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Laurie J Goodyear - One of the best experts on this subject based on the ideXlab platform.
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role of amp activated protein kinase in exercise capacity whole body Glucose homeostasis and Glucose Transport in skeletal muscle insight from analysis of a transgenic mouse model
Diabetes Research and Clinical Practice, 2007Co-Authors: Nobuharu Fujii, Michael F Hirshman, Erin M Kane, Lauren Peter, Matthew M Seifert, Schuyler Winstead, Laurie J GoodyearAbstract:To examine the role of muscle AMP-activated protein kinase (AMPK) in maximal exercise capacity, whole body Glucose homeostasis, and Glucose Transport in skeletal muscle, we generated muscle-specific transgenic mice carrying cDNAs of inactive AMPK alpha2 (alpha2i TG). Fed blood Glucose was slightly higher in alpha2i TG mice compared to wild type littermates, however, the difference was not statistically significant. In alpha2i TG mice, Glucose tolerance was slightly impaired in male, but not in female mice, compared to wild type littermates. Maximal exercise capacity was dramatically reduced in alpha2i TG mice, suggesting that AMPK alpha2 has a critical role in skeletal muscle during exercise. We confirmed that known insulin-independent stimuli of Glucose Transport including mitochondrial respiration inhibition, hyperosmolarity, and muscle contraction increased both AMPK alpha1 and alpha2 activities in isolated EDL muscle in wild type mice. While, alpha2 activation was severely blunted and alpha1 activation was only slightly reduced in alpha2i TG mice by these insulin independent stimuli compared to wild type mice. Mitochondrial respiration inhibition-induced Glucose Transport was fully inhibited in isolated EDL muscles in alpha2i TG mice. However, contraction- or hyperosmolarity-induced Glucose Transport was nearly normal. These results suggest that AMPK alpha2 activation is essential for some, but not all insulin-independent Glucose Transport.
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amp activated protein kinase and the regulation of Glucose Transport
American Journal of Physiology-endocrinology and Metabolism, 2006Co-Authors: Nobuharu Fujii, Niels Jessen, Laurie J GoodyearAbstract:The AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by acute increases in the cellular [AMP]/[ATP] ratio. In skeletal and/or cardiac muscle, AMPK activity is increased by stimuli such as exercise, hypoxia, ischemia, and osmotic stress. There are many lines of evidence that increasing AMPK activity in skeletal muscle results in increased rates of Glucose Transport. Although similar to the effects of insulin to increase Glucose Transport in muscle, it is clear that the underlying mechanisms for AMPK-mediated Glucose Transport involve proximal signals that are distinct from that of insulin. Here, we discuss the evidence for AMPK regulation of Glucose Transport in skeletal and cardiac muscle and describe research investigating putative signaling mechanisms mediating this effect. We also discuss evidence that AMPK may play a role in enhancing muscle and whole body insulin sensitivity for Glucose Transport under conditions such as exercise, as well as the use of the AMPK activator AICAR to reverse insulin-resistant conditions. The identification of AMPK as a novel Glucose Transport mediator in skeletal muscle is providing important insights for the treatment and prevention of type 2 diabetes.
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amp activated protein kinase α2 activity is not essential for contraction and hyperosmolarity induced Glucose Transport in skeletal muscle
Journal of Biological Chemistry, 2005Co-Authors: Nobuharu Fujii, Michael F Hirshman, Erin M Kane, Lauren Peter, Matthew M Seifert, Laurie J GoodyearAbstract:Abstract To examine the role of AMP-activated protein kinase (AMPK) in muscle Glucose Transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive α2 (α2i TG) and α1 (α1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In α2i TG mice basal α2 activity was barely detectable, whereas basal α1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK α2 activity in α2i TG mice, whereas α1 activation was attenuated by only 30–50%. Glucose Transport was measured in vitro using isolated EDL muscles from α2i TG mice. AICAR- and rotenone-stimulated Glucose Transport was fully inhibited in α2i TG mice; however, the lack of AMPK α2 activity had no effect on contraction- or sorbitol-induced Glucose Transport. Similar to these observations in vitro, contraction-stimulated Glucose Transport, assessed in vivo by 2-deoxy-d-[3H]Glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in α2i TG mice. Thus, AMPK α2 activation is essential for some, but not all, insulin-independent Glucose Transport. Muscle contraction- and hyperosmolarity-induced Glucose Transport may be regulated by a redundant mechanism in which AMPK α2 is one of multiple signaling pathways.
