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Mitochondrial respiration and redox coupling in articular chondrocytes.

Lane RS, Fu Y, Matsuzaki S, Kinter M, Humphries KM, Griffin TM - Arthritis Res. Ther. (2015)

Bottom Line: However, the production of nitric oxide and superoxide and the expression and activity of SOD2 were significantly reduced after 5 days in galactose culture.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.

View Article: PubMed Central - PubMed

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.

ABSTRACT

Introduction: 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.

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Replacing glucose with galactose reduces glycolysis and upregulates mitochondrial respiration. (A) Cell viability was not altered by 1 or 5 days of galactose culture (n = 6). Five days of galactose culture (B) significantly reduced lactate production (n = 6) and (C) trended toward a decrease in lactate dehydrogenase (LDH) activity (n = 4), indicating a reduction in non-oxidative glycolytic flux. The reduction in glycolysis after 5 days in galactose culture was offset by (D) an increase in the basal rate of cellular oxygen consumption (n = 6), which was associated with a near maximal rate of oxygen consumption, as indicated by (E) the ratio of coupled to uncoupled respiration approaching 100 (n = 4). The increase in mitochondrial respiration did not correspond to (F) an increase in the expression of genetic mediators of mitochondrial biogenesis (TFAM and PGC1A) after 1 day of galactose culture (n = 4) or an (G) increased abundance of mitochondrial proteins (ATP5B and VDAC1) after 5 days in galactose culture (n = 3). (H) However, 5 days of galactose culture significantly increased the expression of the mitochondrial electron transport chain and Krebs cycle enzyme succinate dehydrogenase (SDH) (n = 4). These metabolic changes were not able to maintain cellular metabolic homeostasis after 5 days of galactose culture, as indicated by (I) an increased ratio of NAD+ to NADH (n = 4) and (J) a decrease in the cellular energy charge (n = 3). Bars represent mean ± standard error of the mean. *P <0.05 and **P <0.01 between glucose and galactose.
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Fig1: Replacing glucose with galactose reduces glycolysis and upregulates mitochondrial respiration. (A) Cell viability was not altered by 1 or 5 days of galactose culture (n = 6). Five days of galactose culture (B) significantly reduced lactate production (n = 6) and (C) trended toward a decrease in lactate dehydrogenase (LDH) activity (n = 4), indicating a reduction in non-oxidative glycolytic flux. The reduction in glycolysis after 5 days in galactose culture was offset by (D) an increase in the basal rate of cellular oxygen consumption (n = 6), which was associated with a near maximal rate of oxygen consumption, as indicated by (E) the ratio of coupled to uncoupled respiration approaching 100 (n = 4). The increase in mitochondrial respiration did not correspond to (F) an increase in the expression of genetic mediators of mitochondrial biogenesis (TFAM and PGC1A) after 1 day of galactose culture (n = 4) or an (G) increased abundance of mitochondrial proteins (ATP5B and VDAC1) after 5 days in galactose culture (n = 3). (H) However, 5 days of galactose culture significantly increased the expression of the mitochondrial electron transport chain and Krebs cycle enzyme succinate dehydrogenase (SDH) (n = 4). These metabolic changes were not able to maintain cellular metabolic homeostasis after 5 days of galactose culture, as indicated by (I) an increased ratio of NAD+ to NADH (n = 4) and (J) a decrease in the cellular energy charge (n = 3). Bars represent mean ± standard error of the mean. *P <0.05 and **P <0.01 between glucose and galactose.

Mentions: Culturing chondrocytes in either glucose- or galactose-supplemented media for 1 or 5 days did not alter cell viability (Figure 1A). However, galactose culture did significantly alter chondrocyte metabolism. After 1 day in galactose culture, lactate production decreased 54%, from 17.3 to 8.0 μmol per 106 cells (P <0.0001). After 5 days of galactose culture, both lactate production and maximal LDH activity were substantially reduced. Lactate production decreased by 92% (P <0.0001; Figure 1B), and LDH activity was reduced by 28% (P = 0.051; Figure 1C). These results are consistent with a substantial reduction in glycolytic flux and a reduced reliance on glycolysis for cellular ATP production. Galactose treatment, however, was not equivalent to complete glycolytic inhibition. Culturing chondrocytes for 1 day in 2-deoxy-D-glucose, a glucose analog that inhibits glycolysis, caused a modest 8% increase in cell death compared with galactose culture.Figure 1


Mitochondrial respiration and redox coupling in articular chondrocytes.

