Cellular depletion of atypical PKC lambda is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells

Clare Stretton, Ashleigh Evans, Harinder S. Hundal

    Research output: Contribution to journalArticle

    21 Citations (Scopus)

    Abstract

    Stretton C, Evans A, Hundal HS. Cellular depletion of atypical PKC lambda is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells. Am J Physiol Endocrinol Metab 299: E402-E412, 2010. First published June 8, 2010; doi:10.1152/ajpendo.00171.2010.-Atypical protein kinase C (aPKC) isoforms (lambda and zeta) have been implicated in the control of insulin-stimulated glucose uptake in adipose and skeletal muscle, but their precise role in this process remains unclear, especially in light of accumulating evidence showing that, in response to numerous stimuli, including insulin and lipids such as ceramide, activation of aPKCs acts to negatively regulate key insulin-signaling molecules, such as insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB)/cAMP-dependent PKC (Akt). In this study, we have depleted PKC lambda in L6 skeletal muscle cells using RNA interference and assessed the effect this has upon insulin action. Muscle cells did not express detectable amounts of PKC zeta. Depletion of PKC lambda (> 95%) had no significant effect on the expression of proteins participating in insulin signaling [i.e., insulin receptor, IRS-1, phosphatidylinositol 3-kinase (PI 3-kinase), PKB, or phosphate and tensin homolog deleted on chromosome 10] or those involved in glucose transport [ Akt substrate of 160 kDa, glucose transporter (GLUT) 1, or GLUT4]. However, PKC lambda-depleted muscle cells exhibited greater activation of PKB/Akt and phosphorylation of its downstream target glycogen synthase kinase 3, in the basal state and displayed greater responsiveness to submaximal doses of insulin with respect to p85-PI 3-kinase/IRS-1 association and PKB activation. The increase in basal and insulin-induced signaling resulted in an associated enhancement of basal and insulin-stimulated glucose transport, both of which were inhibited by the PI 3-kinase inhibitor wortmannin. Additionally, like RNAi-mediated depletion of PKC lambda, overexpression of a dominant-negative mutant of PKC zeta induced a similar insulin-sensitizing effect on PKB activation. Our findings indicate that aPKCs are likely to play an important role in restraining proximal insulin signaling events but appear dispensable with respect to insulin-stimulated glucose uptake in cultured L6 muscle cells.

    Original languageEnglish
    Pages (from-to)E402-E412
    Number of pages11
    JournalAmerican Journal of Physiology, Endocrinology and Metabolism
    Volume299
    Issue number3
    DOIs
    Publication statusPublished - Sep 2010

    Keywords

    • protein kinase C lambda
    • glucose uptake
    • insulin receptor substrate 1
    • ribonucleic acid interference
    • protein kinase B/adenosine 3 ',5 '-cyclic monophosphate-associated kinase
    • PROTEIN-KINASE-C
    • PLECKSTRIN HOMOLOGY DOMAIN
    • RECEPTOR SUBSTRATE-1 IRS-1
    • SIGNAL-TRANSDUCTION
    • PHOSPHOINOSITIDE 3-KINASE
    • INDUCED PHOSPHORYLATION
    • B-ALPHA
    • ZETA
    • ACTIVATION
    • TRANSPORT