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metabolic stress and altered Glucose Transport activation of amp activated protein kinase as a unifying coupling mechanism
Diabetes, 2000Co-Authors: Tatsuya Hayashi, Lee A Witters, Michael F Hirshman, Nobuharu Fujii, Susan A Habinowski, Laurie J GoodyearAbstract:5'AMP-activated protein kinase (AMPK) can be activated in response to cellular fuel depletion and leads to switching off ATP-consuming pathways and switching on ATP-regenerating pathways in many cell types. We have hypothesized that AMPK is a central mediator of insulin-independent Glucose Transport, which enables fuel-depleted muscle cells to take up Glucose for ATP regeneration under conditions of metabolic stress. To test this hypothesis, rat epitrochlearis muscles were isolated and incubated in vitro under several conditions that evoke metabolic stress accompanied by intracellular fuel depletion. Rates of Glucose Transport in the isolated muscles were increased by all of these conditions, including contraction (5-fold above basal), hypoxia (8-fold), 2,4-dinotrophenol (11-fold), rotenone (7-fold), and hyperosmolarity (8-fold). All of these stimuli simultaneously increased both alpha1 and alpha2 isoform-specific AMPK activity. There was close correlation between alpha1 (r2 = 0.72) and alpha2 (r2 = 0.67) AMPK activities and the rate of Glucose Transport, irrespective of the metabolic stress used, all of which compromised muscle fuel status as judged by ATP, phosphocreatine, and glycogen content. 5-Aminoimidazole-4-carboxamide ribonucleoside, a pharmacological AMPK activator that is metabolized to an AMP-mimetic ZMP, also increased both Glucose Transport and AMPK activity but did not change fuel status. Insulin stimulated Glucose Transport by 6.5-fold above basal but did not affect AMPK activity. These results suggest that the activation of AMPK may be a common mechanism leading to insulin-independent Glucose Transport in skeletal muscle under conditions of metabolic stress.
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evidence for 5 amp activated protein kinase mediation of the effect of muscle contraction on Glucose Transport
Diabetes, 1998Co-Authors: Tatsuya Hayashi, Michael F Hirshman, Emily J Kurth, William W Winder, Laurie J GoodyearAbstract:The intracellular signaling proteins that lead to exercise-stimulated Glucose Transport in skeletal muscle have not been identified, although it is clear that there are separate signaling mechanisms for exercise- and insulinstimulated Glucose Transport. We have hypothesized that the 5′AMP-activated protein kinase (AMPK) functions as a signaling intermediary in exercise-stimulated Glucose uptake. This hypothesis was based on recent studies showing the following: 1 ) muscle contraction increases AMPK activity and 2 ) perfusion of rat hindlimb skeletal muscles with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a compound that results in increased AMPK activity, increased insulin-stimulated Glucose uptake. In the current study, isolated rat epitrochlearis muscles were treated to contract in vitro (via electrical stimulation for 10 min) and/or incubated in the absence or presence of AICAR (2 mmol/l), insulin (1 μmol/1), or wortmannin (100 nmolA). Both contraction and AICAR significantly increased AMPK activity, while the enzyme was not activated by insulin. AICAR, contraction, and insulin all increased 3- O -methylGlucose (3MG) Transport by threefold to fivefold above basal. The phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin completely blocked insulin-stimulated Transport, but did not inhibit AICAR- or contraction-stimulated Transport. The increase in Glucose Transport with the combination of maximal AICAR plus maximal insulin treatments was partially additive, suggesting that these stimuli increase Glucose Transport by different mechanisms. In contrast, there was no additive effect on Glucose Transport with the combination of AICAR plus contraction. These data suggest that AICAR and contraction stimulate Glucose Transport by a similar insulin-independent signaling mechanism and are consistent with the hypothesis that AMPK is involved in exercise-stimulated Glucose uptake.
