A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle

2012 ◽  
Vol 447 (1) ◽  
pp. 137-147 ◽  
Author(s):  
Yu-Chiang Lai ◽  
Yang Liu ◽  
Roxane Jacobs ◽  
Mark H. Rider
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Glucose Uptake ◽  
Insulin Action ◽  
Glycogen Synthase ◽  
Insulin Signalling ◽  
Glycogen Synthesis ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Akt Inhibitor

PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle

2008 ◽  
Vol 294 (1) ◽  
pp. E28-E35 ◽  
Author(s):  
Michale Bouskila ◽  
Michael F. Hirshman ◽  
Jørgen Jensen ◽  
Laurie J. Goodyear ◽  
Kei Sakamoto
Keyword(s):  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Muscle Glycogen ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Wild Type ◽  
Muscle Glycogen Synthesis ◽  
Mutant Forms

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating glycogen synthase kinase (GSK) 3 through phosphorylation. Insulin also promotes glucose uptake and glucose 6-phosphate (G-6- P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphorylation of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knockin mice in which wild-type GSK3α and -β genes are replaced with mutant forms (GSK3α/βS21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3α/βS21A/S21A/S9A/S9Amice, glycogen content in different muscles from these mice was similar compared with wild-type mice. Basal and epinephrine-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knockin mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G-6- P, the rate of [14C]glucose incorporation into glycogen, and an increase in total glycogen content were similar between wild-type and GSK3 knockin mice. Injection of glucose containing 2-deoxy-[3H]glucose and [14C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knockin mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G-6- P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.

scholarly journals Inhibition of the Protein Tyrosine Phosphatase SHP-1 Increases Glucose Uptake in Skeletal Muscle Cells by Augmenting Insulin Receptor Signaling and GLUT4 Expression

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 4581-4588 ◽  
Author(s):  
Sébastien Bergeron ◽  
Marie-Julie Dubois ◽  
Kerstin Bellmann ◽  
Michael Schwab ◽  
Nancy Larochelle ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Expression ◽  
Glucose Uptake ◽  
Protein Tyrosine Phosphatase ◽  
Insulin Action ◽  
Tyrosine Phosphatase ◽  
Glycogen Synthesis ◽  
Glut4 Translocation ◽  
Protein Tyrosine ◽  
Glut4 Expression

The protein tyrosine phosphatase (PTPase) Src-homology 2-domain-containing phosphatase (SHP)-1 was recently reported to be a novel regulator of insulin's metabolic action. In order to examine the role of this PTPase in skeletal muscle, we used adenovirus (AdV)-mediated gene transfer to express an interfering mutant of SHP-1 [dominant negative (DN)SHP-1; mutation C453S] in L6 myocytes. Expression of DNSHP-1 increased insulin-induced Akt serine-threonine kinase phosphorylation and augmented glucose uptake and glycogen synthesis. Pharmacological inhibition of glucose transporter type 4 (GLUT4) activity using indinavir and GLUT4 translocation assays revealed an important role for this transporter in the increased insulin-induced glucose uptake in DNSHP-1-expressing myocytes. Both GLUT4 mRNA and protein expression were also found to be increased by DNSHP-1 expression. Furthermore, AdV-mediated delivery of DNSHP-1 in skeletal muscle of transgenic mice overexpressing Coxsackie and AdV receptor also enhanced GLUT4 protein expression. Together, these findings confirm that SHP-1 regulates muscle insulin action in a cell-autonomous manner and further suggest that the PTPase negatively modulates insulin action through down-regulation of both insulin signaling to Akt and GLUT4 translocation, as well as GLUT4 expression.

