2型糖尿病小鼠骨骼肌细胞受体后胰岛素信号转导蛋白的下降调节

目的研究2型糖尿病小鼠骨骼肌细胞受体后胰岛素信号转导蛋白:胰岛素受体底物-1(IRS-1)、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(PKB,Akt)的蛋白表达和磷酸化情况。方法雌性C57BL/6J小鼠予高脂、高糖膳食,制成2型糖尿病动物模型,提取完整的骨骼肌用胰岛素刺激2、15和30min。Westernblot技术检测IRS-1、PI3-Kp85α、PKB的蛋白水平,免疫沉淀技术检测IRS-1酪氨酸磷酸化水平。结果对照组、2型糖尿病模型组骨骼肌细胞的信号转导蛋白IRS-1、PKB未发现数量上的不同,2型糖尿病模型组的PI3Kp85的蛋白比对照组明显减少(P<0·05),在基础状态下,对照组和2型糖尿病模型组的IRS-1、PKB磷酸化水平相似,但2型糖尿病模型组胰岛素刺激后的这些蛋白磷酸化反应曲线上升幅度较之对照组明显降低。结论2型糖尿病小鼠骨骼肌细胞存在受体后胰岛素信号传导蛋白的下降调节。     

GDF11在棕榈酸诱导骨骼肌细胞胰岛素抵抗中的作用研究

目的探讨GDF11对棕榈酸诱导骨骼肌细胞胰岛素抵抗的影响。方法用棕榈酸构建骨骼肌细胞胰岛素抵抗模型,分为对照组、GDF11干预组、棕榈酸干预组和GDF11联合棕榈酸干预组。CCK-8检测细胞活力,2NBDG检测细胞葡萄糖摄取。实时荧光定量PCR检测肌管标志基因(desmin、myogenin),胰岛素介导葡萄糖摄取相关基因(GLUT-4、IRS-1)及PGC-1α的表达。Western blot检测PGC-1α蛋白水平的表达。结果不同浓度GDF11对骨骼肌细胞活力无明显影响。与对照组相比,棕榈酸干预组葡萄糖摄取及GLUT-4、IRS-1、PGC-1α的表达明显降低(P<0.05)。与棕榈酸干预组相比,GDF11联合棕榈酸干预组葡萄糖摄取及GLUT-4、IRS-1、PGC-1α的表达无明显变化。结论棕榈酸可成功诱导骨骼肌细胞胰岛素抵抗,而GDF11对骨骼肌细胞胰岛素抵抗没有明显改善作用。     

罗格列酮对胰岛素抵抗大鼠骨骼肌蛋白激酶B表达的影响

目的 :探讨胰岛素增敏剂罗格列酮对胰岛素抵抗大鼠骨骼肌蛋白激酶B(PKB)表达的影响。方法 :应用高脂饲料喂养复制胰岛素抵抗大鼠模型。应用Westernblotting方法检测骨骼肌中PKB的表达。结果 :持续高脂饲料喂养组大鼠产生了明显胰岛素抵抗 ,骨骼肌PKB的表达较正常对照组显著降低 (OD值 8.2 4±s 0 .11vs 9.36± 0 .18,P <0 .0 1) ,罗格列酮治疗组和正常饲料替代组大鼠胰岛素抵抗明显改善 ,PKB的表达较持续高脂饲料喂养组大鼠明显增加 (OD值分别为 8.89± 0 .0 8,8.4 0± 0 .0 9vs 8.2 4± 0 .11,P <0 .0 1)。结论 :高脂喂养的大鼠胰岛素抵抗的产生与骨骼肌胰岛素刺激的PKB表达降低有明显关系 ,罗格列酮能显著增加已经降低了的胰岛素抵抗大鼠骨骼肌中PKB的表达 ,部分恢复受损的胰岛素信号转导 ,进而明显改善胰岛素抵抗 ,这可能也是罗格列酮减轻胰岛素抵抗的作用机制之一。     

