格列喹酮对糖尿病肾脏疾病改善作用的效果观察

目的评价格列喹酮用于糖尿病肾脏疾病(DKD)治疗的临床疗效及安全性。方法将60例DKD患者随机分为格列喹酮组和胰岛素组,比较两组治疗前后尿白蛋白/肌酐比值(ACR)、尿β2微球蛋白(β2-MG)、血糖控制及胰岛功能指标的变化和差异。结果治疗24周后,两组ACR均较治疗前降低(P0.05)。24周时,格列喹酮组ACR低于胰岛素组[5.28(2.74,8.85)vs 11.87(7.18,19.35)mg/mmol,P=0.001]。格列喹酮组尿β2微球蛋白较治疗前下降,且低于胰岛素组[(0.64±0.40)vs(1.24±0.90)mg/L(P0.01)]。两组血糖控制程度相当,但格列喹酮组低血糖事件少于胰岛素组。治疗后格列喹酮组IR改善优于胰岛素组。结论格列喹酮改善DKD的疗效可能与其治疗后肾小球滤过、肾小管重吸收功能的改善、更少低血糖风险及IR的改善等作用相关。     

RAS阻断剂对人前脂肪细胞分化和胰岛素敏感性的影响

目的 探讨肾素-血管紧张素系统(RAS)阻断剂对体外原代培养的人内脏和外周来源的前脂肪细胞分化和胰岛素敏感性的影响. 方法 自16例行电切开腹部手术的健康成年女性腹部皮下和网膜分离前脂肪细胞.分为4组,即无干预的正常对照(NC)组、噻唑烷二酮类约物吡格列酮(Pioglitazone)组、血管紧张素转换酶抑制剂(ACEI)类药物贝那普利(Benazapril)组和血管紧张素Ⅱ受体拮抗剂(ARB)类药物替米沙坦(Telmisartan)组,诱导分化共14 d.观察细胞活力、细胞内脂质含量和前脂肪细胞分化标志物--甘油-3-磷酸脱氧酶的活性.细胞的胰岛素敏感性通过葡萄糖消耗试验测定. 结果 对照组皮下来源的前脂肪细胞活力高于网膜,脂质含量反而低于网膜来源细胞,二者胰岛素敏感性无差别.与对照组相比,贝那普利、替米沙坦和吡格列酮均能明显提高网膜和皮下来源前脂肪细胞的细胞活力和脂质含量,并提高细胞的胰岛素敏感性;其中,替米沙坦组网膜前脂肪细胞的上述各指标均高于吡格列酮组.在网膜,替米沙坦和吡格列酬组的葡萄糖消耗量分别为(5.567±1.612)mmol/L和(4.418±1.572)mmol/L,P=0.020;皮下则相反,两组葡萄糖消耗量分别为(5.335±1.461)mmol/L和(7.506±1.615)mmol/L,P<0.01. 结论 RAS阻断剂(替米沙坦和贝那普利)可促进人前脂肪细胞分化并改善细胞的胰岛素敏感性,且较之吡格列酮在内脏发挥优势性作用.     

