Adrenaline potentiates insulin-stimulated PKB activation in the rat fast-twitch epitrochlearis muscle without affecting IRS-1-associated PI 3-kinase activity

We have previously shown in the rat slow-twitch soleus muscle that adrenaline greatly potentiates insulin-stimulated protein kinase B (PKB) phosphorylation without having an effect alone. However, insulin signalling capacity through the PKB pathway is higher in soleus than in fast-twitch muscles, whereas adrenaline activates phosphorylase more strongly in epitrochlearis. Therefore, the aim of the present study was to investigate the interaction between adrenaline and insulin signalling in the fast-twitch epitrochlearis muscle. Insulin increased insulin receptor substrate-1 (IRS-1)-associated phosphoinositide (PI) 3-kinase activity threefold, and adrenaline did not influence basal or insulin-stimulated PI 3-kinase activity. Insulin but not adrenaline increased PKB activity and phosphorylation of Ser(473) and Thr(308). It is interesting to note that adrenaline potentiated insulin-stimulated PKB activity and PKB Ser(473) and Thr(308) phosphorylation. These effects were mimicked by dibutyryl-cyclic adenosine monophosphate (db-cAMP). Adrenaline and db-cAMP increased glycogen synthase kinase (GSK)-3beta Ser(9) phosphorylation independently of PKB activation and enhanced insulin-stimulated GSK-3beta Ser(9) phosphorylation. Although adrenaline increased GSK-3 phosphorylation (inhibiting activity), phosphorylation of its target sites on glycogen synthase was increased, and adrenaline blocked insulin-stimulated glycogen synthase dephosphorylation of Ser(641) and Ser(645,649,653,657), glycogen synthase activation and glycogen synthesis. Insulin-stimulated glucose transport was not influenced by adrenaline despite the increased PKB activation. In conclusion, as in the slow-twitch soleus muscle, adrenaline potentiates insulin-stimulated PKB activation in the fast-twitch glycolytic epitrochlearis muscle without increasing IRS-1-associated PI 3-kinase activity. Furthermore, adrenaline induces phosphorylation of a pool of GSK-3 that is not involved in the regulation of glycogen metabolism. These results indicate that the combination of adrenaline and insulin may activate novel signalling molecules rather than just summing up their effects on linear pathways.     

Improved insulin‐stimulated glucose uptake and glycogen synthase activation in rat skeletal muscles after adrenaline infusion: role of glycogen content and PKB phosphorylation

Aim: Effects of in vivo adrenaline infusion on subsequent insulin‐stimulated glucose uptake and glycogen synthase activation was investigated in slow‐twitch (soleus) and fast‐twitch (epitrochlearis) muscles. Furthermore, role of glycogen content and Protein kinase B (PKB) phosphorylation for modulation insulin sensitivity was investigated. Methods: Male Wistar rats received adrenaline from osmotic mini pumps (≈150 μg kg^?1 h^?1) for 1 or 12 days before muscles were removed for in vitro studies. Results: Glucose uptake at physiological insulin concentration was elevated in both muscles after 1 and 12 days of adrenaline infusion. Insulin‐stimulated glycogen synthase activation was also improved in both muscles. This elevated insulin sensitivity occurred despite the muscles were exposed to hyperglycaemia in vivo. After 1 day of adrenaline infusion, glycogen content was reduced in both muscles; insulin‐stimulated PKB ser⁴⁷³ phosphorylation was increased in both muscles only at the highest insulin concentration. After 12 days of adrenaline infusion, glycogen remained low in epitrochlearis, but returned to normal level in soleus; insulin‐stimulated PKB phosphorylation was normal in both muscles. Conclusion: Insulin‐stimulated glucose uptake and glycogen synthase activation were increased after adrenaline infusion. Increased insulin‐stimulated glucose uptake and glycogen synthase activation after adrenaline infusion cannot be explained by a reduction in glycogen content or an increase in PKB phosphorylation. The mechanisms for the improved insulin sensitivity after adrenaline treatment deserve particular attention as they occur in conjunction with hyperglycaemia.     

