Exercise-induced AMPK activity in skeletal muscle: Role in glucose uptake and insulin sensitivity

The energy/fuel sensor 5′-AMP-activated protein kinase (AMPK) is viewed as a master regulator of cellular energy balance due to its many roles in glucose, lipid, and protein metabolism. In this review we focus on the regulation of AMPK activity in skeletal muscle and its involvement in glucose metabolism, including glucose transport and glycogen synthesis. In addition, we discuss the plausible interplay between AMPK and insulin signaling regulating these processes.     

The effect of hyperglycaemia on glucose disposal and insulin signal transduction in skeletal muscle

Skeletal muscle is an important tissue for the proper maintenance of glucose homeostasis as it accounts for the major portion of glucose disposal following infusion or ingestion of glucose. Thus, cellular mechanisms regulating glucose uptake in skeletal muscle have a major impact on whole-body glucose homeostasis. Glucose transport into skeletal muscle is a rate-limiting step for glucose utilization under physiological conditions and a site of insulin resistance in patients with non-insulin-dependent diabetes mellitus (NIDDM). Defects in insulin signalling have been coupled to impaired glucose uptake in skeletal muscle from NIDDM patients. Although the exact aetiology is unclear, genetic and environmental (high-energy diets combined with a sedentary lifestyle) factors contribute to the onset of NIDDM. Furthermore, hyperglycaemia is linked with insulin resistance. This chapter will consider mechanisms for glucose disposal in skeletal muscle, potential sites of insulin resistance in skeletal muscle in NIDDM patients and the impact of hyperglycaemia on insulin action.     

脂肪因子Omentin-1、Visfatin体外表达与老年2型糖尿病胰岛素抵抗的关系

目的探讨脂肪因子Omentin-1、Visfatin与老年2型糖尿病(T2DM)胰岛素抵抗(IR)的内在联系。方法复苏、传代及诱导分化老年人源T2DM前脂肪细胞,构建Omentin-1、Visfatin过表达载体,进行载体转化、培养和提取;以3个不同过表达梯度(1.0、2.5、5.0μg)转染传代脂肪细胞,以无转染组为对照;Q-PCR检测Omentin-1、Visfatin、胰岛素受体底物(IRS)-1/2、磷脂酰肌醇-3激酶[PI3K(P85a)]mRNA表达,Western印迹检测Omentin-1、Visfatin、IRS-1、IRS-2、PI3K(P85a)蛋白表达及IRS-1/2酪氨酸磷酸化水平,[~3H]-2-脱氧-D-葡萄糖摄取测定细胞葡萄糖摄取率的变化。结果 (1)Omentin-1、Visfatin mRNA及蛋白表达随转染浓度梯度升高而表达量增加,所构建的Omentin-1、Visfatin过表达载体有效;(2)随Omentin-1表达增加,IRS-1及PI3K(P85a)mRNA和蛋白表达及IRS-1磷酸化程度均出现明显增加,IRS-2未出现明显变化;(3)随Visfatin表达增加,IRS-1/2、PI3K(P85a)mRNA、蛋白表达及IRS-1/2磷酸化程度未出现明显变化;(4)脂肪细胞的葡萄糖摄取率随Omentin-1表达增加而升高,而与Visfatin过表达浓度变化无明显关系。结论 Omentin-1表达可能通过某种途径激活了胰岛素受体后信号传导通路,导致了IRS-1和PI3K活化和表达,从而发挥其胰岛素增敏作用;Visfatin在老年T2DM血清中的高表达,很可能只是一种肥胖、T2DM糖脂代谢紊乱的补偿机制,与T2DM IR的发生没有必然联系。     