    Cite this

    @article{ed347b2618094f62bd9f0993cc64bb58,
    title = "Cellular depletion of atypical PKC lambda is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells",
    abstract = "Stretton C, Evans A, Hundal HS. Cellular depletion of atypical PKC lambda is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells. Am J Physiol Endocrinol Metab 299: E402-E412, 2010. First published June 8, 2010; doi:10.1152/ajpendo.00171.2010.-Atypical protein kinase C (aPKC) isoforms (lambda and zeta) have been implicated in the control of insulin-stimulated glucose uptake in adipose and skeletal muscle, but their precise role in this process remains unclear, especially in light of accumulating evidence showing that, in response to numerous stimuli, including insulin and lipids such as ceramide, activation of aPKCs acts to negatively regulate key insulin-signaling molecules, such as insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB)/cAMP-dependent PKC (Akt). In this study, we have depleted PKC lambda in L6 skeletal muscle cells using RNA interference and assessed the effect this has upon insulin action. Muscle cells did not express detectable amounts of PKC zeta. Depletion of PKC lambda (> 95{\%}) had no significant effect on the expression of proteins participating in insulin signaling [i.e., insulin receptor, IRS-1, phosphatidylinositol 3-kinase (PI 3-kinase), PKB, or phosphate and tensin homolog deleted on chromosome 10] or those involved in glucose transport [ Akt substrate of 160 kDa, glucose transporter (GLUT) 1, or GLUT4]. However, PKC lambda-depleted muscle cells exhibited greater activation of PKB/Akt and phosphorylation of its downstream target glycogen synthase kinase 3, in the basal state and displayed greater responsiveness to submaximal doses of insulin with respect to p85-PI 3-kinase/IRS-1 association and PKB activation. The increase in basal and insulin-induced signaling resulted in an associated enhancement of basal and insulin-stimulated glucose transport, both of which were inhibited by the PI 3-kinase inhibitor wortmannin. Additionally, like RNAi-mediated depletion of PKC lambda, overexpression of a dominant-negative mutant of PKC zeta induced a similar insulin-sensitizing effect on PKB activation. Our findings indicate that aPKCs are likely to play an important role in restraining proximal insulin signaling events but appear dispensable with respect to insulin-stimulated glucose uptake in cultured L6 muscle cells.",
    keywords = "protein kinase C lambda, glucose uptake, insulin receptor substrate 1, ribonucleic acid interference, protein kinase B/adenosine 3 ',5 '-cyclic monophosphate-associated kinase, PROTEIN-KINASE-C, PLECKSTRIN HOMOLOGY DOMAIN, RECEPTOR SUBSTRATE-1 IRS-1, SIGNAL-TRANSDUCTION, PHOSPHOINOSITIDE 3-KINASE, INDUCED PHOSPHORYLATION, B-ALPHA, ZETA, ACTIVATION, TRANSPORT",
    author = "Clare Stretton and Ashleigh Evans and Hundal, {Harinder S.}",
    year = "2010",
    month = "9",
    doi = "10.1152/ajpendo.00171.2010",
    language = "English",
    volume = "299",
    pages = "E402--E412",
    journal = "American Journal of Physiology, Endocrinology and Metabolism",
    issn = "0193-1849",
    publisher = "American Physiological Society",
    number = "3",

    }

    TY - JOUR

    T1 - Cellular depletion of atypical PKC lambda is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells

    AU - Stretton, Clare

    AU - Evans, Ashleigh

    AU - Hundal, Harinder S.

    PY - 2010/9

    Y1 - 2010/9

    N2 - Stretton C, Evans A, Hundal HS. Cellular depletion of atypical PKC lambda is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells. Am J Physiol Endocrinol Metab 299: E402-E412, 2010. First published June 8, 2010; doi:10.1152/ajpendo.00171.2010.-Atypical protein kinase C (aPKC) isoforms (lambda and zeta) have been implicated in the control of insulin-stimulated glucose uptake in adipose and skeletal muscle, but their precise role in this process remains unclear, especially in light of accumulating evidence showing that, in response to numerous stimuli, including insulin and lipids such as ceramide, activation of aPKCs acts to negatively regulate key insulin-signaling molecules, such as insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB)/cAMP-dependent PKC (Akt). In this study, we have depleted PKC lambda in L6 skeletal muscle cells using RNA interference and assessed the effect this has upon insulin action. Muscle cells did not express detectable amounts of PKC zeta. Depletion of PKC lambda (> 95%) had no significant effect on the expression of proteins participating in insulin signaling [i.e., insulin receptor, IRS-1, phosphatidylinositol 3-kinase (PI 3-kinase), PKB, or phosphate and tensin homolog deleted on chromosome 10] or those involved in glucose transport [ Akt substrate of 160 kDa, glucose transporter (GLUT) 1, or GLUT4]. However, PKC lambda-depleted muscle cells exhibited greater activation of PKB/Akt and phosphorylation of its downstream target glycogen synthase kinase 3, in the basal state and displayed greater responsiveness to submaximal doses of insulin with respect to p85-PI 3-kinase/IRS-1 association and PKB activation. The increase in basal and insulin-induced signaling resulted in an associated enhancement of basal and insulin-stimulated glucose transport, both of which were inhibited by the PI 3-kinase inhibitor wortmannin. Additionally, like RNAi-mediated depletion of PKC lambda, overexpression of a dominant-negative mutant of PKC zeta induced a similar insulin-sensitizing effect on PKB activation. Our findings indicate that aPKCs are likely to play an important role in restraining proximal insulin signaling events but appear dispensable with respect to insulin-stimulated glucose uptake in cultured L6 muscle cells.