Improved insulin-stimulated glucose uptake and glycogen synthase activation in rat skeletal muscles after adrenaline infusion: role of glycogen content and PKB phosphorylation

AIM: Effects of in vivo adrenaline infusion on subsequent insulin-stimulated glucose uptake and glycogen synthase activation was investigated in slow-twitch (soleus) and fast-twitch (epitrochlearis) muscles. Furthermore, role of glycogen content and Protein kinase B (PKB) phosphorylation for modulation insulin sensitivity was investigated. METHODS: Male Wistar rats received adrenaline from osmotic mini pumps ( approximately 150 microg kg(-1) h(-1)) for 1 or 12 days before muscles were removed for in vitro studies. RESULTS: Glucose uptake at physiological insulin concentration was elevated in both muscles after 1 and 12 days of adrenaline infusion. Insulin-stimulated glycogen synthase activation was also improved in both muscles. This elevated insulin sensitivity occurred despite the muscles were exposed to hyperglycaemia in vivo. After 1 day of adrenaline infusion, glycogen content was reduced in both muscles; insulin-stimulated PKB ser(473) phosphorylation was increased in both muscles only at the highest insulin concentration. After 12 days of adrenaline infusion, glycogen remained low in epitrochlearis, but returned to normal level in soleus; insulin-stimulated PKB phosphorylation was normal in both muscles. CONCLUSION: Insulin-stimulated glucose uptake and glycogen synthase activation were increased after adrenaline infusion. Increased insulin-stimulated glucose uptake and glycogen synthase activation after adrenaline infusion cannot be explained by a reduction in glycogen content or an increase in PKB phosphorylation. The mechanisms for the improved insulin sensitivity after adrenaline treatment deserve particular attention as they occur in conjunction with hyperglycaemia.     

Caffeine activates preferentially α1-isoform of 5'AMP-activated protein kinase in rat skeletal muscle

Aim: Caffeine activates 5′AMP‐activated protein kinase (AMPK), a signalling intermediary implicated in the regulation of glucose, lipid and energy metabolism in skeletal muscle. Skeletal muscle expresses two catalytic α subunits of AMPK, α1 and α2, but the isoform specificity of caffeine‐induced AMPK activation is unclear. The aim of this study was to determine which α isoform is preferentially activated by caffeine in vitro and in vivo using rat skeletal muscle. Methods: Rat epitrochlearis muscle was isolated and incubated in vitro in the absence or presence of caffeine. In another experiment, the muscle was dissected after intravenous injection of caffeine. Isoform‐specific AMPK activity, the phosphorylation status of AMPKα Thr¹⁷² and acetyl‐CoA carboxylase (ACC) Ser⁷⁹, the concentrations of ATP, phosphocreatine (PCr) and glycogen, and 3‐O‐methyl‐d ‐glucose (3MG) transport activity were estimated. Results: Incubation of isolated epitrochlearis muscle with 1 mm of caffeine for 15 min increased AMPKα1 activity, but not AMPKα2 activity; concentrations of ATP, PCr and glycogen were not affected. Incubation with 3 mm of caffeine activated AMPKα2 and reduced PCr and glycogen concentrations. Incubation with 1 mm of caffeine increased the phosphorylation of AMPK and ACC and enhanced 3MG transport. Intravenous injection of caffeine (5 mg kg^?1) predominantly activated AMPKα1 and increased 3MG transport without affecting energy status. Conclusion: Our results suggest that of the two α isoforms of AMPK, AMPKα1 is predominantly activated by caffeine via an energy‐independent mechanism and that the activation of AMPKα1 increases glucose transport and ACC phosphorylation in skeletal muscle.     

Involvement of phosphatidylinositol 5-phosphate in insulin-stimulated glucose uptake in the L6 myotube model of skeletal muscle

The phosphoinositide phospholipid PtdIns5P has previously been implicated in insulin-stimulated translocation of the glucose transporter GLUT4 into the plasma membrane of adipocytes, but its potential role in glucose transport in muscle has not been explored. The involvement of PtdIns5P in insulin-stimulated glucose uptake was therefore investigated in myotubes of the skeletal muscle cell line L6. Stimulation with insulin produced a transient increase in PtdIns5P, which was abolished by the over-expression of the highly active PtdIns5P 4-kinase PIP4Kα. PIP4Kα over-expression also abolished both the enhanced glucose uptake and the robust peak of PtdIns(3,4,5)P ₃ production stimulated by insulin in myotubes. Delivery of exogenous PtdIns5P into unstimulated myotubes increased Akt phosphorylation, promoted GLUT4 relocalisation from internal membrane to plasma membrane fractions and its association with plasma membrane lawns and also stimulated glucose uptake in a tyrosine kinase and phosphoinositide 3-kinase (PI 3-kinase)-dependent fashion. Our results are consistent with a role for insulin-stimulated PtdIns5P production in regulating glucose transport by promoting PI 3-kinase signalling.     