Defects in insulin signal transduction in liver and muscle of pregnant rats

Summary Pregnancy is known to induce insulin resistance, but the exact molecular mechanism involved is unknown. In the present study, we have examined the levels and phosphorylation state of the insulin receptor and of insulin receptor substrate 1(IRS-1), as well as the association between IRS-1 and phosphatidylinositol 3-kinase (PI 3-kinase) in the liver and muscle of pregnant rats (day 20 of gestation) by immunoprecipitation and immunoblotting with anti-insulin receptor, anti-IRS-1, anti-PI 3-kinase and anti-phosphotyrosine antibodies. There were no changes in the insulin receptor concentration in the liver and muscle of pregnant rats. However, insulin stimulation of receptor autophosphorylation, as determined by immunoblotting with antiphosphotyrosine antibody, was reduced by 30 ± 6 % (p < 0.02) in muscle and 36 ± 5 % (p < 0.01) in liver at day 20 of gestation. IRS-1 protein levels decreased by 45 ± 6 % (p < 0.002) in liver and by 56 ± 9 % (p < 0.002) in muscle of pregnant rats. In samples previously immunoprecipitated with anti-IRS-1 antibody and blotted with antiphosphotyrosine antibody, the insulin-stimulated IRS-1 phosphorylation levels in the muscle and liver of pregnant rats decreased by 70 ± 9 % (p < 0.01) and 75 ± 8 % (p < 0.01), respectively. The insulin-stimulated IRS-1 association with PI 3-kinase decreased by 81 ± 6 % in muscle (p < 0.01) and 79 ± 11 % (p < 0.01) in the liver during pregnancy. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in pregnancy. [Diabetologia (1997) 40: 179–186] Received: 3 April 1996 and in revised form 5 November 1996     

Effects of electroacupuncture on mRNA expressions of insulin-receptor substrates 1 and 2 in the endometrium of PCOS rats and insulin sensitivity

<正>Objective To observe the effects of electroacupuncture(EA)on gene expressions of insulin receptor substrate 1(IRS1)and insulin receptor substrate 2(IRS2)in the endometrium and insulin sensitivity(IS)of polycystic ovary syndrome(PCOS)rats,thereby providing basic evidence of clinical application of EA for improving     

Effect of hyperglycemia on signal transduction in skeletal muscle from diabetic Goto-Kakizaki rats

We determined basal and insulin-stimulated responses on signaling intermediates in soleus skeletal muscle from male Wistar and diabetic Goto-Kakizaki (GK) rats. Rats were infused with glucose (5 or 20 mm) for 3 h, followed by a continuous infusion of saline or insulin (3 U/kg.h) for 20 min. Under euglycemic and hyperglycemic conditions, basal and insulin-stimulated action on phosphatidylinositol (PI) 3-kinase, protein kinase B/Akt, and ERK were reduced in GK rats, whereas insulin-stimulated protein kinase C (PKC)zeta activity was not altered. Interestingly, basal PKCzeta activity was increased under hyperglycemic conditions in GK and Wistar rats. This finding of increased PKCzeta activity was confirmed in vitro in isolated soleus muscle exposed to high extracellular glucose, and occurred concomitant with an increase in PI-dependent kinase 1 (PDK-1) activity. The glucose effects were not specific to PKCzeta, because an increase in phosphorylation of PKCalpha/beta and PKCdelta, but not PKCtheta, in isolated soleus muscle exposed to 25 mm glucose was observed. In conclusion, insulin signaling defects in diabetic GK rats are not corrected by an acute normalization of glycemia. Interestingly, acute hyperglycemia leads to a parallel increase in PDK-1, PKCalpha/beta, PKCdelta, and PKCzeta phosphorylation/activity via a PI 3-kinase-protein kinase B/Akt-independent mechanism. The long-term consequence of elevated PDK-1 and PKC phosphorylation/activity should be considered in the context of diabetes mellitus, as hyperglycemia is a clinical feature of this disease.     

