Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle.
OBJECTIVE
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-beta) and determine protein abundance of ATPsyn-beta 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-beta using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps.


RESULTS
Seven phosphorylation sites were identified on ATPsyn-beta 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 approximately 30% higher phosphorylation of ATPsyn-beta at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-beta protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-beta by approximately 50% in lean and healthy, but not insulin-resistant, individuals.


CONCLUSIONS
These data demonstrate that ATPsyn-beta is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-beta together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-beta may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.
Højlund
K
K
Center for Metabolic Biology, School of Life Sciences, Arizona State University, PO Box 87370, Tempe, AZ 85287, USA. k.hojlund@dadlnet.dk
Yi
Z
Z
Lefort
N
N
Langlais
P
P
Bowen
B
B
Levin
K
K
Beck-Nielsen
H
H
Mandarino
L J
LJ
D000328:Adult; D001665:Binding Sites; D002384:Catalysis; D015331:Cohort Studies; D005260:Female; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007333:Insulin Resistance; D008297:Male; D008875:Middle Aged; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D009132:Muscles / Q000378:metabolism*; D010766:Phosphorylation; D014443:Tyrosine / Q000737:chemistry
C506526:ATP5F1B protein, human; D007328:Insulin; D014443:Tyrosine; D025261:Mitochondrial Proton-Translocating ATPases