Greater filamin C,GSK3α, and GSK3β serine phosphorylation in insulin-stimulated isolated skeletal muscles of calorie restricted 24 month-old rats

Moderate calorie restriction (CR) can improve insulin-stimulated Akt phosphorylation and glucose uptake in muscles from 24 month-old rats, but the specific Akt substrates linking CR-effects on Akt to glucose uptake and other cellular processes are uncertain. We probed CR's influence on site-specific phosphorylation of five Akt substrates (AS160^Ser588, TBC1D1^Thr596, FLNc^Ser2213, GSK3α^Ser21, and GSK3β^Ser9) in predominantly fast-twitch (epitrochlearis) and predominantly slow-twitch (soleus) muscles. We observed no CR-effect on phosphorylation of AS160^Ser588 or TBC1D1^Thr596, but there was a CR-induced increase in insulin-stimulated FLNc^Ser2213, GSK3α^Ser21, and GSK3β^Ser9 phosphorylation for both muscles. These results indicate that CR does not uniformly affect insulin-mediated phosphorylation of Akt substrates in fast- or slow-twitch muscles from 24 month-old rats.     

Over-expression of NYGGF4 (PID1) inhibits glucose transport in skeletal myotubes by blocking the IRS1/PI3K/AKT insulin pathway

IntroductionDefects in insulin-stimulated glucose uptake in muscle are the important early events in the pathogenesis of insulin resistance. NYGGF4 (also named PID1) is a recently discovered gene which is suggested to be associated with obesity-associated insulin resistance. In this study, we aimed to investigate the effects of NYGGF4 on glucose uptake and insulin signaling in rat skeletal muscle cells.MethodsRat L6 myoblasts were transfected with either an empty vector or an NYGGF4-expressing vector and induced to differentiate into mature L6 skeletal myotubes. Glucose uptake was determined by measuring uptake of 2-deoxy-d-[³H] glucose. Immunoblotting was performed to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4). Immunoblotting was also used to measure phosphorylation and total protein levels of the insulin signaling proteins including insulin receptor (IR), insulin receptor substrate 1 (IRS1), Akt, extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun-N-terminal kinase (JNK).ResultsNYGGF4 over-expression in L6 skeletal myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS1 and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, or JNK.ConclusionsOver-expression of NYGGF4 inhibits glucose transport in skeletal myotubes by blocking the IRS1/PI3K/AKT insulin pathway. These observations highlight the potential role of NYGGF4 in glucose homeostasis and the development of insulin resistance in obesity.     

Calphostin C is an inhibitor of contraction, but not insulin-stimulated glucose transport, in skeletal muscle.

Wortmannin selectively impairs insulin-stimulated glucose transport in skeletal muscle. To search for an inhibitor specific for contraction-stimulated glucose transport, we screened a number of calmodulin and PKC inhibitors for their ability to impair contraction- and insulin-stimulated 2-deoxyglucose uptake in incubated rat soleus muscles. In concentrations that did not reduce contraction-induced force output, among calmodulin inhibitors W-7 inhibited both contraction- and insulin-stimulated glucose transport by up to 50% (P < 0.05), while Calmidazolium impaired only insulin-stimulated glucose transport (P < 0.05), and Trifluoperazine and Phenoxybenzamine did not influence glucose transport. In concentrations that did not reduce force generation, among PKC inhibitors Calphostin C specifically inhibited contraction-stimulated glucose transport (P < 0.05), whereas insulin-stimulated transport was impaired by Rottlerin and Bisindolylmaleimide I (P < 0.05), and both contraction- and insulin-stimulated glucose transport were inhibited by RO-31-8220 (P < 0.05). Calphostin C did not reduce contraction-induced increase in AMP-activated protein kinase (AMPK) activity. In conclusion, we have identified specific inhibitors of both contraction- and insulin-stimulated glucose transport. Both calmodulin and different isoenzymes of the PKC family may be involved in contraction- and insulin-stimulated glucose transport. Calphostin C does not influence glucose transport during contractions via stimulation of AMPK. Calphostin C may be used to unravel signal transduction in contraction-stimulated glucose transport.     