胰岛素促进结肠平滑肌细胞合成干细胞因子的信号转导途径

目的 探讨胰岛素(Ins)促进结肠平滑肌细胞(SMC)表达干细胞因子(SCF)的细胞内信号传导途径.方法 分离培养SD大鼠结肠SMC,采用α-actin免疫荧光鉴定.分3大组进行实验.①Ins浓度梯度组:SMC添加不同浓度Ins(0、2.5、5、20、40和80 mg/L),培养16 h.②Ins时间梯度组:SMC+Ins(40 mg/L),分别培养0、8、16和24 h.③信号通路阻断组:SMC加LY-294002或PD-98059预处理后,再予以40 mg/L的Ins诱导16h.四甲基偶氮唑盐(MTT)法检测结肠SMC增殖情况,Western印迹和反转录PCR检测SCF的变化.结果 ①Ins在5 mg/L浓度即能显著促进SMC增殖,其效应与20、40和80 mg/L时差异无统计学意义(P值均＜0.05).②与未添加Ins组(即空白对照组)比较,低中剂量Ins(2.5、5和20 mg/L)即有促进结肠SMC表达SCFmRNA和SCF蛋白的作用(P＜0.05),这种作用在Ins 40 mg/L时达到最大(P＜0.05),80 mg/L时与40mg/L时差异无统计学意义(P＞0.05).③与0h时相比,40mg/L的Ins作用于SMC 8 h后SCF表达增加(P＜0.05),16 h后达峰值(P＜0.05),24 h与16 h差异无统计学意义(P＞0.05).④与空白对照组和溶剂对照组相比,LY-294002、PD-98059处理16 h,均能减少Ins诱导的SCF的表达(P值均＜0.05).结论 Ins能促进SMC增殖;在一定范围内呈剂量与时间依赖性诱导结肠SMC合成SCF,该效应可能与PI3K与MAPK两条信号通路有关.     

胰岛素对大鼠骨骼肌细胞脂联素受体基因表达的影响

目的了解体外胰岛素对原代培养大鼠骨骼肌细胞脂联素受体1表达的影响。方法体外原代培养骨骼肌细胞,应用SYBRGreenⅠ染料建立一种快速、可靠的实时定量PCR,对其主要要素进行优化。观察不同胰岛素浓度不同作用时间下,大鼠骨骼肌细胞脂联素受体基因表达水平的动态变化。结果建立敏感、特异、快速检测脂联素受体1mRNA的实时定量PCR方法,随着胰岛素浓度的增加,脂联素受体1表达逐渐降低。在较低浓度（胰岛素浓度〈1nmol/L）时,脂联素受体1表达的降低无统计学意义,当胰岛素浓度增加到10nmol/L及以上时,骨骼肌细胞脂联素受体1表达的降低有统计学意义（P〈0.05）,这种抑制作用1h后出现,24h后达到高峰。结论成功地建立SYBRGreenⅠ实时定量PCR检测脂联素受体基因的表达方法,体外高胰岛素对骨骼肌细胞脂联素受体1mRNA表达有抑制作用,并呈时间和剂量依赖性。     

饮酒对大鼠骨骼肌胰岛素受体及其底物表达和磷酸化的影响

观察摄入不同剂量的酒精5个月后,大鼠骨骼肌胰岛素刺激后葡萄糖摄取能力和胰岛素受体(IR)、IR底物(IRS)1及IRS-2的表达及胰岛素刺激后酪氨酸磷酸化水平的变化,发现饮酒可降低骨骼肌胰岛素刺激后糖摄取,同时伴有IR、IRS-1和IRS-2表达及酪氨酸磷酸化水平代偿性上调。     

Down-regulation of insulin receptor substrates (IRS)-1 and IRS-2 and Src homologous and collagen-like protein Shc gene expression by insulin in skeletal muscle is not associated with insulin resistance or type 2 diabetes.

