title: Extracellular nicotinamide phosphoribosyltransferase (NAMPT/visfatin) inhibits insulin-like growth factor-1 signaling and proteoglycan synthesis in human articular chondrocytes.
abstract: BACKGROUND
  Obesity is one of the major risk factors for the development of osteoarthritis (OA). Although the mechanical factors appear to be critical, recent studies have suggested a role for adipokines in cartilage degradation. Chondrocytes from osteoarthritic cartilage respond poorly to insulin-like growth factor-1 (IGF-1) and the molecular mechanism(s) involved is not clearly understood. The purpose of the present study was to determine the role of extracellular nicotinamide phosphoribosyltransferase (eNAMPT/visfatin), a newly described adipokine, in regulating IGF-1 function in chondrocytes.
  
  
  METHODS
  Human articular chondrocytes isolated from normal ankle cartilage were pretreated with eNAMPT (0.1 to 5.0 μg/ml) overnight followed by stimulation with IGF-1 (50 ng/ml) for 24 hours, and proteoglycan synthesis was measured by [35S]sulfate incorporation. Chondrocytes were pretreated with eNAMPT overnight followed by IGF-1 for 10 minutes, and the cell lysates were immunoblotted for various signaling proteins that are activated by IGF-1 using phosphospecific antibodies. In addition, chondrocytes were pretreated with mitogen-activated protein kinase kinase inhibitor (U0126) prior to stimulation with eNAMPT and IGF-1.
  
  
  RESULTS
  Pretreatment of chondrocytes with eNAMPT inhibited IGF-1-stimulated proteoglycan synthesis in a dose-dependent manner. Treatment of chondrocytes with eNAMPT inhibited IGF-1-induced phosphorylation of signaling molecules, including insulin receptor substrate-1 and AKT. Interestingly, pretreatment of chondrocytes with eNAMPT did not inhibit IGF-1-mediated phosphorylation of the IGF-1 receptor; however, it stimulated a sustained phosphorylation of the extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway. Inhibition of the ERK/MAPK signaling pathway restored IGF-1-mediated insulin receptor substrate-1 and AKT phosphorylation.
  
  
  CONCLUSIONS
  Our study demonstrates that eNAMPT/visfatin inhibits IGF-1 function in articular chondrocytes by activating the ERK/MAPK pathway independent of the IGF-1 receptor. Since eNAMPT levels are elevated in the synovial fluid of OA patients, the signaling pathway activated by eNAMPT could contribute to IGF-1 resistance in OA.
authors.affiliation: Department of Internal Medicine, Section of Molecular Medicine, Wake Forest School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, USA. ryammani@wakehealth.edu
authors.forename: Raghunatha R
authors.initials: RR
authors.lastname: Yammani
authors.forename: Richard F
authors.initials: RF
authors.lastname: Loeser
mesh_terms: D054392:Adipokines / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D002358:Cartilage, Articular / Q000166:cytology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D054409:Nicotinamide Phosphoribosyltransferase / Q000494:pharmacology*; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011509:Proteoglycans / Q000096:biosynthesis*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D054392:Adipokines; D002070:Butadienes; D024043:Collagen Type II; D004791:Enzyme Inhibitors; D055504:Insulin Receptor Substrate Proteins; D009570:Nitriles; D011509:Proteoglycans; C113580:U 0126; D007334:Insulin-Like Growth Factor I; D054409:Nicotinamide Phosphoribosyltransferase; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases