title: Mitochondrial respiration and redox coupling in articular chondrocytes.
abstract: BACKGROUND
  Chondrocytes rely primarily on glycolysis to meet cellular energy needs, but recent studies implicate impaired mitochondrial function in osteoarthritis (OA) pathogenesis. Our objectives were to investigate the ability of chondrocytes to upregulate mitochondrial respiration when challenged with a nutrient stress and determine the effect on mediators of chondrocyte oxidative homeostasis.
  
  
  METHODS
  Primary bovine chondrocytes were isolated and cultured in alginate beads. Mitochondrial respiration was stimulated by culturing cells with galactose-supplemented media for a period of 1 or 5 days. Metabolic flexibility was assessed by measuring metabolite and enzymatic biomarkers of glycolytic and mitochondrial metabolism. Oxidative homeostasis was assessed by measuring (1) cellular glutathione content and redox homeostasis, (2) rates of nitric oxide and superoxide production, and (3) the abundance and activity of cellular anti-oxidant proteins, especially the mitochondrial isoform of superoxide dismutase (SOD2). The regulatory role of hypoxia-inducible factor 2α (HIF-2α) in mediating the metabolic and redox responses was evaluated by chemical stabilization with cobalt chloride (CoCl2).
  
  
  RESULTS
  After 5 days of galactose culture, lactate production and lactate dehydrogenase activity were reduced by 92% (P<0.0001) and 28% (P=0.051), respectively. Conversely, basal oxygen consumption increased 35% (P=0.042) without increasing mitochondrial content. Glutathione redox homeostasis was unaffected by galactose culture. However, the production of nitric oxide and superoxide and the expression and activity of SOD2 were significantly reduced after 5 days in galactose culture. Nuclear protein expression and gene expression of HIF-2α, a transcription factor for SOD2, were significantly downregulated (more than twofold; P<0.05) with galactose culture. CoCl2-mediated stabilization of HIF-2α during the initial galactose response phase attenuated the reduction in SOD2 (P=0.028) and increased cell death (P=0.003).
  
  
  CONCLUSIONS
  Chondrocyte metabolic flexibility promotes cell survival during a nutrient stress by upregulating mitochondrial respiration and reducing the rate of reactive nitrogen and oxygen species production. These changes are coupled to a substantial reduction in the expression and activity of the mitochondrial anti-oxidant SOD2 and its pro-catabolic transcription factor HIF-2α, suggesting that an improved understanding of physiologic triggers of chondrocyte metabolic flexibility may provide new insight into the etiology of OA.
authors.affiliation: Free Radical Biology and Aging Program, Oklahoma Medical Research Foundation, MS 21, 825 NE 13th Street, Oklahoma City, OK, 73104, USA. rachel-lane@ouhsc.edu.|Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 853, Oklahoma City, OK, 73104, USA. rachel-lane@ouhsc.edu.
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Lane
authors.affiliation: Free Radical Biology and Aging Program, Oklahoma Medical Research Foundation, MS 21, 825 NE 13th Street, Oklahoma City, OK, 73104, USA. yao-fu@omrf.org.|Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 853, Oklahoma City, OK, 73104, USA. yao-fu@omrf.org.
authors.forename: Yao
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Free Radical Biology and Aging Program, Oklahoma Medical Research Foundation, MS 21, 825 NE 13th Street, Oklahoma City, OK, 73104, USA. satoshi-matsuzaki@omrf.org.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Matsuzaki
authors.affiliation: Free Radical Biology and Aging Program, Oklahoma Medical Research Foundation, MS 21, 825 NE 13th Street, Oklahoma City, OK, 73104, USA. michael-kinter@omrf.org.|Department of Geriatric Medicine, Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences Center, 975 NE 10th Street, BRC-1303, Oklahoma City, OK, 73104, USA. michael-kinter@omrf.org.
authors.forename: Michael
authors.initials: M
authors.lastname: Kinter
authors.affiliation: Free Radical Biology and Aging Program, Oklahoma Medical Research Foundation, MS 21, 825 NE 13th Street, Oklahoma City, OK, 73104, USA. kenneth-humphries@omrf.org.|Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 853, Oklahoma City, OK, 73104, USA. kenneth-humphries@omrf.org.|Department of Geriatric Medicine, Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences Center, 975 NE 10th Street, BRC-1303, Oklahoma City, OK, 73104, USA. kenneth-humphries@omrf.org.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Humphries
authors.affiliation: Free Radical Biology and Aging Program, Oklahoma Medical Research Foundation, MS 21, 825 NE 13th Street, Oklahoma City, OK, 73104, USA. tim-griffin@omrf.org.|Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 853, Oklahoma City, OK, 73104, USA. tim-griffin@omrf.org.|Department of Geriatric Medicine, Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences Center, 975 NE 10th Street, BRC-1303, Oklahoma City, OK, 73104, USA. tim-griffin@omrf.org.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Griffin
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000235:genetics*; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D002417:Cattle; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D013058:Mass Spectrometry; D008928:Mitochondria / Q000502:physiology*; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010084:Oxidation-Reduction; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D012313:RNA; D000099162:Endothelial PAS Domain-Containing Protein 1