Effects of gender and prior swim exercise on glucose uptake in isolated skeletal muscles from mice

The purpose of this study was to characterize the effects of prior swim exercise on glucose uptake in isolated skeletal muscles of mice. Male and female mice (C57BL/6) performing 180 min of swimming had significantly decreased glycogen concentration compared to resting controls in soleus, extensor digitorum longus (EDL), and epitrochlearis muscles, regardless of gender. Glucose uptake by isolated muscles was measured using [(3)H]-2-deoxyglucose without insulin or with 180 pmol/l insulin (20, 75, or 200 min post-exercise and sedentary) or 12,000 pmol/l (20 or 200 min post-exercise and sedentary) in the soleus and EDL and without insulin or with 12,000 pmol/l insulin (20 or 200 min post-exercise and sedentary) in the epitrochlearis. Glucose uptake was higher (P < or = 0.01) for female versus male mice at each insulin concentration in the soleus and EDL, but not the epitrochlearis. Although prolonged (180 min) swim exercise did not alter subsequent glucose uptake, a shorter duration exercise protocol (60 min) tested in male mice (20 min post-exercise) led to a 1.5-fold increase in insulin-independent glucose uptake in EDL muscles. However, insulin-stimulated (180 pmol/l) glucose uptake was not altered by 60 min exercise in EDL or soleus. In light of these results, swim exercise is not recommended to evaluate the exercise-induced improvement in insulin-stimulated glucose uptake of muscles of C57BL/6 mice.     

Elevated free fatty acid levels inhibit glucose phosphorylation in slow-twitch rat skeletal muscle

The effect of increased free fatty acid concentrations on glucose metabolism in rat skeletal muscle was investigated at several different steps in glucose metabolism including glucose transport, glucose phosphorylation, glucose oxidation and glycogen synthesis. In isolated soleus (slow-twitch) muscles, insulin-stimulated (100 μml^-1) glucose phosphorylation, but not glucose transport, was inhibited by 26 and 22% in the presence of 1.0 and 2.0 mM oleate, respectively (P< 0.01). Regardless of oleate concentration (0.3 or 2.0 mM), insulin-stimulated glucose 6-phosphate levels were elevated to the same extent over the non-insulin-stimulated levels in soleus muscles {P < 0.01). Insulin-stimulated glucose oxidation was inhibited by 44% in soleus muscles exposed to 2.0 mM oleate (P < 0.05), whereas the rate of glucose incorporation into glycogen was not altered. In insulin-stimulated epitrochlearis (fast-twitch) muscles, elevated concentrations of oleate had no effect on the rates of glucose transport or glucose phosphorylation, or on the level of glucose 6-phosphate. These data suggest that increased free fatty acid availability decreases glucose utilization by selectively inhibiting glucose phosphorylation and oxidation in slow-twitch, but not fast-twitch skeletal muscle.     

Calphostin C is an inhibitor of contraction,but not insulin-stimulated glucose transport,in skeletal muscle

Wortmannin selectively impairs insulin-stimulated glucose transport in skeletal muscle. To search for an inhibitor specific for contraction-stimulated glucose transport, we screened a number of calmodulin and PKC inhibitors for their ability to impair contraction- and insulin-stimulated 2-deoxyglucose uptake in incubated rat soleus muscles. In concentrations that did not reduce contraction-induced force output, among calmodulin inhibitors W-7 inhibited both contraction- and insulin-stimulated glucose transport by up to 50% (P < 0.05), while Calmidazolium impaired only insulin-stimulated glucose transport (P < 0.05), and Trifluoperazine and Phenoxybenzamine did not influence glucose transport. In concentrations that did not reduce force generation, among PKC inhibitors Calphostin C specifically inhibited contraction-stimulated glucose transport (P < 0.05), whereas insulin-stimulated transport was impaired by Rottlerin and Bisindolylmaleimide I (P < 0.05), and both contraction- and insulin-stimulated glucose transport were inhibited by RO-31-8220 (P < 0.05). Calphostin C did not reduce contraction-induced increase in AMP-activated protein kinase (AMPK) activity. In conclusion, we have identified specific inhibitors of both contraction- and insulin-stimulated glucose transport. Both calmodulin and different isoenzymes of the PKC family may be involved in contraction- and insulin-stimulated glucose transport. Calphostin C does not influence glucose transport during contractions via stimulation of AMPK. Calphostin C may be used to unravel signal transduction in contraction-stimulated glucose transport.     

