal muscle. The serum levels of TG, total cholesterol and free fatty acid (FFA) were also measured by ELISA. The expression levels of AMP-activated protein kinase (AMPK) and lipid metabolism-related proteins were determined by Western blot. Mouse myoblast C2C12 cells were used as anmodel to further investigate the effects of Exe.As compared with the/mice treated with saline, 4-week Exe treatment did not reduce body weight, FBG, food intake and fat content in/mice (>0.05). However, serum FFA was decreased (<0.05). Oil red O staining and the quantification of TG showed that 4-week Exe treatment significantly attenuated the ectopic lipid accumulation in the skeletal muscle of/mice (<0.05). The results of Western blot showed that the levels of phosphorylated AMPK (p-AMPK) and lipolysis-related proteins were up-regulated, while the lipid synthesis-related proteins were down-regulated by Exe (<0.05). Treatment with Exe alleviated the lipid accumulation in the C2C12 cells induced by sodium palmate (<0.05), and the effects of Exe on the levels of p-AMPK and lipid metabolism-related proteins in the C2C12 cells were consistent with those in the/mice (<0.05). Treatment with Exe also up-regulated the protein expression of glucose transporter 4 and improved the ability of glucose uptake in the C2C12 cells (<0.05).Short-term Exe treatment attenuates the ectopic lipid accumulation in skeletal muscle of/mice by up-regulating lipolysis-related proteins and down-regulating lipid synthesis-related proteins, which is independent on body weight loss.

        Glucagon-like peptide-1; Exenatide; Skeletal muscle; Lipids; Body weight

        R 587.1; R363.2

        A

        10.3969/j.issn.1000-4718.2020.11.001

        1000-4718(2020)11-1921-07

        2020-03-05

        2020-07-01

        國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81970741; No.81670782);國(guó)家自然科學(xué)基金青年科學(xué)基金資助項(xiàng)目(No. 81300705);“廣東特支計(jì)劃”科技青年拔尖人才(No.2016TQ03R590);廣州市科技計(jì)劃珠江新星專項(xiàng)(No.201610010175);中央高?;緲I(yè)務(wù)費(fèi)青年教師重點(diǎn)培育項(xiàng)目(No.16ykzd12)

        Tel: 020-85252073; E-mail: xufen3@mail.sysu.edu.cn

        (責(zé)任編輯:余小慧,羅森)

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