吡格列酮预防非肥胖糖尿病鼠胰岛β细胞凋亡的机制研究

目的 探讨吡格列酮预防非肥胖糖尿病小鼠胰岛β细胞凋亡的机制.方法 (1)将4周龄NOD雌鼠分为吡格列酮(21只)及对照(21只)组,分别摄食含0.02％吡格列酮的混合饲料和普通营养饲料.观察52周龄的累积糖尿病发病率.(2)各组取12周龄未发病NOD鼠(n=15)胰腺,HE染色观察胰岛炎;TUNEL+SABC法检测胰岛β细胞凋亡.(3)ELISA法测定血清、脾细胞培养上清IFN-γ和IL-4水平及培养脾细胞核因子PPARγ、NF-κB活性.结果 (1)30、52周龄时,吡格列酮及对照组发病率分别为57.1％和76.2％ 、76.2％和90.5％(均P＞0.05);15周龄时,吡格列酮及对照组发病率分别为4.8％和33.3％(P=0.045).(2) 12周龄时,吡格列酮组正常胰岛和胰岛周围炎比例(14.73％,26.02％)高于对照组(5.69％,15.72％;均P＜0.01),胰岛内炎比例(59.25％)则低于对照组(78.59％,P＜0.01);吡格列酮组胰岛β细胞凋亡率(6.17％±3.62％)低于对照组( 10.62％±4.43％,P=0.008).(3)12周龄NOD鼠吡格列酮组血清IFN-γ水平[(561.05 ±78.61) pg/ml]显著低于对照组[(666.43±28.42) pg/ml,P=0.045];在培养的脾细胞上清中,吡格列酮组IFN-γ水平[(605.84±65.60) pg/ml]显著低于对照组[(692.20±44.98)pg/ml,P=0.041].(4)在培养的脾细胞中,吡格列酮组PPARγ活性(0.06±0.01)高于对照组(0.03±0.01,P=0.013),NF-κB活性(0.03±0.01)较对照显著降低(0.08±0.01,P=0.001).结论 吡格列酮活化PPARγ,抑制NF-κB活性,血清和脾细胞上清IFN-γ下降,Th细胞向Th1方向分化减少,NOD鼠胰岛炎减轻、胰岛β细胞凋亡减少.     

叉头状转录因子O1与吡格列酮干预对肝癌HepG-2细胞葡萄糖消耗的影响

目的研究叉头状转录因子O1（FoxO1）及吡格列酮干预对高胰岛素诱导的肝癌HepG-2细胞葡萄糖消耗的影响及机制。方法1×10^-6mol／L胰岛素培养HepG．2细胞24h后,诱发培养基葡萄糖消耗减少建立细胞模型,将细胞分为对照组、空白质粒组、FoxOlsiRNA载体组、1×10^-5mol／L吡格列酮组。以普通培养基培养的细胞作为空白对照组。37℃、5％CO2培养箱中孵育24h,葡萄糖氧化酶法检测培养基中葡萄糖含量,逆转录-聚合酶链反应（RT—PCR）检测FoxO1mRNA和过氧化物酶体增殖物激活受体-γ（PPAR-γ）mRNA表达,Westernblot法检测PPAR-γ蛋白表达。将FoxO1mRNA、PPAR-γ mRNA、PPAR-γ蛋白表达与葡萄糖消耗量进行相关分析和曲线拟合。采用单因素方差分析及多样本均数两两比较进行统计学分析。结果高胰岛素培养24h较未诱导细胞培养基的葡萄糖消耗降低[分别为（1．36±0．03）和（2．93±0．05）mmol／L,P〈0．01],细胞Fox01mRNA升高（分别为0．513±0．016和0．425±0．011,P〈0．05）,PPAR-γmRNA降低（分别为0．260±0．025和0．441±0．012,P〈0．05）,PPAR-γ蛋白降低（分别为0．312±0．032和0．600±0．046,P〈0．05）。在抑制FoxO1和吡格列酮干预后此趋势有所缓解,逐渐接近于空白对照组。FoxO1mRNA、PPAR-γ mRNA和PPAR-γ蛋白表达与葡萄糖消耗量高度相关。结论FoxO1表达升高或降低对葡萄糖代谢产生不利影响;增强PPAR-γ表达可有效恢复高胰岛素诱导的高糖,增加肝细胞的胰岛素敏感性。     

吡格列酮对L6肌细胞脂联素和葡萄糖转运蛋白4表达的影响及其机制研究

建立棕榈酸诱导的大鼠L6肌细胞胰岛素抵抗模型后,以吡格列酮、c-Jun氨基末端激酶(JNK)抑制剂SP600125、p38丝裂原活化蛋白激酶(p38MAPK)抑制剂SB203580进行干预.应用Western 印迹法检测脂联素、葡萄糖转运蛋白4(GLUT4)蛋白表达和JNK、p38MAPK磷酸化水平.结果显示,吡格列酮可显著增加胰岛素抵抗状态下L6细胞p38MAPK磷酸化、脂联素和GLUT4蛋白表达(P＜0.05或P＜0.01),降低JNK磷酸化水平(P＜0.01).阻断p38MAPK信号通路后,吡格列酮上调L6细胞GULT4蛋白表达的效应显著降低(P＜0.01),而阻断JNK信号通路却无显著影响(P＞0.05).     