To examine whether altered gene expression of insulin receptor substrates (IRS)-1 and IRS-2 and Src homologous and collagen-like protein Shc is an inherited trait and is associated with muscle insulin resistance or type 2 diabetes, we measured mRNA levels of these genes by a relative quantitative RT-PCR method in muscle biopsies taken before and after an insulin clamp from 12 monozygotic twin pairs discordant for type 2 diabetes and 12 control subjects. Insulin-stimulated glucose uptake was decreased both in the diabetic and nondiabetic twin, compared with healthy control subjects (5.2 +/- 0.7 and 8.5 +/- 0.8 vs. 11.4 +/- 0.9 mg/kg x min(-1); P < 0.01 and P < 0.02, respectively). Basal mRNA levels of IRS-1, IRS-2, and Shc were similar in the diabetic and nondiabetic twins as well as in the control subjects. Insulin decreased mRNA expression of IRS-1 by 72% (from 0.75 +/- 0.06 to 0.21 +/- 0.04 relative units; P < 0.001), IRS-2 by 71% (from 0.55 +/- 0.10 to 0.16 +/- 0.08 relative units; P < 0.03), and Shc by 25% (from 0.95 +/- 0.04 to 0.71 +/- 0.04 relative units; P < 0.01) vs. baseline as demonstrated in the control subjects. The postclamp Shc mRNA level was slightly higher in the diabetic twins (P = 0.05) but similar in the nondiabetic twins, as compared with the control subjects, whereas postclamp IRS-1 and IRS-2 mRNA levels were similar between the study groups. There was an inverse correlation between postclamp Shc mRNA concentration and glucose uptake (r = -0.53, P = 0.01; n = 22) in the controls and nondiabetic twins. However, the decrease in Shc gene expression by insulin was not significantly different between the study groups. In conclusion, because insulin down-regulates IRS-1, IRS-2, and Shc gene expression in skeletal muscle in diabetic and nondiabetic monozygotic twins and control subjects to the same extent, it is unlikely that expression of these genes is an inherited trait or contributes to skeletal muscle insulin resistance.     

Agonist-specific differential changes of cellular signal transduction pathways in senescent human diploid fibroblasts

Changes in the signal transduction efficiency of senescent cells led us to compare the signaling events induced by two mitogenic agonists, platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) in presenescent and senescent or near-senescent human diploid fibroblasts. When the changes in intracellular [Ca(2+)](i) were analyzed, both PDGF and LPA generated a rhythmic increase in [Ca(2+)](i) in presenescent cells. The frequency of calcium response was reduced and desensitized in PDGF-stimulated senescent cells, while response to a LPA-induced calcium signal was also reduced in frequency, though its magnitude was unaltered. PDGF treatment increased the fibrous actin (F-actin) level in presenescent cells but not in senescent cells in contrast to a reduced but visible increase in F-actin in LPA-treated senescent cells. The effect of PDGF on phospholipase D (PLD) activation was also reduced significantly, as a ca. 60-80% reduction of PLD activity was observed in PDGF-stimulated cells but only a little reduction in LPA-induced cells. Agonist-specific differential changes of cellular signaling events caused a differential effect on DNA synthesis after growth factor stimulation. We observed a dramatic (80-90%) reduction of [3H]thymidine incorporation into DNA in the PDGF-stimulated near-senescent cells. LPA resulted in a 2-3-fold increase in thymidine incorporation even in the near-senescent cells. These differences in the responses of senescent or near-senescent cells to PDGF- and LPA-stimulation raised questions about the differential changes of the respective signaling apparatuses induced by aging. Since PDGF signaling event was affected greatly by aging, we further examined the protein contents involved in PDGF signal transduction pathway. PDGF receptor (PDGFR), protein kinase C-alpha (PKC-alpha), phospholipase C-gamma1 (PLC-gamma1), and PLD1 were examined by Western blot analysis. The protein levels of PKC-alpha and PLC-gamma1 were unchanged, but those of PLD1 and PDGFR were reduced with age. The reduced content of PDGFR protein may be one of the important contributors to the failure of PDGF-stimulated signal transduction in human senescent fibroblasts. Our results strongly suggest that age-dependent agonist-specific changes in signaling events might be in charge of the functional deterioration of senescent cells through imbalance of signal responses.     