巨噬细胞条件培养基对胰岛素调节骨骼肌细胞作用的影响

目的:探讨常氧和缺氧培养的巨噬细胞条件培养基对胰岛素调节骨骼肌葡萄糖转运子4(GLUT4)作用的影响。方法:常氧和缺氧培养巨噬细胞,提取条件培养基孵育C2C12GLUT4myc骨骼肌细胞,用偶联抗体的吸光度法测定细胞膜上GLUT4myc的含量,Real-timePCR法测定巨噬细胞TNF-αmRNA的表达,ELISA法测定巨噬细胞条件培基中TNF-α的含量。结果:常氧和缺氧培养的巨噬细胞条件培养基削弱胰岛素刺激的骨骼肌细胞GLUT4myc转位(P<0.01),但两种条件培养基对骨骼肌中胰岛素作用的影响差异无统计学意义;缺氧培养的巨噬细胞TNF-αmRNA和蛋白表达均高于常氧培养的巨噬细胞(P<0.05或P<0.01)。结论:常氧培养和缺氧培养的巨噬细胞条件培养基均直接造成骨骼肌细胞胰岛素抵抗,缺氧的巨噬细胞条件培养基并不能加重骨骼肌胰岛素抵抗。     

脂联素影响骨骼肌胰岛素抵抗模型中葡萄糖转运蛋白4表达的研究

目的：通过建立骨骼肌L6细胞胰岛素抵抗模型,探讨骨骼肌L6细胞中脂联素（ APN）的分泌,以及脂联素对骨骼肌胰岛素抵抗模型中葡萄糖转运蛋白4（GLUT4）表达的影响。方法（1）体外培养大鼠L6成肌细胞,诱导分化后,给予不同浓度的棕榈酸（PA）,测定不同时间细胞培养上清液葡萄糖浓度,观察PA对L6细胞摄取葡萄糖的影响,建立胰岛素抵抗模型;（2）根据实验条件的不同分为三组：NC组（正常对照组,即骨骼肌L6细胞组）;IR组（胰岛素抵抗模型组）;IR＋PIO组（胰岛素抵抗模型＋吡格列酮组）。应用Western blot方法分别测定上述三组APN和GLUT4表达水平。结果（1）0．4 mmol· L^-1的棕榈酸在作用12、24、36 h以及0．6～0．8 mmol· L^-1棕榈酸作用8～36 h后,细胞培养上清液中葡萄糖含量,明显高于对照组。胰岛素抵抗模型建立;（2）Western blot结果显示：①与NC组比较,IR组APN和GLUT4表达均减少,差异有统计学意义;②与IR组比较,IR＋PIO组其APN和GLUT4表达均增加,差异有统计学意义。结论（1）大鼠L6成肌细胞培养并诱导分化后,经过一定条件下PA刺激,可以建立胰岛素抵抗模型;（2）大鼠L6细胞可分泌和表达脂联素,骨骼肌源性脂联素上调L6细胞GLUT4的表达;（3）吡格列酮作为PPAR-γ激动剂,可增加大鼠L6细胞脂联素的分泌,进而改善胰岛素敏感性。     

褪黑素通过Akt抑制胰岛素抵抗HepG2细胞糖内生

目的探讨褪黑素(melatonin,MLT)对胰岛素抵抗(insulin resistance,IR)肝HepG2细胞葡萄糖内生的影响及其机制。方法HepG2 IR细胞模型采用高糖(25 mmol·L-1)联合高胰岛素(1μmol·L-1)培养诱导建立。MLT(10 nmol·L-1)处理模型细胞6 h后检测糖消耗及糖原含量,GSK-3β、Akt和FoxO1蛋白磷酸化水平检测采用Western blot,免疫荧光法检测FoxO1蛋白核外排情况。结果IR HepG2细胞经MLT处理后,葡萄糖的摄取和糖原合成增加,p-GSK-3β和p-Akt蛋白水平分别增高约66%和48%,FoxO1磷酸化水平明显提高且细胞质含量增加。结论MLT可能通过Akt/GSK-3β及Akt/FoxO1信号通路促进胰岛素抵抗HepG2细胞的糖原合成和抑制糖异生,从而改善糖代谢。     

倍他福林对胰岛素抵抗HepG2细胞模型的作用及其初步机制

目的:探讨倍他福林(betaphrine)对胰岛素抵抗的作用及其机制.方法:将HepG2细胞置于5×10-7mol·L-1胰岛素培养液中16 h,观察高胰岛素对HepG2细胞葡萄糖消耗量的影响.模型建立后,培养液中加入倍他福林共同孵育,观察倍他福林对胰岛素抵抗细胞模型葡萄糖消耗量以及培养液中甘油的含量.并用Western blotting法测定各组细胞内葡萄糖转运蛋白-4(GLUT-4)的表达.结果:与正常对照组相比,模型组细胞葡萄糖消耗量减少,培养液中甘油的含量增加,细胞内葡萄糖转运蛋白-4的表达减少.与模型组相比,倍他福林作用组细胞葡萄糖消耗量增加,培养液中甘油的含量减少,细胞内葡萄糖转运蛋白-4的表达增加.结论:倍他福林可改善胰岛素抵抗HepG2细胞模型初步的胰岛素抵抗性.     