PPARγ和糖皮质激素受体对肝细胞糖代谢的影响

以地塞米松、糖皮质激素受体(GR)阻断剂RU486和吡格列酮孵育HepG2细胞24或48h,测定培养液中的葡萄糖浓度.结果显示,48h时10 mol/L RU486阻断地塞米松的升糖作用,但RU486+吡格列酮组较吡格列酮组培养液中葡萄糖浓度升高(P＜0.05),提示PPARγ和GR在肝细胞糖代谢方面可能存在相互作用.     

曲格列酮对胰岛β细胞胰岛素分泌的影响及机制研究

研究曲格列酮对胰岛β细胞(MIN6细胞株)胰岛素分泌的影响,并探讨其机制.10μmol/L曲格列酮短期抑制大鼠胰岛和MIN6细胞的葡萄糖刺激的胰岛素分泌(GSIS,P<0.01),增加AMP活化的蛋白激酶(AMPK)、乙酰辅酶A羧化酶(ACC)的磷酸化水平(均P<0.01),而AMPK抑制剂复合物C可使其AMPK、ACC的磷酸化水平以及胰岛素分泌完全恢复.     

吡格列酮对胰岛素抵抗大鼠血浆同型半胱氨酸水平的影响

目的　研究吡格列酮对高脂饮食诱导的胰岛素抵抗大鼠血浆同型半胱氨酸 (Hcy)水平的影响。方法　将 2 4只Wistar大鼠随机分为 3组 :对照组给予普通饲料喂养 ;高脂组喂高脂饲料诱导胰岛素抵抗动物模型 ;盐酸吡格列酮组在喂饲高脂饲料的同时 ,灌胃给盐酸吡格列酮 10mg·kg-1·d-1。各组喂食 11周时 ,分别做空腹血糖、葡萄糖耐量试验和胰岛素耐量试验后处死动物。测定血糖和胰岛素水平 ,用HOMA模型公式计算胰岛素抵抗指数 (HOMAIR)。喂食 11周后测定血浆Hcy水平。结果　试验后 3组间体重变化、空腹胰岛素、空腹血糖、HOMAIR均有显著差异 (P <0 0 5 )。其中吡格列酮组空腹胰岛素、空腹血糖、HOMAIR均显著低于高脂组 (P <0 0 1)。试验后 3组间血浆Hcy也有显著差异 ,高脂组 (35 7± 14 1) μmol/L高于对照组 (9 95± 2 4 0 ) μmol/L和吡格列酮组 (8 8± 1 39) μmol/L。相关分析显示血糖曲线下面积、空腹胰岛素、空腹血糖、HOMAIR、内脏脂肪含量均与血浆Hcy水平显著相关 (P <0 0 5 )。进一步多元逐步回归分析显示仅空腹血糖 (r =0 5 0 4 ,P =0 0 31)和HOMAIR与血浆Hcy相关 (r=0 30 2 ,P =0 0 4 6 )。结论　高脂诱导的胰岛素抵抗与大鼠血浆Hcy水平升高有关 ,吡格列酮可能通过减轻胰岛素抵抗降低血浆H     

