title: Increased abundance of the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1) in patients with obesity and type 2 diabetes: evidence for altered adiponectin signalling.
abstract: OBJECTIVE
  The adiponectin signalling pathway is largely unknown, but recently the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1), has been shown to interact directly with adiponectin receptor (ADIPOR)1. APPL1 is present in C2C12 myoblasts and mouse skeletal muscle, but its presence in human skeletal muscle has not been investigated.
  
  
  METHODS
  Samples from type 2 diabetic, and lean and non-diabetic obese participants were analysed by: immunoprecipitation and western blot; HPLC-electrospray ionisation (ESI)-mass spectrometry (MS) analysis; peak area analysis by MS; HPLC-ESI-MS/MS/MS analysis; and RT-PCR analysis of APPL1 mRNA.
  
  
  RESULTS
  Immunoprecipitation and western blot indicated a band specific to APPL1. Tryptic digestion and HPLC-ESI-MS analysis of whole-muscle homogenate APPL1 unambiguously identified APPL1 with 56% sequence coverage. Peak area analysis by MS validated western blot results, showing APPL1 levels to be significantly increased in type 2 diabetic and obese as compared with lean participants. Targeted phosphopeptide analysis by HPLC-ESI-MS/MS/MS showed that APPL1 was phosphorylated specifically on Ser(401). APPL1 mRNA expression was significantly increased in obese and type 2 diabetic participants as compared with lean participants. After bariatric surgery in morbidly obese participants with subsequent weight loss, skeletal muscle APPL1 abundance was significantly reduced (p < 0.05) in association with an increase in plasma adiponectin (p < 0.01), increased levels of ADIPOR1 (p < 0.05) and increased muscle AMP-activated protein kinase (AMPK) phosphorylation (p < 0.05).
  
  
  CONCLUSIONS
  APPL1 abundance is significantly higher in type 2 diabetic muscle; APPL1 is phosphorylated in vivo on Ser(401). Improvements in hyperglycaemia and hypoadiponectinaemia following weight loss are associated with reduced skeletal muscle APPL1, and increased plasma adiponectin levels and muscle AMPK phosphorylation.
authors.affiliation: Department of Physiology, University of Texas Health Science Center, San Antonio, TX, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Holmes
authors.forename: Z
authors.initials: Z
authors.lastname: Yi
authors.forename: E
authors.initials: E
authors.lastname: De Filippis
authors.forename: R
authors.initials: R
authors.lastname: Berria
authors.forename: S
authors.initials: S
authors.lastname: Shahani
authors.forename: P
authors.initials: P
authors.lastname: Sathyanarayana
authors.forename: V
authors.initials: V
authors.lastname: Sherman
authors.forename: K
authors.initials: K
authors.lastname: Fujiwara
authors.forename: C
authors.initials: C
authors.lastname: Meyer
authors.forename: C
authors.initials: C
authors.lastname: Christ-Roberts
authors.forename: H
authors.initials: H
authors.lastname: Hwang
authors.forename: J
authors.initials: J
authors.lastname: Finlayson
authors.forename: L Q
authors.initials: LQ
authors.lastname: Dong
authors.forename: L J
authors.initials: LJ
authors.lastname: Mandarino
authors.forename: M
authors.initials: M
authors.lastname: Bajaj
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D052242:Adiponectin / Q000235:genetics / Q000378:metabolism*; D000328:Adult; D015153:Blotting, Western; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000378:metabolism*; D004586:Electrophoresis; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D013058:Mass Spectrometry; D009765:Obesity / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: C464801:APPL1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D052242:Adiponectin