Lane RS, Fu Y, Matsuzaki S, Kinter M, Humphries KM, Griffin TM - Arthritis Res. Ther. (2015)

Replacing glucose with galactose reduces glycolysis and upregulates mitochondrial respiration. (A) Cell viability was not altered by 1 or 5 days of galactose culture (n = 6). Five days of galactose culture (B) significantly reduced lactate production (n = 6) and (C) trended toward a decrease in lactate dehydrogenase (LDH) activity (n = 4), indicating a reduction in non-oxidative glycolytic flux. The reduction in glycolysis after 5 days in galactose culture was offset by (D) an increase in the basal rate of cellular oxygen consumption (n = 6), which was associated with a near maximal rate of oxygen consumption, as indicated by (E) the ratio of coupled to uncoupled respiration approaching 100 (n = 4). The increase in mitochondrial respiration did not correspond to (F) an increase in the expression of genetic mediators of mitochondrial biogenesis (TFAM and PGC1A) after 1 day of galactose culture (n = 4) or an (G) increased abundance of mitochondrial proteins (ATP5B and VDAC1) after 5 days in galactose culture (n = 3). (H) However, 5 days of galactose culture significantly increased the expression of the mitochondrial electron transport chain and Krebs cycle enzyme succinate dehydrogenase (SDH) (n = 4). These metabolic changes were not able to maintain cellular metabolic homeostasis after 5 days of galactose culture, as indicated by (I) an increased ratio of NAD+ to NADH (n = 4) and (J) a decrease in the cellular energy charge (n = 3). Bars represent mean ± standard error of the mean. *P <0.05 and **P <0.01 between glucose and galactose.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Replacing glucose with galactose reduces glycolysis and upregulates mitochondrial respiration. (A) Cell viability was not altered by 1 or 5 days of galactose culture (n = 6). Five days of galactose culture (B) significantly reduced lactate production (n = 6) and (C) trended toward a decrease in lactate dehydrogenase (LDH) activity (n = 4), indicating a reduction in non-oxidative glycolytic flux. The reduction in glycolysis after 5 days in galactose culture was offset by (D) an increase in the basal rate of cellular oxygen consumption (n = 6), which was associated with a near maximal rate of oxygen consumption, as indicated by (E) the ratio of coupled to uncoupled respiration approaching 100 (n = 4). The increase in mitochondrial respiration did not correspond to (F) an increase in the expression of genetic mediators of mitochondrial biogenesis (TFAM and PGC1A) after 1 day of galactose culture (n = 4) or an (G) increased abundance of mitochondrial proteins (ATP5B and VDAC1) after 5 days in galactose culture (n = 3). (H) However, 5 days of galactose culture significantly increased the expression of the mitochondrial electron transport chain and Krebs cycle enzyme succinate dehydrogenase (SDH) (n = 4). These metabolic changes were not able to maintain cellular metabolic homeostasis after 5 days of galactose culture, as indicated by (I) an increased ratio of NAD+ to NADH (n = 4) and (J) a decrease in the cellular energy charge (n = 3). Bars represent mean ± standard error of the mean. *P <0.05 and **P <0.01 between glucose and galactose.
Mentions: Culturing chondrocytes in either glucose- or galactose-supplemented media for 1 or 5 days did not alter cell viability (Figure 1A). However, galactose culture did significantly alter chondrocyte metabolism. After 1 day in galactose culture, lactate production decreased 54%, from 17.3 to 8.0 μmol per 106 cells (P <0.0001). After 5 days of galactose culture, both lactate production and maximal LDH activity were substantially reduced. Lactate production decreased by 92% (P <0.0001; Figure 1B), and LDH activity was reduced by 28% (P = 0.051; Figure 1C). These results are consistent with a substantial reduction in glycolytic flux and a reduced reliance on glycolysis for cellular ATP production. Galactose treatment, however, was not equivalent to complete glycolytic inhibition. Culturing chondrocytes for 1 day in 2-deoxy-D-glucose, a glucose analog that inhibits glycolysis, caused a modest 8% increase in cell death compared with galactose culture.Figure 1

Bottom Line: However, the production of nitric oxide and superoxide and the expression and activity of SOD2 were significantly reduced after 5 days in galactose culture.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.

View Article: PubMed Central - PubMed

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.

ABSTRACT

Introduction: 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.

Show MeSH
Related in: MedlinePlus