Amira Klip - One of the best experts on this subject based on the ideXlab platform.
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triiodothyronine acutely stimulates Glucose Transport into l6 muscle cells without increasing surface glut4 glut1 or glut3
Thyroid, 2012Co-Authors: Silvania Da Silva Teixeira, Amira Klip, Akhilesh K Tamrakar, Francemilson Goulartsilva, Caroline Serranonascimento, Maria Tereza NunesAbstract:Background: Thyroid hormones (THs) act genomically to stimulate Glucose Transport by elevating Glucose Transporter (Slc2a) expression and Glucose utilization by cells. However, nongenomic effects of THs are now emerging. Here, we assess how triiodothyronine (T3) acutely affects Glucose Transport and the content of GLUT4, GLUT1, and GLUT3 at the surface of muscle cells, and possible interactions between T3 and insulin action. Methods: Differentiated L6 myotubes transfected with myc-tagged Slc2a4 (L6-GLUT4myc) or Slc2a1 (L6-GLUT1myc) and wild-type L6 myotubes were studied in the following conditions: control, hypothyroid (Tx), Tx plus T3, Tx plus insulin, and Tx plus insulin and T3. Results: Glucose uptake and GLUT4 content at the cell surface decreased in the Tx group relative to controls. T3 treatment for 30 minutes increased Glucose Transport into L6-GLUT4myc cells without altering surface GLUT4 content, which increased only thereafter. The total amount of GLUT4 protein remained unchanged among the group...
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an inhibitor of p38 mitogen activated protein kinase prevents insulin stimulated Glucose Transport but not Glucose Transporter translocation in 3t3 l1 adipocytes and l6 myotubes
Journal of Biological Chemistry, 1999Co-Authors: Gary Sweeney, Romel Somwar, Toolsie Ramlal, Allen Volchuk, Atsunori Ueyama, Amira KlipAbstract:Abstract The precise mechanisms underlying insulin-stimulated Glucose Transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated Glucose Transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated Glucose Transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 μm). Basal Glucose uptake was not affected. Moreover, SB203580 added only during the Transport assay did not inhibit basal or insulin-stimulated Transport. SB203580 did not inhibit insulin-stimulated translocation of the Glucose Transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of Glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of Glucose Transporters without stimulating Glucose Transport. We propose that insulin stimulates two independent signals contributing to stimulation of Glucose Transport: phosphatidylinositol 3-kinase leads to Glucose Transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited Glucose Transporter at the membrane.
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multiple roles of phosphatidylinositol 3 kinase in regulation of Glucose Transport amino acid Transport and Glucose Transporters in l6 skeletal muscle cells
Endocrinology, 1995Co-Authors: Theodoros Tsakiridis, H E Mcdowell, T Walker, C P Downes, Harinder S Hundal, Mladen Vranic, Amira KlipAbstract:Phosphatidylinositol 3-kinase (PI3k) activity is required for the insulin stimulation of Glucose Transport in adipocytes and Chinese hamster ovary cells. Wortmannin (WM), an inhibitor of PI3k, inhibits the stimulation of Glucose Transport by insulin and the gain of Glucose Transporters at the cell surface. However, the effect of inhibition of PI3k on the maintenance of the basal and the insulin-stimulated Glucose Transport and on the intracellular donor pool of Glucose Transporters has not been clarified. Here we show that in L6 skeletal muscle cells in culture WM significantly inhibits the basal PI3k activity (by 40%), decreases the levels of phosphatidylinositol 3,4-phosphate and 3,4,5-phosphate (by about 50%) and abolishes the activation of the enzyme by insulin. WM inhibited the basal rate of Transport of Glucose (by 45%) and of amino acids through system A (by 25%) and abolished their stimulation by insulin. Insulin caused a transient increase in PI3k activity and PI3k products that returned to basal...