    AB - Stretton C, Evans A, Hundal HS. Cellular depletion of atypical PKC lambda is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells. Am J Physiol Endocrinol Metab 299: E402-E412, 2010. First published June 8, 2010; doi:10.1152/ajpendo.00171.2010.-Atypical protein kinase C (aPKC) isoforms (lambda and zeta) have been implicated in the control of insulin-stimulated glucose uptake in adipose and skeletal muscle, but their precise role in this process remains unclear, especially in light of accumulating evidence showing that, in response to numerous stimuli, including insulin and lipids such as ceramide, activation of aPKCs acts to negatively regulate key insulin-signaling molecules, such as insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB)/cAMP-dependent PKC (Akt). In this study, we have depleted PKC lambda in L6 skeletal muscle cells using RNA interference and assessed the effect this has upon insulin action. Muscle cells did not express detectable amounts of PKC zeta. Depletion of PKC lambda (> 95%) had no significant effect on the expression of proteins participating in insulin signaling [i.e., insulin receptor, IRS-1, phosphatidylinositol 3-kinase (PI 3-kinase), PKB, or phosphate and tensin homolog deleted on chromosome 10] or those involved in glucose transport [ Akt substrate of 160 kDa, glucose transporter (GLUT) 1, or GLUT4]. However, PKC lambda-depleted muscle cells exhibited greater activation of PKB/Akt and phosphorylation of its downstream target glycogen synthase kinase 3, in the basal state and displayed greater responsiveness to submaximal doses of insulin with respect to p85-PI 3-kinase/IRS-1 association and PKB activation. The increase in basal and insulin-induced signaling resulted in an associated enhancement of basal and insulin-stimulated glucose transport, both of which were inhibited by the PI 3-kinase inhibitor wortmannin. Additionally, like RNAi-mediated depletion of PKC lambda, overexpression of a dominant-negative mutant of PKC zeta induced a similar insulin-sensitizing effect on PKB activation. Our findings indicate that aPKCs are likely to play an important role in restraining proximal insulin signaling events but appear dispensable with respect to insulin-stimulated glucose uptake in cultured L6 muscle cells.

    KW - protein kinase C lambda

    KW - glucose uptake

    KW - insulin receptor substrate 1

    KW - ribonucleic acid interference

    KW - protein kinase B/adenosine 3 ',5 '-cyclic monophosphate-associated kinase

    KW - PROTEIN-KINASE-C

    KW - PLECKSTRIN HOMOLOGY DOMAIN

    KW - RECEPTOR SUBSTRATE-1 IRS-1

    KW - SIGNAL-TRANSDUCTION

    KW - PHOSPHOINOSITIDE 3-KINASE

    KW - INDUCED PHOSPHORYLATION

    KW - B-ALPHA

    KW - ZETA

    KW - ACTIVATION

    KW - TRANSPORT

    U2 - 10.1152/ajpendo.00171.2010

    DO - 10.1152/ajpendo.00171.2010

    M3 - Article

    C2 - 20530734

    VL - 299

    SP - E402-E412

    JO - American Journal of Physiology, Endocrinology and Metabolism

    JF - American Journal of Physiology, Endocrinology and Metabolism

    SN - 0193-1849

    IS - 3

    ER -