title: Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2+ and cPKC.
abstract: 2,4-Dinitrophenol (DNP) uncouples the mitochondrial oxidative chain from ATP production, preventing oxidative metabolism. The consequent increase in energy demand is, however, contested by cells increasing glucose uptake to produce ATP via glycolysis. In L6 skeletal muscle cells, DNP rapidly doubles glucose transport, reminiscent of the effect of insulin. However, glucose transport stimulation by DNP does not require insulin receptor substrate-1 phosphorylation and is wortmannin insensitive. We report here that, unlike insulin, DNP does not activate phosphatidylinositol 3-kinase, protein kinase B/Akt, or p70 S6 kinase. However, chelation of intra- and extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM in conjunction with EGTA inhibited DNP-stimulated glucose uptake by 78.9 +/- 3.5%. Because Ca2+-sensitive, conventional protein kinase C (cPKC) can activate glucose transport in L6 muscle cells, we examined whether cPKC may be translocated and activated in response to DNP in L6 myotubes. Acute DNP treatment led to translocation of cPKCs to plasma membrane. cPKC immunoprecipitated from plasma membranes exhibited a twofold increase in kinase activity in response to DNP. Overnight treatment with 4-phorbol 12-myristate 13-acetate downregulated cPKC isoforms alpha, beta, and gamma and partially inhibited (45.0 +/- 3.6%) DNP- but not insulin-stimulated glucose uptake. Consistent with this, the PKC inhibitor bisindolylmaleimide I blocked PKC enzyme activity at the plasma membrane (100%) and inhibited DNP-stimulated 2-[3H]deoxyglucose uptake (61.2 +/- 2.4%) with no effect on the stimulation of glucose transport by insulin. Finally, the selective PKC-beta inhibitor LY-379196 partially inhibited DNP effects on glucose uptake (66.7 +/- 1.6%). The results suggest interfering with mitochondrial ATP production acts on a signal transduction pathway independent from that of insulin and partly mediated by Ca2+ and cPKCs, of which PKC-beta likely plays a significant role.
authors.affiliation: Programme in Cell Biology, Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada M5S 1A8.
authors.forename: Z A
authors.initials: ZA
authors.lastname: Khayat
authors.forename: T
authors.initials: T
authors.lastname: Tsakiridis
authors.forename: A
authors.initials: A
authors.lastname: Ueyama
authors.forename: R
authors.initials: R
authors.lastname: Somwar
authors.forename: Y
authors.initials: Y
authors.lastname: Ebina
authors.forename: A
authors.initials: A
authors.lastname: Klip
mesh_terms: D019297:2,4-Dinitrophenol / Q000494:pharmacology; D019325:3-O-Methylglucose / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000493:pharmacokinetics; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D014475:Uncoupling Agents / Q000494:pharmacology*
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D014475:Uncoupling Agents; D019325:3-O-Methylglucose; D011493:Protein Kinase C; D005947:Glucose; D019297:2,4-Dinitrophenol; D002118:Calcium