The chondrocytes of articular cartilage use remarkably little O2 compared with most other animal cells. According to results summarized by Stockwell (1), articular cartilage consumes only 2–5% as much O2 per cell as liver or kidney, for example. There is a metabolic explanation for this striking difference. Whereas the majority of animal cells, including the chondrocytes of avian growth cartilage (2), derive their energy by using oxygen for mitochondrial oxidative phosphorylation, mammalian articular chondrocytes do not appear to use this pathway. Instead, carbohydrate breakdown in articular cartilage is dominated by a near-quantitative conversion of glucose to lactate by the Embden-Meyerhof-Parnas (E-M-P) pathway of glycolysis (3–5), a sequence of reactions in which no O2 is consumed.
There are several lines of evidence that support this finding. Although malate dehydrogenase, isocitrate dehydrogenase, and succinate dehydrogenase have been detected in articular or articular epiphyseal cartilage by extraction (6) or cytochemically (7, 8), experiments with 14C-labeled glucose suggest that little pyruvate enters the Krebs cycle (9, 10), and release of CO2 from articular cartilage is small (11). Moreover, mitochondria are sparse in articular chondrocytes, occupying only 1–2% of the intracellular volume (12), compared with ∼15–20% in more typical animal cells (for example, liver ), and in situ, they appear to lack certain cytochromes (14). Cytochrome oxidase has been found to be absent or barely detectable in cells of the surface or midzone of rabbit articular cartilage (7). Oxidative metabolism by articular chondrocytes has been observed, but only after several days of culture in monolayer (3, 15), possibly because dedifferentiation appears linked to an increase in oxidative metabolism (14).
Based on the above evidence, the current view is that the substrate-level phosphorylations of the E-M-P pathway form the principal source of energy (ATP) in articular cartilage (15) and that mitochondrial oxidative phosphorylation makes only a minor contribution to energy supply (15–17). A positive correlation between glycolytic rate and the concentration of ATP in bovine articular cartilage has been noted (5).
Even so, oxygen (O2) plays a role in chondrocyte physiology which is not well understood. Oxygen concentration may influence inflammation associated with cartilage injury and disease (18), and despite the low specific O2 consumption noted above, many studies have shown that articular cartilage responds to O2 in its surroundings. Although not lethal for many days, imposition of anoxia immediately prevents articular chondrocytes from carrying out one of their principal functions, namely, the production of extracellular matrix macromolecules. Thus, at low O2 concentrations, rates of incorporation of sulfate into the large aggregating proteoglycan aggrecan fall steeply (19–21), and assembly of aggrecan into the matrix is affected (22). Hyperoxia has been shown to be as deleterious for matrix synthesis, and indeed for cell survival, as severe hypoxia (20, 21, 23). But the picture obtained from culture experiments is not entirely clear-cut, and there are reports of increased glucose breakdown and lactate production by rabbit articular chondrocytes or cartilage explants after 6–7 days under low O2 concentrations (3, 24). Moreover, in contrast with tissues in which a positive Pasteur effect occurs, in articular cartilage glycolysis decreases at O2 concentrations less than ∼5% in the gas phase, and intracellular levels of ATP are lower (5).
In short-term (4.5-hour) experiments with bovine articular cartilage, we found that rates of glucose utilization and lactate production were markedly greater under aerobic conditions than under anoxia (5), indicating that carbon flux through glycolysis was stimulated by the presence of O2. The effect of O2 on 35S-sulfate incorporation into matrix proteoglycans was even more pronounced (5). Given that glycolysis uses no oxygen (as mentioned above), this result was surprising, and its mechanism unclear. To further examine the connection between O2 supply and glycolysis in articular cartilage, we assumed that O2 functions as an oxidant (electron acceptor) here as it does elsewhere and asked the question: can other oxidants reproduce the effect of O2 and stimulate glycolysis in this tissue? Accordingly, we investigated the effects of reducible substrates on the rate of glycolysis in bovine articular cartilage under anoxic and/or aerobic conditions. If the added substrate is reduced by the cartilage, it functions as an oxidant under the conditions of the test.
The ability of cartilage to reduce externally supplied substrates other than O2 was first reported 70 years ago. Kuwabara (25), in 1932, observed that excised cartilage from several sources (rabbit epiphysis, chick sternum, calf and rabbit costal cartilage) decolorized methylene blue (MEB) under anoxic conditions. Lutwak-Mann (26) noted a “slow, thermolabile reduction of methylene blue” by calf articular cartilage in anaerobic medium, and Bywaters (27) mentioned a similar reaction in equine articular cartilage; however, since then the phenomenon has attracted little attention. Taking these early experiments as a starting point, we examined the response of bovine articular cartilage to a range of reducible compounds (electron acceptors, hence oxidants) including the dyes MEB and 2,6-dichlorophenol-indophenol (DCIP), the iron (III) complex ferricyanide (FECY), and the keto-acids oxaloacetate and pyruvate. We measured lactate production as the primary index of glycolytic flux, a procedure whose validity for this tissue has been critically considered elsewhere (5). In some experiments glucose uptake was recorded at the same time as was lactate production. Incorporation of 35S-sulfate into proteoglycans of the cartilage matrix was also measured as an indication of the broader metabolic and biosynthetic activity of the chondrocytes.