Effect of exercise on insulin action in human skeletal muscle

1989 ◽  
Vol 66 (2) ◽  
pp. 876-885 ◽  
Author(s):  
E. A. Richter ◽  
K. J. Mikines ◽  
H. Galbo ◽  
B. Kiens
Keyword(s):  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Insulin Action ◽  
Human Skeletal Muscle ◽  
Vastus Lateralis ◽  
Femoral Vein ◽  
Glycogen Synthesis ◽  
Vastus Lateralis Muscle ◽  
Glycogen Concentration ◽  
Local Contraction

The effect of 1 h of dynamic one-legged exercise on insulin action in human muscle was studied in 6 healthy young men. Four hours after one-legged knee extensions, a three-step sequential euglycemic hyperinsulinemic clamp combined with arterial and bilateral femoral vein catheterization was performed. Increased insulin action on glucose uptake was found in the exercised compared with the rested thigh at mean plasma insulin concentrations of 23, 40, and 410 microU/ml. Furthermore, prior contractions directed glucose uptake toward glycogen synthesis and increased insulin effects on thigh O2 consumption and at some insulin concentrations on potassium exchange. In contrast, no change in insulin effects on limb exchange of free fatty acids, glycerol, alanine or tyrosine were found after exercise. Glycogen concentration in rested vastus lateralis muscle did not increase measurably during the clamp even though indirect estimates indicated net glycogen synthesis. In contrast, in exercised muscle estimated and biopsy-verified increases in muscle glycogen concentration agreed. Local contraction-induced increases in insulin sensitivity and responsiveness play an important role in postexercise recovery of human skeletal muscle.

Glucose infusion causes insulin resistance in skeletal muscle of rats without changes in Akt and AS160 phosphorylation

2007 ◽  
Vol 293 (5) ◽  
pp. E1358-E1364 ◽  
Author(s):  
Andrew J. Hoy ◽  
Clinton R. Bruce ◽  
Anna Cederberg ◽  
Nigel Turner ◽  
David E. James ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glucose Infusion ◽  
Metabolic Effects ◽  
Synthesis Rate ◽  
Muscle Sample

Hyperglycemia is a defining feature of Type 1 and 2 diabetes. Hyperglycemia also causes insulin resistance, and our group (Kraegen EW, Saha AK, Preston E, Wilks D, Hoy AJ, Cooney GJ, Ruderman NB. Am J Physiol Endocrinol Metab Endocrinol Metab 290: E471–E479, 2006) has recently demonstrated that hyperglycemia generated by glucose infusion results in insulin resistance after 5 h but not after 3 h. The aim of this study was to investigate possible mechanism(s) by which glucose infusion causes insulin resistance in skeletal muscle and in particular to examine whether this was associated with changes in insulin signaling. Hyperglycemia (∼10 mM) was produced in cannulated male Wistar rats for up to 5 h. The glucose infusion rate required to maintain this hyperglycemia progressively lessened over 5 h (by 25%, P < 0.0001 at 5 h) without any alteration in plasma insulin levels consistent with the development of insulin resistance. Muscle glucose uptake in vivo (44%; P < 0.05) and glycogen synthesis rate (52%; P < 0.001) were reduced after 5 h compared with after 3 h of infusion. Despite these changes, there was no decrease in the phosphorylation state of multiple insulin signaling intermediates [insulin receptor, Akt, AS160 (Akt substrate of 160 kDa), glycogen synthase kinase-3β] over the same time course. In isolated soleus strips taken from control or 1- or 5-h glucose-infused animals, insulin-stimulated 2-deoxyglucose transport was similar, but glycogen synthesis was significantly reduced in the 5-h muscle sample (68% vs. 1-h sample; P < 0.001). These results suggest that the reduced muscle glucose uptake in rats after 5 h of acute hyperglycemia is due more to the metabolic effects of excess glycogen storage than to a defect in insulin signaling or glucose transport.

scholarly journals Insulin-Like Growth Factor I Circumvents Defective Insulin Action in Human Myotonic Dystrophy Skeletal Muscle Cells1