葛根素对胰岛素抵抗大鼠骨骼肌中蛋白激酶B表达影响

目的　建立胰岛素抵抗模型。观察葛根素注射液对模型大鼠骨骼肌丝氨酸 /苏氨酸蛋白激酶B(PKB/Akt)表达的影响。方法　选取 3 0只SD大鼠 ,随机设为正常对照组和模型组。应用高脂饮食制备胰岛素抵抗大鼠模型。并在 8wk后将其分为葛根素组 (腹腔注射葛根素注射液 10 0mg·kg- 1·d- 1)及病理对照组。实验结束时采用Western Blotting免疫印迹法检测葛根素组骨骼肌中PKB的表达水平。结果　 (1)高脂饮食喂养后 8wk ,模型组出现腹型肥胖 ,高血糖、高胰岛素血症 ,胰岛素抵抗指数 (HOMA IR)升高 ,胰岛素敏感指数 (ISI)下降 (P <0 0 1) ,表现为胰岛素抵抗 ;(2 )葛根素治疗后 4wk与病理对照组相比 :空腹血糖、胰岛素、胰岛素抵抗指数降低 ,胰岛素敏感指数上升 ,内脏脂肪重量及占体重百分比下降 ,骨骼肌中PKB表达水平提高 (P <0 0 1)。结论　葛根素可增加PKB表达并改善胰岛素抵抗 ,该作用可能与其增强胰岛素生物效应 ,减弱脂毒性对胰岛素信号通路抑制等机制有关     

厄贝沙坦对高糖诱导肾小管上皮细胞转分化中GSK-3β表达的影响

目的探讨糖原合成酶激酶-3β(glycogen synthase ki-nase-3β,GSK-3β)在高糖诱导肾小管上皮细胞转分化中的表达及血管紧张素Ⅱ受体1拮抗剂(AT1Ra)厄贝沙坦对其活性的影响。方法体外培养人近端肾小管上皮细胞(HKC),分为正常糖组、甘露醇对照组、高糖组、高糖+厄贝沙坦干预组。采用免疫细胞化学观察磷酸化GSK-3β(p-GSK-3β)在肾小管细胞表达情况;Western blot检测GSK-3β、p-GSK-3β、E-钙粘蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)表达水平;逆转录-聚合酶链反应(RT-PCR)检测GSK-3β和TGF-β1mRNA表达。结果高糖组较正常糖及甘露醇对照组p-GSK-3β、α-SMA蛋白及TGF-β1mRNA表达增高,E-cadherin表达降低。高糖刺激细胞12hp-GSK-3β表达增强,24h达到高峰。厄贝沙坦能够下调高糖诱导的p-GSK-3β、α-SMA蛋白及TGF-β1mRNA表达,同时逆转E-cadherin下降水平。结论GSK-3β可能参与了高糖诱导的肾小管上皮细胞转分化过程,厄贝沙坦抑制该过程可能是通过调节GSK-3β的活性而实现的。     

丹皮酚对PA诱导的大鼠血管内皮细胞的胰岛素抵抗和氧化应激的拮抗作用

目的研究丹皮酚在棕榈酸(PA)诱导胰岛素抵抗中对葡萄糖摄取、氧化应激中的改善作用。方法通过PA诱导建立主动脉内皮细胞(RAECs)胰岛素抵抗模型,通过MTT法等实验研究了丹皮酚对PA处理后细胞活性以及葡萄糖摄取的影响,通过荧光法检测了RAECs中ROS产物水平,通过ELISA和蛋白免疫实验研究了丹皮酚对PA处理后RAECs炎症反应和细胞凋亡的影响。结果丹皮酚对RAECs细胞活性没有影响,可明显提高其葡萄糖摄取能力。荧光素酶实验结果显示,PA处理后RAECs细胞内ROS产物水平明显升高,丹皮酚可明显逆转这些改变。丹皮酚可降低PA诱导后TNF-α、 IL-6和IL-1β的水平,并可明显降低促凋亡因子Bax表达和Cleaved Caspase-9/Caspase-9比值,并增高抗凋亡因子Bcl-2的表达水平。结论丹皮酚可明显提高PA诱导后葡萄糖摄取能力,降低RAECs细胞内ROS水平,降低细胞炎症反应和细胞凋亡水平。     