Increased insulin-stimulated glucose uptake in athletes: the importance of GLUT4 mRNA,GLUT4 protein and fibre type composition of skeletal muscle

In the present study the expression of GLUT4 and fibre type composition were examined in biopsies from skeletal muscle in seven male athletes and eight male sedentary subjects. Estimated maximal oxygen uptake was increased in the trained group when compared with the sedentary group (74.0 ± 3.9 vs. 42.9±5.1 ml kg^-1 min^-1; P < 0.01). A biopsy of vastus lateralis muscle was taken in the fasting state, 36 h after the last bout of exercise. A second muscle biopsy was obtained following 4 h of a hyperinsulinaemic (2 mU kg^-1 min^-1), euglycaemic clamp. The rate of insulin-stimulated glucose uptake was increased in the trained subjects (17.34±0.53 vs. 13.53±0.79 mg kg^-1 min^-1, P < 0.01). In parallel, the steady state levels of GLUT4 protein and mRNA per DNA were higher in muscle biopsies obtained in the basal state from athletes than in sedentary controls, 21 and 71% respectively (P < 0.05). In the total group of participants, GLUT4 protein per DNA in the basal state and insulin-stimulated glucose uptake rate correlated positively, (r = 0.51, P = 0.05). In the insulin-stimulated state we did not find any significant correlation between GLUT4 protein per DNA and glucose uptake rate (r = 0.13, n.s.). No significant relationships between GLUT4 protein abundance per DNA and muscle fibre type distribution were observed. A significantly negative correladon was found between type 2B fibre area and insulin-stimulated glucose uptake (r =–0.63, P < 0.05). In conclusion, the abundance of GLUT4 protein and mRNA, respectively, is increased in skeletal muscle from endurance trained subjects compared to sedentary subjects. However, factors other than GLUT4 immunoreactive protein abundance seem to be determinant for the increased insulin-stimulated whole body glucose uptake in endurance trained subjects.     

Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic

Insulin stimulates glucose uptake into muscle and adipose tissues through glucose transporter 4 (GLUT4). GLUT4 cycles between the intracellular compartments and the plasma membrane. GLUT4 traffic-regulating insulin signals are largely within the insulin receptor-insulin receptor substrate-phosphatidylinositol 3-kinase (IR-IRS-PI3K) axis. In muscle cells, insulin signal bifurcates downstream of the PI3K into one arm leading to the activation of the Ser/Thr kinases Akt and atypical protein kinase C, and another leading to the activation of Rho family protein Rac1 leading to actin remodelling. Activated Akt inactivates AS160, a GTPase-activating protein for Rab family small G proteins. Here we review the roles of Rab and Rho proteins, particularly Rab substrates of AS160 and Rac1, in insulin-stimulated GLUT4 traffic. We discuss: (1) how distinct steps in GLUT4 traffic may be regulated by discrete Rab proteins, and (2) the importance of Rac1 activation in insulin-induced actin remodelling in muscle cells, a key element for the net gain in surface GLUT4.     