Nobuharu Fujii - One of the best experts on this subject based on the ideXlab platform.
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sucrose nonfermenting ampk related kinase snark mediates contraction stimulated Glucose Transport in mouse skeletal muscle
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Taro Toyoda, Nobuharu Fujii, Michelle M Jung, Amee Rathod, Janwillem R Middelbeek, Sarah J Lessard, Jonas T Treebak, Katsuya Tsuchihara, Hiroyasu Esumi, Erik A RichterAbstract:The signaling mechanisms that mediate the important effects of contraction to increase Glucose Transport in skeletal muscle are not well understood, but are known to occur through an insulin-independent mechanism. Muscle-specific knockout of LKB1, an upstream kinase for AMPK and AMPK-related protein kinases, significantly inhibited contraction-stimulated Glucose Transport. This finding, in conjunction with previous studies of ablated AMPKα2 activity showing no effect on contraction-stimulated Glucose Transport, suggests that one or more AMPK-related protein kinases are important for this process. Muscle contraction increased sucrose nonfermenting AMPK-related kinase (SNARK) activity, an effect blunted in the muscle-specific LKB1 knockout mice. Expression of a mutant SNARK in mouse tibialis anterior muscle impaired contraction-stimulated, but not insulin-stimulated, Glucose Transport. Whole-body SNARK heterozygotic knockout mice also had impaired contraction-stimulated Glucose Transport in skeletal muscle, and knockdown of SNARK in C2C12 muscle cells impaired sorbitol-stimulated Glucose Transport. SNARK is activated by muscle contraction and is a unique mediator of contraction-stimulated Glucose Transport in skeletal muscle.
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role of amp activated protein kinase in exercise capacity whole body Glucose homeostasis and Glucose Transport in skeletal muscle insight from analysis of a transgenic mouse model
Diabetes Research and Clinical Practice, 2007Co-Authors: Nobuharu Fujii, Michael F Hirshman, Erin M Kane, Lauren Peter, Matthew M Seifert, Schuyler Winstead, Laurie J GoodyearAbstract:To examine the role of muscle AMP-activated protein kinase (AMPK) in maximal exercise capacity, whole body Glucose homeostasis, and Glucose Transport in skeletal muscle, we generated muscle-specific transgenic mice carrying cDNAs of inactive AMPK alpha2 (alpha2i TG). Fed blood Glucose was slightly higher in alpha2i TG mice compared to wild type littermates, however, the difference was not statistically significant. In alpha2i TG mice, Glucose tolerance was slightly impaired in male, but not in female mice, compared to wild type littermates. Maximal exercise capacity was dramatically reduced in alpha2i TG mice, suggesting that AMPK alpha2 has a critical role in skeletal muscle during exercise. We confirmed that known insulin-independent stimuli of Glucose Transport including mitochondrial respiration inhibition, hyperosmolarity, and muscle contraction increased both AMPK alpha1 and alpha2 activities in isolated EDL muscle in wild type mice. While, alpha2 activation was severely blunted and alpha1 activation was only slightly reduced in alpha2i TG mice by these insulin independent stimuli compared to wild type mice. Mitochondrial respiration inhibition-induced Glucose Transport was fully inhibited in isolated EDL muscles in alpha2i TG mice. However, contraction- or hyperosmolarity-induced Glucose Transport was nearly normal. These results suggest that AMPK alpha2 activation is essential for some, but not all insulin-independent Glucose Transport.