吡格列酮对胰岛素抵抗HepG2细胞胰岛素受体底物表达的影响

目的探讨毗格列酮对胰岛索抵抗（IR）HepG2细胞胰岛素受体底物（IRS）蛋白表达的影响。方法胰岛素抵抗HepG2细胞模型建立后,培养液中加入吡格列酮共同孵育,观察吡格列酮对模型细胞葡萄糖掺入率的影响;应用免疫细胞化学染色法观察吡格列酮对IR HepG2细胞IRS-1、IRS-2表达的影响。结果与模型细胞组比较,1×10^-5mol／L吡格列酮显著提高了HepG2细胞的葡萄糖掺入率（P〈0．01）,使IRHepG2细胞IRS-1、IRS-2蛋白的表达显著增加（P〈0．05）。结论吡格列酮的胰岛素增敏作用可能与胰岛素信号转导分子IRS-1、IRS-2蛋白的表达增强有关。     

受体相互作用蛋白140对吡格列酮改善胰岛β细胞糖脂毒性损伤的影响

目的探讨过氧化物酶增殖活化受体-γ(PPAR-γ)激动剂吡格列酮对胰岛β细胞糖脂毒性损伤的保护及受体相互作用蛋白140(RIP140)在其中的介导机制。方法将胰岛β细胞株MIN6细胞分为NC组、高糖高脂组、吡格列酮干预组。稳定过表达RIP140的MIN6细胞(O-RIP140-MIN6)和过表达绿色荧光蛋白(GFP)的MIN6细胞(GFP-MIN6)。分别予高糖高脂(25 rmmol/L葡萄糖+500μmol/L棕榈酸)和/或10/μmol/L吡格列酮干预。利用MTT分别检测各组细胞增殖率、流式细胞仪检测凋亡率、RT-PCR检测RIP140 mRNA、Western blot检测B淋巴细胞瘤-2(Bcl-2)的表达、硫代巴比妥酸法检测丙二醛(MDA)水平及黄嘌呤氧化酶法检测超氧化合物歧化酶(SOD)含量。结果 NC组、高糖高脂组及吡格列酮干预组MTT吸光值分别为:24 h(1.80±0.04)、(0.95±0.04)及(0.97±0.03);48 h(2.70±0.11)、(1.04±0.06)及(1.30±0.03)。NC组与高糖高脂组比较,差异有统计学意义(24 h:t=25.94,P0.01,48 h:t=24.00,P0.01)。高糖高脂组与吡格列酮干预组比较,差异有统计学意义(48 h:t=9.37,P0.01)。各组间的24 h凋亡率分别为(2.93±0.66)%、(48.08±3.95)%(vs NC组,t=19.54,P0.01)及(31.38±3.92)%(vs高糖高脂组,t=5.20,P0.01)。Bcl-2相对表达量分别为(1.14±0.06)、(0.42±0.02)(vs NC组,t=20.52,P0.01)及(0.86±0.04)(vs高糖高脂组,t=17.71,P0.01)。RIP140表达量分别为(1.13±0.11)、(2.34±0.21)(vs NC组,t=9.69,P0.01)及(1.63±0.13)(vs高糖高脂组(t=5.03,P0.01);高糖高脂组与NC组比较,MDA[(10.13±0.47vs(5.00±0.26)nmol/mg,t=16.57,P0.01]、SOD[(5.15±1.07)协(12.25±1.25)nmol/mg,t=7.51,P0.01]比较,差异均有统计学意义。高糖高脂组与吡格列酮干预组比较,MDA[(10.13±0.47)vs(7.83±0.36)nmol/mg,t=6.77,P0.01]、SOD[(5.15±1.07)v5(8.74±0.59)nmol/mg,t=5.16,P0.01)差异有统计学意义。O-RIP140-MIN6和GFP-MIN6细胞分别给予高糖高脂及吡格列酮处理后,两组MTT吸光值:24 h(1.04±0.07)vs(1.40±0.16)(t=5.01,P0.01),48 h(1.16±0.13)vs(1.98±0.14)(t=10.73,P0.01)。凋亡率为(41.95±4.88)%vs(31.26±2.86)%(t=2.97,P0.05)、Bcl-2相对表达为(0.22±0.04)vs(0.76±0.03)(t=21.54,P0.01),SOD为(7.53±0.71)vs(9.62±0.43)nmol/mg(t=4.36,P0.05),MDA为(10.23±0.28)vs(8.15±0.38)nmol/mg(t=7.63,P0.01)。结论高糖高脂促进胰岛β细胞损伤,吡格列酮通过下调RIP140表达来抑制高糖高脂对胰岛β细胞的损伤。     