Endocrinology ◽  
1999 ◽  
Vol 140 (9) ◽  
pp. 4244-4250 ◽  
Author(s):  
Denis Furling ◽  
André Marette ◽  
Jack Puymirat
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Growth Factor ◽  
Insulin Receptor ◽  
Glucose Uptake ◽  
Insulin Action ◽  
Factor I ◽  
Impaired Insulin ◽  
Igf I ◽  
Impaired Insulin Action

Abstract Primary human skeletal muscle cell cultures derived from muscles of a myotonic dystrophy (DM) fetus provided a model in which both resistance to insulin action described in DM patient muscles and the potential ability of insulin-like growth factor I (IGF-I) to circumvent this defect could be investigated. Basal glucose uptake was the same in cultured DM cells as in normal myotubes. In DM cells, a dose of 10 nm insulin produced no stimulatory effect on glucose uptake, and at higher concentrations, stimulation of glucose uptake remained significantly lower than that in normal myotubes. In addition, basal and insulin-mediated protein synthesis were both significantly reduced compared with those in normal cells. In DM myotubes, insulin receptor messenger RNA expression and insulin receptor binding were significantly diminished, whereas the expression of GLUT1 and GLUT4 glucose transporters was not affected. These results indicate that impaired insulin action is retained in DM cultured myotubes. The action of recombinant human IGF-I (rhIGF-I) was evaluated in this cellular model. We showed that rhIGF-I is able to stimulate glucose uptake to a similar extent as in control cells and restore normal protein synthesis level in DM myotubes. Thus, rhIGF-I is able to bypass impaired insulin action in DM myotubes. This provides a solid foundation for the eventual use of rhIGF-I as an effective treatment of muscle weakness and wasting in DM.

Phosphorylation of eukaryotic initiation factor eIF2Bε in skeletal muscle during sepsis

2002 ◽  
Vol 283 (5) ◽  
pp. E1032-E1039 ◽  
Author(s):  
Thomas C. Vary ◽  
Gina Deiter ◽  
Scot R. Kimball
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Glycogen Synthase ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Sterile Inflammation ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Skeletal Muscle Protein

We reported that the inhibition of protein synthesis in skeletal muscle during sepsis correlated with reduced eukaryotic initiation factor eIF2B activity. The present studies define changes in eIF2Bε phosphorylation in gastrocnemius of septic animals. eIF2B kinase activity was significantly elevated 175% by sepsis compared with sterile inflammation, whereas eIF2B phosphatase activity was unaffected. Phosphorylation of eIF2Bε-Ser535 was significantly augmented over 2-fold and 2.5-fold after 3 and 5 days and returned to control values after 10 days of sepsis. Phosphorylation of glycogen synthase kinase-3 (GSK-3), a potential upstream kinase responsible for the elevated phosphorylation of eIF2Bε, was significantly reduced over 36 and 41% after 3 and 5 days and returned to control values after 10 days of sepsis. The phosphorylation of PKB, a kinase thought to directly phosphorylate and inactivate GSK-3, was significantly reduced ∼50% on day 3, but not on days 5 or 10, postinfection compared with controls. Treatment of septic rats with TNF-binding protein prevented the sepsis-induced changes in eIF2Bε and GSK-3 phosphorylation, implicating TNF in mediating the effects of sepsis. Thus increased phosphorylation of eIF2Bε via activation of GSK-3 is an important mechanism to account for the inhibition of skeletal muscle protein synthesis during sepsis. Furthermore, the study presents the first demonstration of changes in eIF2Bε phosphorylation in vivo.

Skeletal muscle glucose metabolism in obesity-prone and obesity-resistant rats

1993 ◽  
Vol 264 (6) ◽  
pp. R1224-R1228 ◽  
Author(s):  
M. J. Pagliassotti ◽  
K. A. Shahrokhi ◽  
J. O. Hill
Keyword(s):  
Skeletal Muscle ◽  
Body Weight ◽  
Weight Gain ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Glycogen Synthase ◽  
Body Weight Gain ◽  
Glycogen Synthesis ◽  
Insulin Resistant ◽  
Wide Range