A new antidiabetic agent (JTT-501) rapidly stimulates glucose disposal rates by enhancing insulin signal transduction in skeletal muscle

Summary A newly synthesized antidiabetic agent, JTT-501 is an isoxazolidinedione rather than a thiazolidinedione. An oral dose of JTT-501 (100 mg · kg^–1· day^–1) given to 12-week-old male Zucker fatty rats for 7 days led to the amelioration of both hyperinsulinaemia (40 % of non-treated) and hypertriglyceridaemia (23 % of non-treated) as well as a 2.4-fold increased insulin sensitivity as determined by a euglycaemic insulin clamp. In our study, we further evaluated the acute effect of JTT-501 on both the glucose infusion rates (GIR) and insulin signalling in skeletal muscle. Male Sprague-Dawley (SD) rats aged 10 weeks were injected intravenously with JTT-501 (5 mg/kg) and then a euglycaemic insulin clamp was initiated and glucose infusion rates monitored for 150 min. We found that this treatment increased the glucose infusion rate by 33 % during the last 30 min in SD rats. After the clamp had been initiated for 30 min, the insulin-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activities co-immunoprecipitated with insulin receptor substrate 1 (IRS-1) were also enhanced, resulting in increased glycogen synthase activities in the soleus muscles. Treatment with JTT-501 also enhanced the phosphorylation of insulin receptors and insulin receptor-substrate 1 rapidly as well as the phosphatidylinositol 3-kinase activities, which were stimulated by a bolus injection of insulin. Similarly, JTT-501 stimulated the glucose infusion rate by 30 % and enhanced insulin signalling in Zucker fatty rats. In conclusion, a newly developed isoxazolidinedione, JTT-501, rapidly potentiates the insulin sensitivity of skeletal muscle by enhancing insulin signalling and could be useful for the treatment of insulin-resistant diabetic subjects. [Diabetologia (1999) 42: 151–159] Received: 2 June 1998 and in final revised form: 2 October 1998     

Sulfhydryl G proteins and phospholipase A(2)-associated G proteins are involved in adrenergic signal transduction in the rat pineal gland

The rat pineal gland with its circadian noradrenaline-regulated melatonin rhythm is an excellent model for studying adrenergic signal transduction with respect to cAMP and cGMP formation. The stimulatory G(s) proteins play a well-established role in this process. In contrast, the potential roles of the inhibitory G(i) proteins, the functionally unclear other G(o) proteins, and a number of G protein subtypes are not known. The present study examines the effects on beta(1)- and beta(1)-plus-alpha(1)-stimulated cAMP and cGMP formation of a number of G protein modulators in rat pinealocyte suspension cultures. The effects of the nitric oxide donor sodium nitroprusside on cGMP were also examined. The results showed that drugs that activate G proteins of the G(i)/G(o) family, i.e., pertussis toxin, mastoparan, and compound 48/80, had no effect on unstimulated, isoproterenol (beta(1))-stimulated, or combined isoproterenol/phenylephrine (beta(1)-plus()-alpha(1))-stimulated cAMP and cGMP accumulation. However, in this experimental paradigm, the inhibitors of sulfhydryl G proteins (N-ethylmaleimide) and those of phospholipase A2-related G proteins (isotetrandrine) exerted a clear inhibitory effect. Sodium-nitroprusside-stimulated cGMP accumulation was also inhibited. These results confirm a previous report that members of the G(i)/G(o) family, which are present in the rat pineal gland, do not play a major role in adrenergic signal transduction. The new finding that sulfhydryl G proteins and phospholipase A2-associated G proteins exert a clear stimulatory effect on adrenergic signal transduction suggests that they are subtypes of G(s) proteins.     