Indinavir uncovers different contributions of GLUT4 and GLUT1 towards glucose uptake in muscle and fat cells and tissues

AIMS/HYPOTHESIS: Insulin-dependent glucose influx in skeletal muscle and adipocytes is believed to rely largely on GLUT4, but this has not been confirmed directly. We assessed the relative functional contribution of GLUT4 in experimental models of skeletal muscle and adipocytes using the HIV-1 protease inhibitor indinavir. METHODS: Indinavir (up to 100 micro mol/l) was added to the glucose transport solution after insulin stimulation of wild-type L6 muscle cells, L6 cells over-expressing either GLUT4myc or GLUT1myc, 3T3-L1 adipocytes, isolated mouse brown or white adipocytes, and isolated mouse muscle preparations. RESULTS: 100 micro mol/l indinavir inhibited 80% of both basal and insulin-stimulated 2-deoxyglucose uptake in L6GLUT4myc myotubes and myoblasts, but only 25% in L6GLUT1myc cells. Cell-surface density of glucose transporters was not affected. In isolated soleus and extensor digitorum longus muscles, primary white and brown adipocytes, insulin-stimulated glucose uptake was inhibited 70 to 80% by indinavir. The effect of indinavir on glucose uptake was variable in 3T3-L1 adipocytes, averaging 45% and 67% inhibition of basal and maximally insulin-stimulated glucose uptake, respectively. In this cell, fractional inhibition of glucose uptake by indinavir correlated positively with the fold-stimulation of glucose uptake by insulin, and was higher with sub-maximal insulin concentrations. The latter finding coincided with an increase only in GLUT4, but not GLUT1, in plasma membrane lawns. CONCLUSION/INTERPRETATION: Indinavir is a useful tool to assess different functional contributions of GLUT4 to glucose uptake in common models of skeletal muscle and adipocytes.     

C-reactive protein induces phosphorylation of insulin receptor substrate-1 on Ser<Superscript>307</Superscript> and Ser<Superscript>612</Superscript> in L6 myocytes,thereby impairing the insulin signalling pathway that promotes glucose transport

Aims/hypothesis C-reactive protein (CRP) is associated with insulin resistance and predicts development of type 2 diabetes. However, it is unknown whether CRP directly affects insulin signalling action. To this aim, we determined the effects of human recombinant CRP (hrCRP) on insulin signalling involved in glucose transport in L6 myotubes. Materials and methods L6 myotubes were exposed to endotoxin-free hrCRP and insulin-stimulated activation of signal molecules, glucose uptake and glycogen synthesis were assessed. Results We found that hrCRP stimulates both c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2 activity. These effects were paralleled by a concomitant increase in IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹², respectively. The stimulatory effects of hrCRP on IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹² were partially reversed by treatment with specific JNK and ERK1/2 inhibitors, respectively. Exposure of L6 myotubes to hrCRP reduced insulin-stimulated phosphorylation of IRS-1 at Tyr⁶³², a site essential for engaging p85 subunit of phosphatidylinositol-3 kinase (PI-3K), protein kinase B (Akt) activation and glycogen synthase kinase-3 (GSK-3) phosphorylation. These events were accompanied by a decrease in insulin-stimulated glucose transporter (GLUT) 4 translocation to the plasma membrane, glucose uptake and glucose incorporation into glycogen. The inhibitory effects of hrCRP on insulin signalling and insulin-stimulated GLUT4 translocation were reversed by treatment with JNK inhibitor I and the mitogen-activated protein kinase inhibitor, PD98059. Conclusions/interpretation Our data suggest that hrCRP may cause insulin resistance by increasing IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹² via JNK and ERK1/2, respectively, leading to impaired insulin-stimulated glucose uptake, GLUT4 translocation, and glycogen synthesis mediated by the IRS-1/PI-3K/Akt/GSK-3 pathway.     