过表达SOCS3对西格列汀改善胰岛素抵抗作用的影响

目的:探究过表达SOCS3对西格列汀(sitagliptin,SITA)在改善脂质代谢和氧化应激,以减轻棕榈酸(palrnitic acid,PA)介导的HepG2细胞胰岛素抵抗(insulin resistance,IR)中的作用。方法:MTT法检测PA与SITA对肝癌细胞HepG2增殖活性的影响;将HepG2分为4组:Control组,PA组,PA+SITA+pEX-RB-NC组和PA+SITA+pEX-RB-SOCS3组;油红O染色检测细胞中脂质的积聚水平,RT-PCR检测细胞中SREBP1c和PPARα的mRNA表达,DCFH-DA法检测细胞中ROS的水平,RT-PCR与试剂盒检测细胞中CAT与GPx的mRNA表达与酶活性;Western blot试验检测细胞IR通路中p-IRS-1^Ser307/IRS-1、p-AKT^Ser473/AKT、p-GSK3β^Ser9/GSK3β的比值。结果:与Control组相比,SITA能削弱PA诱导的HepG2细胞活力损伤(P＜0.05),且PA组、PA+SITA+pEX-RB-NC组和PA+SITA+pEX-RB-SOCS3组中富含大量红色脂滴,SREBP1c与PPARα的mRNA水平、ROS、CAT与GPx表达及p-IRS-1^Ser307/IRS-1、p-AKT^Ser473/AKT、p-GSK3β^Ser9/GSK3β比值均明显升高(P＜0.05);但与PA组相比,PA+SITA+pEX-RB-NC组除CAT、GPx及p-IRS-1^Ser307/IRS-1、p-AKT^Ser473/AKT、p-GSK3β^Ser9/GSK3β比值升高外(P＜0.05),其他检测指标均显著降低(P＜0.05),PA+SITA+pEX-RB-SOCS3组中上述指标均无明显改变(P＞0.05)。结论:SITA可抑制脂质代谢和氧化应激,从而以减轻PA介导的肝细胞IR,而该作用能够被过表达SOCS3所抵消。     

β-Adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKA

Background and purpose:

Genetic approaches have documented protein kinase B (PKB) as a pivotal regulator of heart function. Insulin strongly activates PKB, whereas adrenaline is not considered a major physiological regulator of PKB in heart. In skeletal muscles, however, adrenaline potentiates insulin-stimulated PKB activation without having effect in the absence of insulin. The purpose of the present study was to investigate the interaction between insulin and β-adrenergic stimulation in regulation of PKB phosphorylation.

Experimental approach:

Cardiomyocytes were isolated from adult rats by collagenase, and incubated with insulin, isoprenaline, and other compounds. Protein phosphorylation was evaluated by Western blot and phospho-specific antibodies.

Key results:

Isoprenaline increased insulin-stimulated PKB Ser⁴⁷³ and Thr³⁰⁸ phosphorylation more than threefold in cardiomyocytes. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3β Ser⁹ phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (β₁-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (β₂-agonist) only potentiated insulin-stimulated PKB phosphorylation by approximately 80%. Dobutamine, but not salbutamol, increased phospholamban Ser¹⁶ phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogue that activates PKA (dibutyryl-cAMP and N⁶-benzoyl-cAMP) increased insulin-stimulated PKB phosphorylation by more than threefold without effect alone. The Epac-specific activator 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (007) increased insulin-stimulated PKB phosphorylation by approximately 50%. Db-cAMP and N⁶-benzoyl-cAMP, but not 007, increased phospholamban Ser¹⁶ phosphorylation.

Conclusions and implications:

β-adrenoceptors are strong regulators of PKB phosphorylation via cAMP and PKA when insulin is present. We hypothesize that PKB mediates important signalling in the heart during β-adrenergic receptors stimulation.     

Inhibition of protein kinase B by Palmitate in the insulin signaling of HepG2 cells and the preventive effect of Arachidonic acid on insulin resistance