Ad libitum access to a high-fat (HF) diet produces a wide range of weight gain in rats. Rats most susceptible to weight gain on such a diet (obesity prone; OP) are more insulin resistant after 4-5 wk of diet exposure than are those most resistant (obesity resistant; OR) to weight gain. To investigate whether skeletal muscle glucose metabolism contributes to insulin resistance in this model, insulin-stimulated glucose metabolism was assessed in the perfused hindquarter of rats exposed to either a low-fat (LF, n = 6) or HF diet for 5 wk. Delineation of OP (n = 6) and OR (n = 6) rats was based on body weight gain. OP rats gained 60% more body weight while eating only 10% more energy than OR rats. Single-pass perfusions were carried out for 2 h in the presence of glucose, insulin, and [U-14C]glucose. Insulin-stimulated glucose uptake (mumol.100 g-1.min-1) was 14.2 +/- 0.9 in LF, 11.1 +/- 0.8 in OR, and 6.2 +/- 0.6 in OP. Glucose oxidation (mumol.100 g-1.min-1) was 1.7 +/- 0.3 and 1.2 +/- 0.3 in LF and OR, respectively, but was 0.2 +/- 0.1 in OP. Net glycogen synthesis was significantly reduced in OP compared with OR and LF despite similar glycogen synthase I activity. Muscle triglyceride concentration was not significantly different in OR and OP rats. These results demonstrate significant defects in skeletal muscle glucose uptake and disposal in rats most susceptible to HF diet-induced obesity. Clearly, the heterogeneous response to a HF diet involves not only body weight gain but also skeletal muscle fuel metabolism.

Invited Review: Intracellular signaling in contracting skeletal muscle

2002 ◽  
Vol 93 (1) ◽  
pp. 369-383 ◽  
Author(s):  
Kei Sakamoto ◽  
Laurie J. Goodyear
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Physical Exercise ◽  
Glucose Uptake ◽  
Intracellular Signaling ◽  
Glycogen Synthase ◽  
Contractile Activity ◽  
Glycogen Synthesis ◽  
Mitogen Activated Protein Kinase ◽  
Molecular Signaling

Physical exercise is a significant stimulus for the regulation of multiple metabolic and transcriptional processes in skeletal muscle. For example, exercise increases skeletal muscle glucose uptake, and, after exercise, there are increases in the rates of both glucose uptake and glycogen synthesis. A single bout of exercise can also induce transient changes in skeletal muscle gene transcription and can alter rates of protein metabolism, both of which may be mechanisms for chronic adaptations to repeated bouts of exercise. A central issue in exercise biology is to elucidate the underlying molecular signaling mechanisms that regulate these important metabolic and transcriptional events in skeletal muscle. In this review, we summarize research from the past several years that has demonstrated that physical exercise can regulate multiple intracellular signaling cascades in skeletal muscle. It is now well established that physical exercise or muscle contractile activity can activate three of the mitogen-activated protein kinase signaling pathways, including the extracellular signal-regulated kinase 1 and 2, the c-Jun NH2-terminal kinase, and the p38. Exercise can also robustly increase activity of the AMP-activated protein kinase, as well as several additional molecules, including glycogen synthase kinase 3, Akt, and the p70 S6 kinase. A fundamental goal of signaling research is to determine the biological consequences of exercise-induced signaling through these molecules, and this review also provides an update of progress in this area.

Increased lipid oxidation but normal muscle glycogen response to epinephrine in humans with IDDM

1996 ◽  
Vol 271 (2) ◽  
pp. E284-E293 ◽  
Author(s):  
N. Cohen ◽  
M. Halberstam ◽  
L. Rossetti ◽  
H. Shamoon
Keyword(s):  
Skeletal Muscle ◽  
Lipid Oxidation ◽  
Glucose Uptake ◽  
Muscle Glycogen ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycogen Metabolism ◽  
Whole Body ◽  
Dependent Diabetes Mellitus ◽  
Skeletal Muscle Glycogen