Gliclazide protects 3T3L1 adipocytes against insulin resistance induced by hydrogen peroxide with restoration of GLUT4 translocation

Increased oxidative stress under hyperglycemia may contribute to progressive deterioration of peripheral insulin sensitivity. In this study, we investigated whether gliclazide, a second-generation sulfonylurea, can protect 3T3L1 adipocytes from insulin resistance induced by oxidative stress, and whether gliclazide can restore insulin-stimulated glucose transporter 4 (GLUT4) translocation under oxidative stress. We incubated 3T3L1 adipocytes in hydrogen peroxide to produce oxidative stress, then administered various concentrations of gliclazide, N-acetylcystein (NAC), or glibenclamide. Cells treated with these drugs were next exposed to insulin, subsequent glucose uptake was measured, and the insulin-stimulated GLUT4 translocation was monitored in living cells. We found that hydrogen peroxide treatment alone suppressed glucose uptake by insulin stimulation to 65.9%+/-7.8% of the corresponding controls (P<.01). However, addition of 0.1 to 10 micromol/L gliclazide to hydrogen peroxide-treated cells dose-dependently restored glucose uptake, with 5 micromol/L gliclazide significantly restoring glucose uptake to 93.3+/-6.6% (P<.01) even under hydrogen peroxide. Treatment with the known anti-oxidant NAC also dose-dependently (0.1-10 mmol/L) restored insulin-induced glucose uptake in the presence of hydrogen peroxide. However, glibenclamide (0.1-10 micromol/L), another second-generation sulfonylurea, failed to improve glucose uptake. Similarly, treatment with 5 micromol/L gliclazide or 10 mmol/L NAC significantly overcome the reduction in insulin-stimulated GLUT4 translocation by hydrogen peroxide (P<.01), whereas 5 micromol/L glibenclamide did not. Therefore our data regarding gliclazide further characterize its mechanism of hypoglycemic effect: the observed improvements in insulin sensitivity and in GLUT4 translocation indicate that gliclazide counters the hydrogen peroxide-induced insulin resistance in 3T3L1 adipocytes and also would further augment the hypoglycemic effect of this drug as insulinotropic sulfonylurea.     

High leptin levels acutely inhibit insulin-stimulated glucose uptake without affecting glucose transporter 4 translocation in l6 rat skeletal muscle cells

Obesity is a major risk factor for the development of insulin resistance, characterized by impaired stimulation of glucose disposal into muscle. The mechanisms underlying insulin resistance are unknown. Here we examine the direct effect of leptin, the product of the obesity gene, on insulin-stimulated glucose uptake in cultured rat skeletal muscle cells. Preincubation of L6 myotubes with leptin (2 or 100 nM, 30 min) had no effect on basal glucose uptake but reduced insulin-stimulated glucose uptake. However, leptin had no effect on the insulin-induced gain in myc-tagged glucose transporter 4 (GLUT4) appearance at the cell surface of L6 myotubes. Preincubation of cells with leptin also had no effect on insulin-stimulated tyrosine phosphorylation of insulin receptor, IRS-1 and IRS-2, phosphatidylinositol 3-kinase activity, or Akt phosphorylation. We have previously shown that insulin regulates glucose uptake via a signaling pathway sensitive to inhibitors of p38 MAP kinase. Here, leptin pretreatment reduced the extent of insulin-stimulated p38 MAP kinase phosphorylation and phosphorylation of cAMP response element binder, a downstream effector of p38 MAP kinase. These results show that high leptin levels can directly reduce insulin-stimulated glucose uptake in L6 muscle cells despite normal GLUT4 translocation. The mechanism of this effect could involve inhibition of insulin-stimulated p38 MAP kinase and GLUT4 activation.     

Peripheral glucose uptake and skeletal muscle GLUT4 content in man: effect of insulin and free fatty acids.