Transglutaminase 2 transamidation activity during first-phase insulin secretion: natural substrates in INS-1E

Transglutaminase 2 (TG2) is a multifunctional protein with Ca²⁺-dependent transamidating and G protein activity. Previously, we reported that tgm2 ?/? mice have an impaired insulin secretion and that naturally occurring TG2 mutations associated with familial, early-onset type 2 diabetes, show a defective transamidating activity. Aim of this study was to get a better insight into the role of TG2 in insulin secretion by identifying substrates of TG2 transamidating activity in the pancreatic beta cell line INS-1E. To this end, we labeled INS-1E that are capable of secreting insulin upon glucose stimulation in the physiologic range, with an artificial acyl acceptor (biotinamido-pentylamine) or donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. In addition, subcellular localization of TG2 in human endocrine pancreas was studied by electron microscopy. Among several TG2’s transamidating substrates in INS-1E, mass spectrometry identified cytoplasmic actin (a result confirmed in human pancreatic islet), tropomyosin, and molecules that participate in insulin granule structure (e.g., GAPDH), glucose metabolism, or [Ca²⁺] sensing (e.g., calreticulin). Physical interaction between TG2 and cytoplasmic actin during glucose-stimulated first-phase insulin secretion was confirmed by co-immunoprecipitation. Electron microscopy revealed that TG2 is localized close to insulin and glucagon granules in human pancreatic islet. We propose that TG2’s role in insulin secretion may involve cytoplasmic actin remodeling and may have a regulative action on other proteins during granule movement. A similar role of TG2 in glucagon secretion is also suggested.     

固醇调节元件结合蛋白1c对骨骼肌细胞胰岛素受体底物1表达调控的影响

目的 探讨固醇调节元件结合蛋白1c（SREBP-1c）对大鼠骨骼肌细胞胰岛素受体底物1（IRS-1）表达调控的影响.方法 采用酶联合消化法取2～3 d SPF级雄性SD大鼠原代骨骼肌细胞,将原代细胞分为对照组（C）、对照＋胰岛素组（C＋I）、高脂组（PA）及高脂＋胰岛素组（PA＋I）.将表达SREBP-1c腺病毒转染L6细胞,根据感染复数（MOI）分为含绿色荧光蛋白阴性载体（GFP）组、MOI值为5、50、100、200组.将靶基因为SREBP-1c的干扰RNA （siRNA）转染L6细胞,并分为空白对照组、阴性siRNA组及SREBP-1c siRNA组.Western blotting和实时定量聚合酶链反应（RT-PCR）检测SREBP-1c、IRS-1、蛋白激酶B（Akt）基因及蛋白表达,油红O染色法检测细胞内脂质沉积情况.多组资料比较采用方差分析,两两比较采用最小显著差异法.结果 与C组相比,PA组SREBP-1c基因和蛋白水平升高（分别为2.72±0.08比1.00±0.18,3.02 ±0.19比1.00±0.05,t=15.240、18.289,均P＜0.05）,IRS-1基因和蛋白水平降低（分别为0.71 ±0.04比1.00 ±0.05,0.82 ±0.04比1.00±0.04,t=-7.960、-6.052,均P＜0.05）,丝氨酸磷酸化IRS-1蛋白表达升高,丝氨酸磷酸化Akt（p-Akt）蛋白表达下降（t=20.987、-5.869,均P＜0.05）.与GFP组相比,MOI值为50、100和200组的SREBP-1c基因和蛋白表达呈剂量依赖性上升（均P＜0.05）,IRS-1基因和蛋白表达水平呈剂量依赖性下降（均P＜0.05）.与空白对照组和阴性siRNA组相比,SREBP-1c siRNA组SREBP-1c基因和蛋白水平降低,IRS-1蛋白表达升高（均P＜0.05）.结论 SREBP-1c可抑制骨骼肌IRS-1胰岛素信号通路,参与肌细胞胰岛素抵抗的发生.