The effects of physiological increments in epinephrine and insulin on glucose production (GP), skeletal muscle glycogen metabolism, and substrate oxidation were studied in eight insulin-dependent diabetes mellitus (IDDM) and nine control subjects. Epinephrine was coinfused for the final 120 min of a 240-min euglycemic, hyperinsulinemic clamp. In both groups, insulin increased glucose uptake, glycogen synthesis, and whole body carbohydrate (CHO) oxidation and inhibited GP (by 70-80%) and lipid oxidation (by approximately 50%), whereas epinephrine antagonized the effect of insulin on glucose uptake and glycogen synthesis. In contrast, GP increased in IDDM subjects (P < 0.02) but remained suppressed by insulin in controls. CHO oxidation fell (1.37 +/- 0.25 vs. 2.08 +/- 0.32 mg.kg-1.min-1) and lipid oxidation increased to baseline in IDDM subjects, with increments in plasma free fatty acids (FFA) and glycerol. In contrast, in controls, plasma FFA and glycerol remained suppressed and lipid oxidation decreased further with epinephrine (P < 0.005). Epinephrine completely reversed insulin's activation of muscle glycogen synthase in both groups. Thus, during hyperinsulinemia, the hepatic response to epinephrine in IDDM subjects may be dependent on activation of lipid oxidation. Skeletal muscle glycogen metabolism is exquisitely sensitive to epinephrine despite the presence of hyperinsulinemia.

Quantitation of glycolysis and skeletal muscle glycogen synthesis in humans

1993 ◽  
Vol 265 (5) ◽  
pp. E761-E769 ◽  
Author(s):  
L. Rossetti ◽  
Y. T. Lee ◽  
J. Ruiz ◽  
S. C. Aldridge ◽  
H. Shamoon ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Muscle Glycogen ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Whole Body ◽  
Skeletal Muscle Glycogen ◽  
Muscle Glycogen Synthesis ◽  
The Difference ◽  
Total Glucose

We measured the net rates of skeletal muscle glycogen synthesis and glycolysis (conversion of [3-3H]glucose to 3H2O) in healthy overnight-fasted volunteers. Two studies were performed. In study 1, seven subjects participated in two paired infusions under basal conditions of either [2-3H]glucose (H2) or [3-3H]glucose (H3). Total glucose uptake (Rd) and rates of whole body 3H2O formation (3H2O Ra) were measured. With H2, Rd and 3H2O Ra were similar. With H3, 3H2O Ra, equal to glycolysis, was 65% of Rd. In study 2, six different subjects underwent a 3-h, 40 mU.m-2 x min-1 euglycemic insulin clamp. [6,6-2H2]glucose was infused throughout and H3 was infused during the last hour of the study. Open muscle biopsies were obtained at 150 and 180 min. Glycogen synthesis was assessed by three independent means: 1) direct measurement, as 3H disintegrations per minute in isolated muscle glycogen per plasma H3 specific activity; 2) extrapolation from the activity of glycogen synthase assayed in the presence of the concentrations of glucose 6-phosphate and UDP-glucose measured in the biopsy; and 3) the difference between Rd and glycolysis. Despite a wide range in Rd [24.5-58.8 mumol.kg fat-free mass (FFM)-1 x min-1] and glycolysis (14.2-26.1), the three methods yielded similar results of 20.0 +/- 3.9, 22.5 +/- 3.7, and 20.6 +/- 3.7 mumol.kg FFM-1 x min-1 and correlated highly with each other (r2 = 0.92-0.96). Our results (study 1) indicate that the rate of plasma tritiated water formation reflects the intracellular detritiation of tritiated glucose. Under hyperinsulinemic conditions (study 2) the net rate of muscle glycogen synthesis can be accurately estimated from the glycogen synthase activity and from the difference between total glucose uptake and glycolysis. Thus, at high physiological plasma insulin concentrations resulting in submaximal stimulation of muscle glycogen synthesis, the latter can be accurately measured in humans.

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