To investigate the relationship between glucose uptake and the content of the insulin regulatable glucose transporter, GLUT4, in skeletal muscle at near physiological insulin concentrations in vivo, we measured the effect of a 3h euglycemic insulin-infusion (40 mU m-2 min-1) on glucose uptake and skeletal muscle GLUT4 content in 10 healthy subjects. We found no correlation (r approximately 0.1) between individual muscle GLUT4 content and insulin-stimulated glucose uptake. Mean GLUT4 content in skeletal muscle was reduced by 19 +/- 6.3% (mean +/- SE, p less than 0.02) after insulin infusion. However, when the same subjects were made insulin resistant by infusion of lipid, as evidenced by a reduction of 16 +/- 7.2% (mean +/- SE, p less than 0.05), in insulin-stimulated glucose uptake, the effect of insulin on GLUT4 content was attenuated and no change in GLUT4 content was observed. Our results show that the total content of skeletal muscle GLUT4 is a poor predictor for in vivo response to near physiological insulin concentrations in healthy human subjects.     

Effects of adenoviral gene transfer of wild-type, constitutively active, and kinase-defective protein kinase C-lambda on insulin-stimulated glucose transport in L6 myotubes

We used adenoviral gene transfer methods to evaluate the role of atypical protein kinase Cs (PKCs) during insulin stimulation of glucose transport in L6 myotubes. Expression of wild-type PKC-lambda potentiated maximal and half-maximal effects of insulin on 2-deoxyglucose uptake, but did not alter basal uptake. Expression of constitutively active PKC-lambda enhanced basal 2-deoxyglucose uptake to virtually the same extent as that observed during insulin treatment. In contrast, expression of kinase-defective PKC-lambda completely blocked insulin-stimulated, but not basal, 2-deoxyglucose uptake. Similar to alterations in glucose transport, constitutively active PKC-lambda mimicked, and kinase-defective PKC-lambda completely inhibited, insulin effects on GLUT4 glucose transporter translocation to the plasma membrane. Expression of kinase-defective PKC-lambda, in addition to inhibition of atypical PKC enzyme activity, was attended by paradoxical increases in GLUT4 and GLUT1 glucose transporter levels and insulin-stimulated protein kinase B enzyme activity. Our findings suggest that in L6 myotubes, 1) atypical PKCs are required and sufficient for insulin-stimulated GLUT4 translocation and glucose transport; and 2) activation of protein kinase B in the absence of activation of atypical PKCs is insufficient for insulin-induced activation of glucose transport.     

Reduction of insulin-stimulated glucose uptake in L6 myotubes by the protein kinase inhibitor SB203580 is independent of p38MAPK activity

Insulin increases glucose uptake through translocation of the glucose transporter GLUT4 to the plasma membrane. We previously showed that insulin activates p38MAPK, and inhibitors of p38MAPKalpha and p38MAPKbeta (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38alpha and/or p38beta. Here we further explore the possible participation of p38MAPK through a combination of molecular strategies. SB203580 reduced insulin stimulation of glucose uptake in L6 myotubes overexpressing an SB203580-resistant p38alpha (drug-resistant p38alpha) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38alpha or p38beta reduced p38MAPK phosphorylation by 70% but had no effect on insulin-stimulated glucose uptake. Gene silencing via isoform-specific small interfering RNAs reduced expression of p38alpha or p38beta by 60-70% without diminishing insulin-stimulated glucose uptake. SB203580 reduced photoaffinity labeling of GLUT4 by bio-LC-ATB-BMPA only in the insulin-stimulated state. Unless low levels of p38MAPK suffice to regulate glucose uptake, these results suggest that the inhibition of insulin-stimulated glucose transport by SB203580 is likely not mediated by p38MAPK. Instead, changes experienced by insulin-stimulated GLUT4 make it susceptible to inhibition by SB203580.     