L6大鼠成肌细胞胰岛素信号传导通路中磷脂酰肌醇3激酶、蛋白激酶B和葡萄糖转运蛋白4的表达

背景：血管紧张素Ⅱ可损伤胰岛素信号中的下游信号分子引起胰岛素抵抗,但其机制不清。 目的：观察血管紧张素Ⅱ对L6大鼠成肌细胞胰岛素信号传导通路中磷脂酰肌醇3激酶、蛋白激酶B和葡萄糖转运蛋白4的影响。 方法：L6细胞培养及诱导分化肌管,根据干预措施不同实验分为对照组、胰岛素组、胰岛素+血管紧张素Ⅱ组及胰岛素+血管紧张素Ⅱ+H89组。采用RT-PCR检测4组磷脂酰肌醇3激酶、蛋白激酶B mRNA表达,免疫荧光检测胰岛素受体底物1、酪氨酸磷酸化胰岛素受体底物1、葡萄糖转运蛋白4表达。 结果与结论：胰岛素组、胰岛素+血管紧张素Ⅱ组及胰岛素+血管紧张素Ⅱ+H89组的磷脂酰肌醇3激酶mRNA表达均较对照组显著升高(P < 0.05)。各组间蛋白激酶B mRNA表达差异无显著性意义(P> 0.05)。相比对照组,其余3组间胰岛素受体底物1、酪氨酸磷酸化胰岛素受体底物1和葡萄糖转运蛋白4(膜蛋白)表达均升高(P < 0.05);胰岛素+血管紧张素Ⅱ+H89组酪氨酸磷酸化胰岛素受体底物1和葡萄糖转运蛋白4表达低于胰岛素组但高于胰岛素+血管紧张素Ⅱ组(P < 0.05)。结果显示,血管紧张素Ⅱ在骨骼肌细胞中通过JAK2-PKA通路引起胰岛素下游信号传导受阻,葡萄糖转运蛋白4表达减少,葡萄糖转运障碍,进而导致胰岛素抵抗。     

Silibinin improves palmitate-induced insulin resistance in C2C12 myotubes by attenuating IRS-1/PI3K/Akt pathway inhibition

The present study investigated the effect of silibinin, the principal potential anti-inflammatory flavonoid contained in silymarin, a mixture of flavonolignans extracted from Silybum marianum seeds, on palmitate-induced insulin resistance in C2C12 myotubes and its potential molecular mechanisms. Silibinin prevented the decrease of insulin-stimulated 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) uptake and the downregulation of glutamate transporter type 4 (GLUT4) translocation in C2C12 myotubes induced by palmitate. Meanwhile, silibinin suppressed the palmitate-induced decrease of insulin-stimulated Akt Ser473 phosphorylation, which was reversed by wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K). We also found that palmitate downregulated insulin-stimulated Tyr632 phosphorylation of insulin receptor substrate 1 (IRS-1) and up-regulated IRS-1 Ser307 phosphorylation. These effects were rebalanced by silibinin. Considering several serine/threonine kinases reported to phosphorylate IRS-1 at Ser307, treatment with silibinin downregulated the phosphorylation of both c-Jun N-terminal kinase (JNK) and nuclear factor-κB kinase β (IKKβ), which was increased by palmitate in C2C12 myotubes mediating inflammatory status, whereas the phosphorylation of PKC-θ was not significantly modulated by silibinin. Collectively, the results indicated that silibinin prevented inhibition of the IRS-1/PI3K/Akt pathway, thus ameliorating palmitate-induced insulin resistance in C2C12 myotubes.     

Effect of diffusion distance on measurement of rat skeletal muscle glucose transport in vitro

The relationships between muscle size, diffusion distance, and glucose uptake were studied using the Type II b epitrochlearis (13 ± 1 mg intact), Type I soleus (25± 1 mg), and mixed Type II a/II b extensor digitorum longus (25 ± 1 mg) from 60–70 g rats. Using intact muscles, the relative rates of 3-O-methyl-glucose uptake in response to 2 mUml^-1 insulin were soleus = epitrochlearis > extensor digitorum longus, a finding inconsistent with the fibre-type compositions and the relative GLUT-4 protein levels (soleus > extensor digitorum longus > epitrochlearis). To test whether these results were influenced by substrate diffusion limitations in the tubular muscles, soleus and extensor digitorum longus were split longitudinally from tendon to tendon into strips of comparable size (13 ± 1 mg) to the epitrochlearis. Insulin-stimulated rates of 3-O-methyl-glucose uptake were significantly enhanced in the split soleus (+120%) and split extensor digitorum longus (+200%), but not in the epitrochlearis, with the relative rates being soleus > extensor digitorum longus > epitrochlearis. Diffusion distances of the split soleus and extensor digitorum longus, as reflected by [¹⁴C]mannitol space equilibration time, were markedly enhanced (by at least 50%) relative to the intact muscles, and were comparable to that of the epitrochlearis. These results indicate that when muscles of different size and/or shape are used for in vitro measurement of glucose transport, the muscle preparations used must have similar diffusion distances for physiologically meaningful comparisons to be made.     

