[Show abstract][Hide abstract] ABSTRACT: Insulin stimulates glycogen synthase (GS) through dephosphorylation of serine residues, and this effect is impaired in skeletal muscle from insulin-resistant [obese and type 2 diabetic (T2DM)] subjects. Exercise also increases GS activity, yet it is not known whether the ability of exercise to affect GS is impaired in insulin-resistant subjects. The objective of this study was to examine the effect of acute exercise on GS phosphorylation and enzyme kinetic properties in muscle from insulin-resistant individuals. Lean normal glucose-tolerant (NGT), obese NGT, and obese T2DM subjects performed 40 min of moderate-intensity cycle exercise (70% of Vo(2max)). GS kinetic properties and phosphorylation were measured in vastus lateralis muscle before exercise, immediately after exercise, and 3.5 h postexercise. In lean subjects, GS fractional activity increased twofold after 40 min of exercise, and it remained elevated after the 3.5-h rest period. Importantly, exercise also decreased GS K(m) for UDP-glucose from ≈0.5 to ≈0.2 mM. In lean subjects, exercise caused significant dephosphorylation of GS by 50-70% (Ser(641), Ser(645), and Ser(645,649,653,657)), and phosphorylation of these sites remained decreased after 3.5 h; Ser⁷ phosphorylation was not regulated by exercise. In obese NGT and T2DM subjects, exercise increased GS fractional activity, decreased K(m) for UDP-glucose, and decreased GS phosphorylation as effectively as in lean NGT subjects. We conclude that the molecular regulatory process by which exercise promotes glycogen synthesis in muscle is preserved in insulin-resistant subjects.
[Show abstract][Hide abstract] ABSTRACT: In the present study, the effects of insulin and contraction on glycogen synthase (GS) kinetic properties and phosphorylation were investigated in epitrochlearis muscles from lean and obese Zucker rats. Total GS activity and protein expression were ~15% lower in epitrochlearis from obese rats compared with lean rats. Insulin-stimulated GS fractional activity and affinity for UDP-glucose were lower (higher K(m)) in muscles from obese rats. GS Ser(641) and Ser(645,649,653,657) phosphorylation was higher in insulin-stimulated muscles from obese rats, which agreed with lower GS activation. Contraction-mediated GS dephosphorylation of Ser(641), Ser(641+645), Ser(645,649,653,657), and Ser(7+10) was normal in muscles from obese Zucker rats, and GS fractional activity increased to similar levels in epitrochlearis muscles from lean and obese rats. GS affinity for UDP glucose was ~0.8, ~0.4, and ~0.1 mM with assay buffers containing 0, 0.17, and 12 mM glucose 6-phosphate, respectively. Contraction increased affinity for UDP-glucose (reduced K(m)) at a physiological concentration of glucose 6-phosphate (0.17 mM) to ~0.2 mM in muscles from both lean and obese rats. Interestingly, in the absence of glucose 6-phosphate in the assay buffer, contraction (and insulin) did not influence GS affinity for UDP-glucose, indicating that affinity is regulated by sensitivity for glucose 6-phosphate. In conclusion, contraction-mediated activation and dephosphorylation of GS were normal in muscles from obese Zucker rats, whereas insulin-mediated GS activation and dephosphorylation were impaired.
Preview · Article · Mar 2012 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: Glycogen synthesis increases after muscle contraction and during insulin stimulation, and insulin-stimulated glycogen synthesis is enhanced after contraction. We hypothesized that the initial glycogen content influences the magnitude of additive effect of contraction on insulin-stimulated glycogen synthesis. Contraction and insulin had full additive effect on rate of glycogen synthesis measured after contraction in muscles with normal and high glycogen content. In muscles with low glycogen, contraction increased insulin-stimulated glycogen synthesis nearly as much as in muscles with normal glycogen, but not to the sum of the two stimuli studied separately; still glycogen synthesis was generally highest in muscles with low glycogen. Glycogen synthase fractional activity inversely correlated with glycogen content and contraction increased glycogen synthase fractional activity. Contraction and insulin additively increased glycogen synthase fractional activity at all glycogen contents. In conclusion, after contraction insulin-stimulated glycogen synthesis was increased by rather similar magnitude at all glycogen contents in concert with increased glycogen synthase activation.
No preview · Article · Jul 2010 · Archives of Physiology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Genetic approaches have documented protein kinase B (PKB) as a pivotal regulator of heart function. Insulin strongly activates PKB, whereas adrenaline is not considered a major physiological regulator of PKB in heart. In skeletal muscles, however, adrenaline potentiates insulin-stimulated PKB activation without having effect in the absence of insulin. The purpose of the present study was to investigate the interaction between insulin and beta-adrenergic stimulation in regulation of PKB phosphorylation.
Cardiomyocytes were isolated from adult rats by collagenase, and incubated with insulin, isoprenaline, and other compounds. Protein phosphorylation was evaluated by Western blot and phospho-specific antibodies.