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amp activated protein kinase and the regulation of Glucose Transport
American Journal of Physiology-endocrinology and Metabolism, 2006Co-Authors: Nobuharu Fujii, Niels Jessen, Laurie J GoodyearAbstract:The AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by acute increases in the cellular [AMP]/[ATP] ratio. In skeletal and/or cardiac muscle, AMPK activity is increased by stimuli such as exercise, hypoxia, ischemia, and osmotic stress. There are many lines of evidence that increasing AMPK activity in skeletal muscle results in increased rates of Glucose Transport. Although similar to the effects of insulin to increase Glucose Transport in muscle, it is clear that the underlying mechanisms for AMPK-mediated Glucose Transport involve proximal signals that are distinct from that of insulin. Here, we discuss the evidence for AMPK regulation of Glucose Transport in skeletal and cardiac muscle and describe research investigating putative signaling mechanisms mediating this effect. We also discuss evidence that AMPK may play a role in enhancing muscle and whole body insulin sensitivity for Glucose Transport under conditions such as exercise, as well as the use of the AMPK activator AICAR to reverse insulin-resistant conditions. The identification of AMPK as a novel Glucose Transport mediator in skeletal muscle is providing important insights for the treatment and prevention of type 2 diabetes.
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amp activated protein kinase α2 activity is not essential for contraction and hyperosmolarity induced Glucose Transport in skeletal muscle
Journal of Biological Chemistry, 2005Co-Authors: Nobuharu Fujii, Michael F Hirshman, Erin M Kane, Lauren Peter, Matthew M Seifert, Laurie J GoodyearAbstract:Abstract To examine the role of AMP-activated protein kinase (AMPK) in muscle Glucose Transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive α2 (α2i TG) and α1 (α1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In α2i TG mice basal α2 activity was barely detectable, whereas basal α1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK α2 activity in α2i TG mice, whereas α1 activation was attenuated by only 30–50%. Glucose Transport was measured in vitro using isolated EDL muscles from α2i TG mice. AICAR- and rotenone-stimulated Glucose Transport was fully inhibited in α2i TG mice; however, the lack of AMPK α2 activity had no effect on contraction- or sorbitol-induced Glucose Transport. Similar to these observations in vitro, contraction-stimulated Glucose Transport, assessed in vivo by 2-deoxy-d-[3H]Glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in α2i TG mice. Thus, AMPK α2 activation is essential for some, but not all, insulin-independent Glucose Transport. Muscle contraction- and hyperosmolarity-induced Glucose Transport may be regulated by a redundant mechanism in which AMPK α2 is one of multiple signaling pathways.
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metabolic stress and altered Glucose Transport activation of amp activated protein kinase as a unifying coupling mechanism
Diabetes, 2000Co-Authors: Tatsuya Hayashi, Lee A Witters, Michael F Hirshman, Nobuharu Fujii, Susan A Habinowski, Laurie J GoodyearAbstract:5'AMP-activated protein kinase (AMPK) can be activated in response to cellular fuel depletion and leads to switching off ATP-consuming pathways and switching on ATP-regenerating pathways in many cell types. We have hypothesized that AMPK is a central mediator of insulin-independent Glucose Transport, which enables fuel-depleted muscle cells to take up Glucose for ATP regeneration under conditions of metabolic stress. To test this hypothesis, rat epitrochlearis muscles were isolated and incubated in vitro under several conditions that evoke metabolic stress accompanied by intracellular fuel depletion. Rates of Glucose Transport in the isolated muscles were increased by all of these conditions, including contraction (5-fold above basal), hypoxia (8-fold), 2,4-dinotrophenol (11-fold), rotenone (7-fold), and hyperosmolarity (8-fold). All of these stimuli simultaneously increased both alpha1 and alpha2 isoform-specific AMPK activity. There was close correlation between alpha1 (r2 = 0.72) and alpha2 (r2 = 0.67) AMPK activities and the rate of Glucose Transport, irrespective of the metabolic stress used, all of which compromised muscle fuel status as judged by ATP, phosphocreatine, and glycogen content. 5-Aminoimidazole-4-carboxamide ribonucleoside, a pharmacological AMPK activator that is metabolized to an AMP-mimetic ZMP, also increased both Glucose Transport and AMPK activity but did not change fuel status. Insulin stimulated Glucose Transport by 6.5-fold above basal but did not affect AMPK activity. These results suggest that the activation of AMPK may be a common mechanism leading to insulin-independent Glucose Transport in skeletal muscle under conditions of metabolic stress.