Elevated plasma levels of free fatty acids (FFAs) may contribute to insulin resistance (IR) that is characteristic of type 2 diabetes mellitus. In this study, we investigated the effects of two fatty acids, palmitate (PA) and arachidonic acid (AA) on glycogenesis under insulin signaling in HepG2cells, a transformed hepatic carcinoma cell line. In the presence of 200 μmol of palmitate, insulin (10⁻⁷ mol/L) stimulation of glycogenesis was inhibited, as evidenced by increased glucose in the medium and decreased intracellular glycogen. Wortmannin (WM), a specific inhibitor of PI3K, dramatically decreased the amount of intracellular glycogen in cells without PA incubation. However, glycogen in PA treated cells was not significantly changed by WM, indicating that PA may also act on PI3K. Interestingly, AA restored the effects of WM inhibition on glycogenesis in PA cells. Western blot analysis demonstrated that PA in the absence of WM increased phosphorylated glycogen synthase (inactive form of GS) and decreased phosphorylated protein kinase B (active form of PKB), causing a reduction of intracellular glycogen. AA, however, reversed the effects of PA on GS and PKB. Furthermore, inhibition of protein kinase C (PKC) by a specific inhibitor chelerythrine chloride (CC) abolished the inhibitory effect of PA on glycogen synthesis by decreasing phosphorylated GS and increasing phosphorylated PKB. However, the effect of CC in the presence of PA disappeared when AA was also present. Our results suggest that there is a disruption of the insulin signaling pathway between PKB and GS when the cells were exposed to PA, contributing to IR. PA may also interrupt the PKC signaling pathway. In contrast, AA could rescue glycogenesis impaired by PA.     

T2DM 小型猪模型骨骼肌、 肝脏及胰腺组织蛋白激酶 B 表达及磷酸化的变化

摘要： 目的 研究高脂高糖饮食联合链脲佐菌素 （STZ） 构建巴马小型猪 2 型糖尿病 （T2DM） 模型的可行性, 并观 察小型猪模型骨骼肌、 肝脏及胰腺组织蛋白激酶 B（PKB）表达及磷酸化变化。方法 将健康雄性巴马小型猪 10 只 随机分为 2 组： 对照组 5 只, 喂养普通饲料; 糖尿病模型组 5 只, 高脂高糖饮食联合 STZ 构建巴马小型猪 T2DM 模 型。检测 2 组小型猪空腹血糖、 空腹胰岛素水平, 并计算胰岛素抵抗指数 （HOMA-IR）。采用 Western blot 法检测骨 骼肌、 肝脏及胰腺组织中 PKB 蛋白表达及磷酸化水平。结果 （1） 高脂高糖饲料喂养 10 个月后, 与对照组比较, 模 型组体质量、 空腹血糖、 空腹胰岛素水平及 HOMA-IR 明显升高 （P＜0.01）。（2） 与对照组相比, 模型组 STZ 给药后空 腹血糖水平进一步升高, 空腹胰岛素水平明显下降 （P＜0.01）。（3） 与对照组比较, 模型组骨骼肌、 肝脏及胰腺组织中 PKB 蛋白表达及磷酸化水平明显降低 （P＜0.05）。结论 高脂高糖饮食联合 STZ 多次腹腔注射可以成功构建 T2DM 小型猪模型, 模型组小型猪骨骼肌、 肝脏及胰腺组织 PKB 蛋白表达及磷酸化水平明显降低     

Berberine improves intralipid-induced insulin resistance in murine

Insulin resistance (IR) is a major metabolic risk factor even before the onset of hyperglycemia. Recently, berberine (BBR) is found to improve hyperglycemia and IR. In this study, we investigated whether BBR could improve IR independent of hyperglycemia. Acute insulin-resistant state was induced in rats by systemic infusion of intralipid (6.6%). BBR was administered via different delivery routes before or after the beginning of a 2-h euglycemic-hyperinsulinemic clamp. At the end of experiment, rats were sacrificed, gastrocnemius muscle was collected for detecting mitochondrial swelling, phosphorylation of Akt and AMPK, as well as the mitochondrial permeability regulator cyclophilin D (CypD) protein expression. We showed that BBR administration markedly ameliorated intralipid-induced IR without affecting blood glucose, which was accompanied by alleviated mitochondrial swelling in skeletal muscle. We used human skeletal muscle cells (HSMCs), AML12 hepatocytes, human umbilical vein endothelial cells, and CypD knockout mice to investigate metabolic and molecular alternations. In either HSMCs or AML12 hepatocytes, BBR (5 μM) abolished palmitate acid (PA)-induced increase of CypD protein levels. In CypD-deficient mice, intralipid-induced IR was greatly attenuated and the beneficial effect of BBR was diminished. Furthermore, we demonstrated that the inhibitory effect of BBR on intralipid-induced IR was mainly mediated by skeletal muscle, but not by intestine, liver, or microvasculature; BBR administration suppressed intralipid-induced upregulation of CypD expression in skeletal muscle. These results suggest that BBR alleviates intralipid-induced IR, which is related to the inhibition of CypD protein expression in skeletal muscle.