Isoprenaline increased insulin-stimulated PKB Ser(473) and Thr(308) phosphorylation more than threefold in cardiomyocytes. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3beta Ser(9) phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (beta(1)-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (beta(2)-agonist) only potentiated insulin-stimulated PKB phosphorylation by approximately 80%. Dobutamine, but not salbutamol, increased phospholamban Ser(16) phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogue that activates PKA (dibutyryl-cAMP and N(6)-benzoyl-cAMP) increased insulin-stimulated PKB phosphorylation by more than threefold without effect alone. The Epac-specific activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (007) increased insulin-stimulated PKB phosphorylation by approximately 50%. Db-cAMP and N(6)-benzoyl-cAMP, but not 007, increased phospholamban Ser(16) phosphorylation.
beta-adrenoceptors are strong regulators of PKB phosphorylation via cAMP and PKA when insulin is present. We hypothesize that PKB mediates important signalling in the heart during beta-adrenergic receptors stimulation.
Full-text · Article · May 2010 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Caffeine and theophylline inhibit phosphatidylinositol 3-kinase (PI3-kinase) activity and insulin-stimulated protein kinase B (PKB) phosphorylation. Insulin-stimulated glucose uptake involves PI3-kinase/PKB, and the aim of the present study was to test the hypothesis that caffeine and theophylline inhibit insulin-stimulated glucose uptake in skeletal muscles.
Rat epitrochlearis muscles and soleus strips were incubated with insulin and different concentrations of caffeine and theophylline for measurement of glucose uptake, force development and PKB phosphorylation. The effect of caffeine was also investigated in muscles stimulated electrically.
Caffeine and theophylline completely blocked insulin-stimulated glucose uptake in both soleus and epitrochlearis muscles at 10 mm. Furthermore, insulin-stimulated PKB Ser(473) and Thr(308) and GSK-3beta Ser(9) phosphorylation were blocked by caffeine and theophylline. Caffeine reduced and theophylline blocked insulin-stimulated glycogen synthase activation. Caffeine stimulates Ca(2+) release and force development increased rapidly to 10-20% of maximal tetanic contraction. Dantrolene (25 microm), a well-known inhibitor of Ca(2+)-release, prevented caffeine-induced force development, but caffeine inhibited insulin-stimulated glucose uptake in the presence of dantrolene. Contraction, like insulin, stimulates glucose uptake via translocation of glucose transporter-4 (GLUT4). Caffeine and theophylline reduced contraction-stimulated glucose uptake by about 50%, whereas contraction-stimulated glycogen breakdown was normal.
Caffeine and theophylline block insulin-stimulated glucose uptake independently of Ca(2+) release, and the likely mechanism is via blockade of insulin-stimulated PI3-kinase/PKB activation. Caffeine and theophylline also reduced contraction-stimulated glucose uptake, which occurs independently of PI3-kinase/PKB, and we hypothesize that caffeine and theophylline also inhibit glucose uptake in skeletal muscles via an additional and hitherto unknown molecule involved in GLUT4 translocation.
No preview · Article · Feb 2010 · Acta Physiologica
[Show abstract][Hide abstract] ABSTRACT: Glycogen content and contraction strongly regulate glycogen synthase (GS) activity, and the aim of the present study was to explore their effects and interaction on GS phosphorylation and kinetic properties. Glycogen content in rat epitrochlearis muscles was manipulated in vivo. After manipulation, incubated muscles with normal glycogen [NG; 210.9 +/- 7.1 mmol/kg dry weight (dw)], low glycogen (LG; 108.1 +/- 4.5 mmol/ kg dw), and high glycogen (HG; 482.7 +/- 42.1 mmol/kg dw) were contracted or rested before the studies of GS kinetic properties and GS phosphorylation (using phospho-specific antibodies). LG decreased and HG increased GS K(m) for UDP-glucose (LG: 0.27 +/- 0.02 < NG: 0.71 +/- 0.06 < HG: 1.11 +/- 0.12 mM; P < 0.001). In addition, GS fractional activity inversely correlated with glycogen content (R = -0.70; P < 0.001; n = 44). Contraction decreased K(m) for UDP-glucose (LG: 0.14 +/- 0.01 = NG: 0.16 +/- 0.01 < HG: 0.33 +/- 0.03 mM; P < 0.001) and increased GS fractional activity, and these effects were observed independently of glycogen content. In rested muscles, GS Ser(641) and Ser(7) phosphorylation was decreased in LG and increased in HG compared with NG. GSK-3beta Ser(9) and AMPKalpha Thr(172) phosphorylation was not modulated by glycogen content in rested muscles. Contraction decreased phosphorylation of GS Ser(641) at all glycogen contents. However, contraction increased GS Ser(7) phosphorylation even though GS was strongly activated. In conclusion, glycogen content regulates GS affinity for UDP-glucose and low affinity for UDP-glucose in muscles with high glycogen content may reduce glycogen accumulation. Contraction increases GS affinity for UDP-glucose independently of glycogen content and creates a unique phosphorylation pattern.
Preview · Article · Dec 2007 · AJP Endocrinology and Metabolism