Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle

K. Hojlund, Z. Yi, N. Lefort, P. Langlais, B. Bowen, K. Levin, H. Beck-Nielsen, L. J. Mandarino

    Research output: Contribution to journalArticle

    39 Citations (Scopus)

    Abstract

    Aims/hypothesis: Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-β) and determine protein abundance of ATPsyn-β and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals. Methods: Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n = 10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-β using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps. Results: Seven phosphorylation sites were identified on ATPsyn-β purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a ∼30% higher phosphorylation of ATPsyn-β at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-β protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-β by ∼50% in lean and healthy, but not insulin-resistant, individuals. Conclusions/interpretation: These data demonstrate that ATPsyn-β is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-β together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-β may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.

    Original languageEnglish (US)
    Pages (from-to)541-551
    Number of pages11
    JournalDiabetologia
    Volume53
    Issue number3
    DOIs
    StatePublished - Mar 2010

    Fingerprint

    Adenosine Triphosphate
    Phosphorylation
    Insulin
    Muscles
    Oxidative Phosphorylation
    Skeletal Muscle
    Proteins
    Type 2 Diabetes Mellitus
    Insulin Resistance
    Volunteers
    Mitochondrial Diseases
    Glucose Clamp Technique
    Tandem Mass Spectrometry
    Immunoblotting
    Catalytic Domain
    Down-Regulation
    Nucleotides
    Obesity
    Biopsy
    Glucose

    Keywords

    • ATP synthase
    • Insulin resistance
    • Mass spectrometry
    • Mitochondrial oxidative phosphorylation
    • Obesity
    • Proteomics
    • Type 2 diabetes

    ASJC Scopus subject areas

    • Internal Medicine
    • Endocrinology, Diabetes and Metabolism
    • Medicine(all)

    Cite this

    Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle. / Hojlund, K.; Yi, Z.; Lefort, N.; Langlais, P.; Bowen, B.; Levin, K.; Beck-Nielsen, H.; Mandarino, L. J.

    In: Diabetologia, Vol. 53, No. 3, 03.2010, p. 541-551.

    Research output: Contribution to journalArticle

    Hojlund, K, Yi, Z, Lefort, N, Langlais, P, Bowen, B, Levin, K, Beck-Nielsen, H & Mandarino, LJ 2010, 'Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle', Diabetologia, vol. 53, no. 3, pp. 541-551. https://doi.org/10.1007/s00125-009-1624-0
    Hojlund, K. ; Yi, Z. ; Lefort, N. ; Langlais, P. ; Bowen, B. ; Levin, K. ; Beck-Nielsen, H. ; Mandarino, L. J. / Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle. In: Diabetologia. 2010 ; Vol. 53, No. 3. pp. 541-551.
    @article{6d922b6ad5cb4518b2666bf87e3646a1,
    title = "Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle",
    abstract = "Aims/hypothesis: Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-β) and determine protein abundance of ATPsyn-β and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals. Methods: Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n = 10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-β using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps. Results: Seven phosphorylation sites were identified on ATPsyn-β purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a ∼30{\%} higher phosphorylation of ATPsyn-β at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-β protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-β by ∼50{\%} in lean and healthy, but not insulin-resistant, individuals. Conclusions/interpretation: These data demonstrate that ATPsyn-β is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-β together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-β may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.",
    keywords = "ATP synthase, Insulin resistance, Mass spectrometry, Mitochondrial oxidative phosphorylation, Obesity, Proteomics, Type 2 diabetes",
    author = "K. Hojlund and Z. Yi and N. Lefort and P. Langlais and B. Bowen and K. Levin and H. Beck-Nielsen and Mandarino, {L. J.}",
    year = "2010",
    month = "3",
    doi = "10.1007/s00125-009-1624-0",
    language = "English (US)",
    volume = "53",
    pages = "541--551",
    journal = "Diabetologia",
    issn = "0012-186X",
    publisher = "Springer Verlag",
    number = "3",

    }

    TY - JOUR

    T1 - Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle

    AU - Hojlund, K.

    AU - Yi, Z.

    AU - Lefort, N.

    AU - Langlais, P.

    AU - Bowen, B.

    AU - Levin, K.

    AU - Beck-Nielsen, H.

    AU - Mandarino, L. J.

    PY - 2010/3

    Y1 - 2010/3

    N2 - Aims/hypothesis: Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-β) and determine protein abundance of ATPsyn-β and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals. Methods: Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n = 10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-β using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps. Results: Seven phosphorylation sites were identified on ATPsyn-β purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a ∼30% higher phosphorylation of ATPsyn-β at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-β protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-β by ∼50% in lean and healthy, but not insulin-resistant, individuals. Conclusions/interpretation: These data demonstrate that ATPsyn-β is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-β together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-β may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.

    AB - Aims/hypothesis: Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-β) and determine protein abundance of ATPsyn-β and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals. Methods: Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n = 10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-β using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps. Results: Seven phosphorylation sites were identified on ATPsyn-β purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a ∼30% higher phosphorylation of ATPsyn-β at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-β protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-β by ∼50% in lean and healthy, but not insulin-resistant, individuals. Conclusions/interpretation: These data demonstrate that ATPsyn-β is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-β together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-β may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.

    KW - ATP synthase

    KW - Insulin resistance

    KW - Mass spectrometry

    KW - Mitochondrial oxidative phosphorylation

    KW - Obesity

    KW - Proteomics

    KW - Type 2 diabetes

    UR - http://www.scopus.com/inward/record.url?scp=77949272758&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=77949272758&partnerID=8YFLogxK

    U2 - 10.1007/s00125-009-1624-0

    DO - 10.1007/s00125-009-1624-0

    M3 - Article

    C2 - 20012595

    AN - SCOPUS:77949272758

    VL - 53

    SP - 541

    EP - 551

    JO - Diabetologia

    JF - Diabetologia

    SN - 0012-186X

    IS - 3

    ER -