Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells

Sophie Turban, Clare Stretton, Olivier Drouin, Charlotte J. Green, Maria L. Watson, Alexander Gray, Fiona Ross, Louise Lantier, Benoit Viollet, D.Grahame Hardie, Andre Marette, Harinder S. Hundal (Lead / Corresponding author)

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    Abstract

    The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GUin L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulatedGUwas sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interferingAMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide- stimulated GU by ~20%. Consistent with this, analysis of GU in muscle cells from a1 /a2 AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ~35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.
    Original languageEnglish
    Pages (from-to)20088-20099
    Number of pages12
    JournalJournal of Biological Chemistry
    Volume287
    Issue number24
    DOIs
    Publication statusPublished - 8 Jun 2012

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    AMP-Activated Protein Kinases
    Metformin
    Muscle Cells
    Protein Kinase C
    Muscle
    Skeletal Muscle
    Cells
    Glucose
    Biguanides
    Phosphorylation
    Phenformin
    Skeletal Muscle Fibers
    AMP-activated protein kinase kinase
    Chemical activation
    Insulin
    Phosphatidylinositol 3-Kinases
    Small Interfering RNA

    Cite this

    Turban, Sophie ; Stretton, Clare ; Drouin, Olivier ; Green, Charlotte J. ; Watson, Maria L. ; Gray, Alexander ; Ross, Fiona ; Lantier, Louise ; Viollet, Benoit ; Hardie, D.Grahame ; Marette, Andre ; Hundal, Harinder S. / Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 24. pp. 20088-20099.
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    title = "Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells",
    abstract = "The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GUin L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulatedGUwas sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-{\ss}-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interferingAMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide- stimulated GU by ~20{\%}. Consistent with this, analysis of GU in muscle cells from a1 /a2 AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ~35{\%} compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90{\%}) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.",
    author = "Sophie Turban and Clare Stretton and Olivier Drouin and Green, {Charlotte J.} and Watson, {Maria L.} and Alexander Gray and Fiona Ross and Louise Lantier and Benoit Viollet and D.Grahame Hardie and Andre Marette and Hundal, {Harinder S.}",
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    Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells. / Turban, Sophie; Stretton, Clare; Drouin, Olivier; Green, Charlotte J.; Watson, Maria L.; Gray, Alexander; Ross, Fiona; Lantier, Louise; Viollet, Benoit; Hardie, D.Grahame; Marette, Andre; Hundal, Harinder S. (Lead / Corresponding author).

    In: Journal of Biological Chemistry, Vol. 287, No. 24, 08.06.2012, p. 20088-20099.

    Research output: Contribution to journalArticle

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    T1 - Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells

    AU - Turban, Sophie

    AU - Stretton, Clare

    AU - Drouin, Olivier

    AU - Green, Charlotte J.

    AU - Watson, Maria L.

    AU - Gray, Alexander

    AU - Ross, Fiona

    AU - Lantier, Louise

    AU - Viollet, Benoit

    AU - Hardie, D.Grahame

    AU - Marette, Andre

    AU - Hundal, Harinder S.

    N1 - Copyright 2012 Elsevier B.V., All rights reserved.

    PY - 2012/6/8

    Y1 - 2012/6/8

    N2 - The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GUin L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulatedGUwas sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interferingAMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide- stimulated GU by ~20%. Consistent with this, analysis of GU in muscle cells from a1 /a2 AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ~35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.

    AB - The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GUin L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulatedGUwas sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interferingAMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide- stimulated GU by ~20%. Consistent with this, analysis of GU in muscle cells from a1 /a2 AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ~35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.

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