Robert V. Farese - One of the best experts on this subject based on the ideXlab platform.
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repletion of atypical protein kinase c following rna interference mediated depletion restores insulin stimulated Glucose Transport
Journal of Biological Chemistry, 2006Co-Authors: Mini P Sajan, Mary L Standaert, J Rivas, Robert V. FareseAbstract:Abstract The role of atypical protein kinase C (aPKC) in insulin-stimulated Glucose Transport in myocytes and adipocytes is controversial. Whereas studies involving the use of adenovirally mediated expression of kinase-inactive aPKC in L6 myocytes and 3T3/L1 and human adipocytes, and data from knock-out of aPKC in adipocytes derived from mouse embryonic stem cells and subsequently derived adipocytes, suggest that aPKCs are required for insulin-stimulated Glucose Transport, recent findings in studies of aPKC knockdown by small interfering RNA (RNAi) in 3T3/L1 adipocytes are conflicting. Moreover, there are no reports of aPKC knockdown in myocytes, wherein insulin effects on Glucose Transport are particularly relevant for understanding whole body Glucose disposal. Presently, we exploited the fact that L6 myotubes and 3T3/L1 adipocytes have substantially different (30% nonhomology) major aPKCs, viz. PKC-ζ in L6 myotubes and PKC-λ in 3T3/L1 adipocytes, that nevertheless can function interchangeably for Glucose Transport. Accordingly, in L6 myotubes, RNAi-targeting PKC-ζ, but not PKC-λ, markedly depleted aPKC and concomitantly inhibited insulin-stimulated Glucose Transport; more importantly, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-λ. Conversely, in 3T3/L1 adipocytes, RNAi constructs targeting PKC-λ, but not PKC-ζ, markedly depleted aPKC and concomitantly inhibited insulin-stimulated Glucose Transport; here again, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-ζ. These findings in knockdown and, more convincingly, rescue studies, strongly support the hypothesis that aPKCs are required for insulin-stimulated Glucose Transport in myocytes and adipocytes.
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sorbitol activates atypical protein kinase c and glut4 Glucose Transporter translocation Glucose Transport through proline rich tyrosine kinase 2 the extracellular signal regulated kinase pathway and phospholipase d
Biochemical Journal, 2002Co-Authors: Mini P Sajan, Mary L Standaert, Gautam Bandyopadhyay, Yoshinori Kanoh, Michael J Quon, Brent C Reed, Ivan Dikic, Robert V. FareseAbstract:Sorbitol, "osmotic stress", stimulates GLUT4 Glucose Transporter translocation to the plasma membrane and Glucose Transport by a phosphatidylinositol (PI) 3-kinase-independent mechanism that reportedly involves non-receptor proline-rich tyrosine kinase-2 (PYK2) but subsequent events are obscure. In the present study, we found that extracellular signal-regulated kinase (ERK) pathway components, growth-factor-receptor-bound-2 protein, son of sevenless (SOS), RAS, RAF and mitogen-activated protein (MAP) kinase/ERK kinase, MEK(-1), operating downstream of PYK2, were required for sorbitol-stimulated GLUT4 translocation/Glucose Transport in rat adipocytes, L6 myotubes and 3T3/L1 adipocytes. Furthermore, sorbitol activated atypical protein kinase C (aPKC) through a similar mechanism depending on the PYK2/ERK pathway, independent of PI 3-kinase and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1). Like PYK2/ERK pathway components, aPKCs were required for sorbitol-stimulated GLUT4 translocation/Glucose Transport. Interestingly, sorbitol stimulated increases in phospholipase D (PLD) activity and generation of phosphatidic acid (PA), which directly activated aPKCs. As with aPKCs and Glucose Transport, sorbitol-stimulated PLD activity was dependent on the ERK pathway. Moreover, PLD-generated PA was required for sorbitol-induced activation of aPKCs and GLUT4 translocation/Glucose Transport. Our findings suggest that sorbitol sequentially activates PYK2, the ERK pathway and PLD, thereby increasing PA, which activates aPKCs and GLUT4 translocation. This mechanism contrasts with that of insulin, which primarily uses PI 3-kinase, D3-PO(4) polyphosphoinositides and PDK-1 to activate aPKCs.