Stretch-stimulated glucose uptake in skeletal muscle is mediated by reactive oxygen species and p38 MAP-kinase

Alternatives to the canonical insulin-stimulated pathway for glucose uptake are exercise- and exogenous reactive oxygen species (ROS)-stimulated glucose uptake. We proposed a model wherein mechanical loading, i.e. stretch, stimulates production of ROS to activate AMP-activated kinase (AMPK) to increase glucose uptake. Immunoblotting was used to measure protein phosphorylation; the fluorochrome probe 2'7'-dichlorofluorescin diacetate was used to measure cytosolic oxidant activity and 2-deoxy- d [1,2-³H]glucose was used to measure glucose uptake. The current studies demonstrate that stretch increases ROS, AMPKα phosphorylation and glucose transport in murine extensor digitorum longus (EDL) muscle (+121%, +164% and +184%, respectively; P < 0.05). We also demonstrate that stretch-induced glucose uptake persists in transgenic mice expressing an inactive form of the AMPKα2 catalytic subunit in skeletal muscle (+173%; P < 0.05). MnTBAP, a superoxide dismutase (SOD) mimetic, N -acteyl cysteine (NAC), a non-specific antioxidant, ebselen, a glutathione mimetic, or combined SOD plus catalase (ROS-selective scavengers) all decrease stretch-stimulated glucose uptake ( P < 0.05) without changing basal uptake ( P > 0.16). We also demonstrate that stretch-stimulated glucose uptake persists in the presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294001 ( P < 0.05) but is diminished by the p38-MAPK inhibitors SB203580 and A304000 ( P > 0.99). These data indicate that stretch-stimulated glucose uptake in skeletal muscle is mediated by a ROS- and p38 MAPK-dependent mechanism that appears to be AMPKα2- and PI3-K-independent.     

Vaspin alleviates dysfunction of endothelial progenitor cells induced by high glucose via PI3K/Akt/eNOS pathway

Improving the dysfunction of endothelial progenitor cell (EPCs) in patients with diabetes mellitus is important for preventing vascular complication. Vaspin, an adipocytokine, has the anti-atherogenic properties rely on its positive effect on nitric oxide (NO) bioavailability. We hypothesis that vaspin may ameliorate high glucose induced dysfunction of EPCs. In rat bone morrow derived EPCs, glucose treatment results in a decrease in the proliferation and migration capacity in a dose dependent manner. These detrimental effects can be alleviated by vaspin. Furthermore, vaspin increased the production of NO and the effect of vaspin on EPCs can be diminished partly by the eNOS inhibitor (_L-NAME). We assessed total eNOS protein expression and Ser¹¹⁷⁷-phospho-eNOS expression and found that vaspin not only induced eNOS protein expression but also up regulate the eNOS activation. Subsequently, we investigated protein kinase B (Akt) activation in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor (LY-2940002). Vaspin increased total Akt and Ser⁴⁷³-phospho-Akt expression and these effects can be blocked by LY-2940002. The results of our study indicate a novel effect of vaspin to regulate eNOS expression and function in EPCs via a PI3K/Akt/eNOS pathway; vaspin may have a protective effect in patients with diabetes to prevent the occurrence of vascular complication.     

Activation of muscarinic M-1 cholinoceptors by curcumin to increase glucose uptake into skeletal muscle isolated from Wistar rats

Curcumin, an active principle contained in rhizome of Curcuma longa, has been mentioned to show merit for diabetes through its anti-oxidative and anti-inflammatory properties. In the present study, we found that curcumin caused a concentration-dependent increase of glucose uptake into skeletal muscle isolated from Wistar rats. This action was inhibited by pirenzepine at concentration enough to block muscarinic M-1 cholinoceptor (M₁-mAChR). In radioligand binding assay, the binding of [³H]-pirenzepine was also displaced by curcumin in a concentration-dependent manner. In the presence of inhibitors for PLC–PI3K pathway, either U73122 (phospholipase C inhibitor) or LY294002 (phosphoinositide 3-kinase inhibitor), curcumin-stimulated glucose uptake into skeletal muscle was markedly reduced. In Western blotting analysis, the membrane protein level of glucose transporter 4 (GLUT4) increased by curcumin was also reversed by blockade of M₁-mAChR or PLC–PI3K pathway in a same manner. In conclusion, the obtained results suggest that curcumin can activate M₁-mAChR at concentrations lower than to scavenge free radicals for increase of glucose uptake into skeletal muscle through PLC-PI3-kinase pathway.