PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.

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.

Background and purpose: 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 b-adrenergic stimulation in regulation of PKB phosphorylation. Experimental approach: 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. Key results: 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-3b Ser 9 phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (b1-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (b2-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-methylcAMP (007) increased insulin-stimulated PKB phosphorylation by approximately 50%. Db-cAMP and N 6 -benzoyl-cAMP, but not 007, increased phospholamban Ser 16 phosphorylation. Conclusions and implications: b-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 b-adrenergic receptors stimulation. Pharmacology (2010) 160, 116-129; doi:10.1111/j.1476-5381.2010 published online 23 March 2010 Keywords: Heart; Akt; GSK-3; phosphorylation; phosphatidylinositol 3-kinase; phoshodiesterase; phospholamban; hypertrophy; rolipram; ERK Abbreviations: DNA-PK, DNA-dependent protein kinase; Epac, exchange protein directly activated by cAMP; ERK, extracellular signal-regulated kinase; GSK-3, glycogen synthase kinase-3; IGF-1, insulin like growth factor-1; mAKAP, muscle-specific A-kinase anchoring protein; MEK, mitogen-activated protein kinase kinase; mTORC2, mammalian target of rapamyosin (mTOR) complex-2; PDK1, phosphoinositide-dependent kinase-1; PTEN, phosphatase and tensin homolog deleted on chromosome 10 British Journal of

Lin FC, Bolling A, Stuenaes JT, Cumming KT, Ingvaldsen A, Lai YC, Ivy JL, Jensen J. Effect of insulin and contraction on glycogen synthase phosphorylation and kinetic properties in epitrochlearis muscles from lean and obese Zucker rats.