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evidence for involvement of protein kinase c pkc zeta and noninvolvement of diacylglycerol sensitive pkcs in insulin stimulated Glucose Transport in l6 myotubes
Endocrinology, 1997Co-Authors: Gautam Bandyopadhyay, Mary L Standaert, Lamar Galloway, Jorge Moscat, Robert V. FareseAbstract:We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likely to play a role in insulin-stimulated Glucose Transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 microM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80-100% losses of all DAG-dependent PKCs (alpha, beta1, beta2, delta, epsilon) and TPA-stimulated Glucose Transport (2-deoxyGlucose uptake); in contrast, there was full retention of PKC-zeta, as well as insulin-stimulated Glucose Transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCbeta2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on Glucose Transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on Glucose Transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate Glucose Transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubstrate inhibited insulin-stimulated Glucose Transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-zeta pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated Glucose Transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated Glucose Transport, LY379196, which inhibits PKC-beta (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated Glucose Transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated Glucose Transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of Glucose Transport, DAG-sensitive PKCs are unlikely participants.
Abram Katz - One of the best experts on this subject based on the ideXlab platform.
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mechanical load plays little role in contraction mediated Glucose Transport in mouse skeletal muscle
The Journal of Physiology, 2007Co-Authors: Marie E Sandstrom, Shijin Zhang, Hakan Westerblad, Abram KatzAbstract:The factors responsible for control of Glucose Transport during exercise are not fully understood. We investigated the role of mechanical load in contraction-mediated Glucose Transport in an isolated muscle preparation. Mouse extensor digitorum longus muscles were stimulated with repeated contractions for 10 min with or without N-benzyl-p-toluene sulphonamide (BTS, an inhibitor of myosin II ATPase) to block crossbridge activity. BTS inhibited force production during repeated contraction to ∼5% of control. In contrast, BTS had little effect on Glucose Transport in the basal state (control = 0.55 ± 0.04; BTS = 0.47 ± 0.09 μmol (20 min)−1 ml−1) or after contraction (control = 2.27 ± 0.15; BTS = 2.10 ± 0.16 μmol (20 min)−1 ml−1). BTS did not significantly alter the contraction-mediated changes in high-energy phosphates, glutathione status (a measure of oxidant status) or AMP-activated protein kinase activity. In conclusion, these data show that mechanical load plays little role in contraction-mediated Glucose Transport. Instead, it is likely that the increased Glucose Transport during contraction is a consequence of the increase in myoplasmic Ca2+ and the subsequent alterations in metabolism, e.g. increased energy turnover and production of reactive oxygen species.
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role of reactive oxygen species in contraction mediated Glucose Transport in mouse skeletal muscle
The Journal of Physiology, 2006Co-Authors: Marie E Sandstrom, Shijin Zhang, Hakan Westerblad, Joseph D Bruton, Jose P Silva, Michael B Reid, Abram KatzAbstract:Exercise increases Glucose Transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction-mediated Glucose Transport. Repeated contractions increased 2-deoxyGlucose (2-DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast-twitch) muscle. N-Acetylcysteine (NAC), a non-specific antioxidant, inhibited contraction-mediated 2-DG uptake by ∼50% (P < 0.05 versus control values), but did not significantly affect basal 2-DG uptake or the uptake induced by insulin, hypoxia or 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, which mimics AMP-mediated activation of AMP-activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction-mediated 2-DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+-dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a ∼25% higher rate of contraction-mediated 2-DG uptake versus muscles from wild-type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction-mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation ∼10-fold, and NAC blocked ∼50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction-mediated activation of Glucose Transport in fast-twitch muscle.