Agent and cell-type specificity in the induction of insulin resistance by HIV protease inhibitors

OBJECTIVE: To test agent and cell-type specificity in insulin resistance induced by prolonged exposure to HIV protease inhibitors (HPI), and to assess its relation to the direct, short-term inhibition of insulin-stimulated glucose uptake. METHODS: Following prolonged (18 h) and short (5-10 min) exposure to HPI, insulin-stimulated glucose transport, protein kinase B (PKB) phosphorylation, and GLUT4 translocation were evaluated in 3T3-L1 adipocytes, fibroblasts, L6 myotubes, and L6 cells overexpressing a myc tag on the first exofacial loop of GLUT4 or GLUT1. RESULTS: Prolonged exposure of 3T3-L1 adipocytes to nelfinavir, but not to indinavir or saquinavir, resulted in increased basal lipolysis but decreased insulin-stimulated glucose transport and PKB phosphorylation. In addition, impaired insulin-stimulated glucose uptake and PKB phosphorylation were also observed in the skeletal muscle cell line L6, and in 3T3-L1 fibroblasts. Interestingly, this coincided with increased basal glucose uptake as well as with elevated total-membrane glucose transporter GLUT1 protein content. In contrast to these unique effects of nelfinavir, the mere presence of any of the agents in the 5 min transport assay inhibited insulin-stimulated glucose-uptake activity. This appeared to be caused by direct and specific interaction of the drugs with GLUT4 fully assembled at the plasma membrane, since insulin-stimulated cell-surface exposure of an exofacial myc epitope on GLUT4 was normal. CONCLUSIONS: Independent mechanisms for HPI-induced insulin resistance exist: prolonged exposure to nelfinavir interferes with insulin signaling and alters cellular metabolism of adipocytes and muscle cells, whereas a direct inhibitory effect on insulin-stimulated glucose uptake may occurs through specific interaction of HPI with GLUT4.     

Rosiglitazone produces insulin sensitisation by increasing expression of the insulin receptor and its tyrosine kinase activity in brown adipocytes

Aims/hypothesis Rosiglitazone is used to treat Type 2 diabetes because it improves insulin sensitivity. However, the specific molecular mechanism by which this compound acts has not yet been explained.Methods We used fetal rat primary brown adipocytes cultured for 24 h with or without 10 µmol/l rosiglitazone and further stimulated for 5 min with 10 nmol/l insulin. Next we measured glucose uptake and GLUT4 translocation and submitted the cells to lysis, immunoprecipitation and immunoblotting in order to measure the insulin signalling cascade.Results Rosiglitazone noticeably activated basal glucose uptake in a manner dependent on p38-mitogen-activated protein kinase. Rosiglitazone also produced a 40% increase in insulin-stimulated glucose uptake as a result of increased GLUT4 translocation to the plasma membrane. This happened without changes in the expression of GLUT4 at the mRNA or protein level. This effect correlated with the potentiation by rosiglitazone of insulin-stimulated Tyr phosphorylation of insulin receptor substrate-1 and to a greater extent of insulin receptor substrate-2. It also correlated with the subsequent activation of phosphatidylinositol 3-kinase and Akt, without changes in protein kinase C activity. Rosiglitazone treatment increased insulin receptor expression and insulin-stimulated Tyr phosphorylation of insulin receptor beta-chain, but decreased insulin-stimulated Ser phosphorylation. It also potentiated insulin-induced Tyr phosphorylation of insulin receptor beta-chain and protein tyrosine phosphatase 1B in co-immunoprecipitates and impaired insulin activation of protein tyrosine phosphatase 1B activity.Conclusions/interpretation At the insulin receptor level, rosiglitazone-induced improvements of insulin sensitivity result from two convergent mechanisms: increased insulin receptor expression and insulin receptor activation.Abbreviations TZDs Thiazolidinediones - IR insulin receptor - PPAR peroxisome proliferating activated receptor - PI phosphatidylinositol - MAPK mitogen-activated protein kinase - PKC protein kinase C - PTP protein tyrosine phosphatase - FAS fatty acid synthase - UCP uncoupling protein - MBP myelin basic protein - MEM minimal essential medium