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- MATERIALS AND METHODS
Oxygen has a powerful influence on cartilage metabolism even though chondrocyte energy is derived mainly from glycolysis (4, 5, 8, 9). Under anoxia, although chondrocytes remained viable for many days (34), rates of both sulfate incorporation and lactate production were low; addition of O2 increased both rates substantially, in a concentration-dependent manner (5, 21). The results of the present study show that this effect was not specific to O2: other oxidants could also stimulate metabolism significantly. When the dyes MEB and DCIP, the inorganic complex FECY, or the keto-acids oxaloacetate and pyruvate were added to anoxic cartilage, lactate production and 35S-sulfate incorporation were greatly increased (Figures 3–6). At the highest availability of MEB, DCIP, or FECY tested (0.1–0.5 mM; Figure 3), these substances increased anoxic glycolysis (lactate production) as effectively as did similar concentrations of O2 (∼0.2 mM dissolved O2 in air-saturated medium at 37°C ). The keto-acids oxaloacetate or pyruvate (or PEP) stimulated anoxic glycolysis to a somewhat greater extent (Figure 5). It should be noted, however, that these compounds were supplied at a far higher concentration (20 mM), and the dose-dependence of their action was not investigated.
When O2 or other oxidants were supplied to anoxic cartilage, 35S-sulfate incorporation was stimulated proportionally more strongly than was lactate production (Figures 4 and 5). Figure 7 shows that the same numeric relationship between lactate production and 35S-sulfate incorporation persisted over a wide range of rates, irrespective of which class of oxidant was tested (reducible dyes and FECY in Figure 7A, keto-acids in Figure 7B, O2 in Figures 7A and B).
Figure 7. Relationship between lactate production and sulfate incorporation in anoxic cartilage supplied with artificial oxidants or O2 (air-saturated medium). The results in A are from Figure 4 and include anoxic, MEB, DCIP, FECY, and aerobic treatments. The results in B are from Figure 5 and include anoxic, OAA, PYR, PEP, and aerobic treatments. Because the 2 variables (lactate production, sulfate incorporation) had broadly comparable experimental error, neither could be regarded as the “independent” variable. Therefore, for each graph, regressions of y on x and of x on y were calculated, and the mean slope of the 2 lines was taken. The mean slopes thus obtained were 1.75 in A and 1.61 in B. The correlation coefficients (r) between lactate production and sulfate incorporation were +0.89 in A and +0.90 in B. See Figures 1 and 5 for definitions.
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Iodoacetamide and 2-deoxyglucose have their principal sites of action at different points in the glycolytic pathway (32, 33). The presence of either inhibitor largely prevented oxaloacetate or pyruvate from stimulating glycolysis under anoxia (Figure 6). From this we conclude that a fully functional glycolytic pathway is needed for rapid anoxic lactate formation in the presence of artificial oxidants, as it is for rapid lactate formation in response to O2 itself (5).
Within the limits of our measurements, neither O2 nor the keto-acids affected the stoichiometry of the glycolytic pathway between glucose and lactate, despite a marked increase in flux. The additional anaerobic glycolytic flux that oxaloacetate, pyruvate, or PEP promoted showed a near-theoretical 2:1 molar ratio of lactate produced:glucose consumed, the same molar ratio as was seen when glycolysis was stimulated with O2 (Table 1). These findings demonstrate that irrespective of the rate of glycolysis or how it is stimulated, a close balance is maintained between the production of NADH in the reaction catalyzed by G3PDH and its consumption in the reaction catalyzed by lactate dehydrogenase. Glucose thus remains the principal carbon source for the glycolytic pathway when it is stimulated by other oxidants in place of O2. The fact that pyruvate or oxaloacetate increased lactate formation even when the cartilage was in glucose-free medium (see Results) showed that these keto-acids did not act by overcoming a restriction on the entry of glucose into the cells. The same reasoning applies in the case of O2 as well (5). Articular chondrocytes in vivo are known to contain glycogen (12), which can evidently support continued lactate formation—albeit at a somewhat lower rate—in the absence of external glucose.
Our results also confirm that stimulation of metabolism occurs as the result of reduction of the dyes MEB and DCIP, the inorganic complex FECY, and the keto-acids oxaloacetate and pyruvate; in undergoing reduction, all function as oxidants (electron acceptors) with respect to cartilage. That MEB, DCIP, and FECY were reduced by live cartilage was evident from very obvious color changes (Figure 1). Oxaloacetate could function as an oxidant for the cartilage if it was reduced to L-malate in the reaction catalyzed by L-malate dehydrogenase (EC 220.127.116.11). Malate was present in the used incubation medium (Figure 2). The cytosolic form of malate dehydrogenase, which would be most accessible to externally supplied oxaloacetate, has been detected at high activity in rat articular cartilage (6) and in rabbit articular chondrocytes grown in monolayer culture (36). There is a precedent for compounds such as oxaloacetate serving as oxidants in vivo: the blood parasite Leishmania uses certain 4-carbon acids (oxaloacetate, malate, fumarate) as electron acceptors, reducing them to succinate by partial reversal of the Krebs cycle as a means of reoxidizing NADH (37). The powerful response elicited by oxaloacetate, the smaller but positive effects of L-malate and fumarate, and the inactivity of succinate (see Results) would be consistent with the notion of a similar process operating here.
Like oxaloacetate, externally supplied pyruvate (or PEP) increased glycolysis in anoxic cartilage (Figures 5 and 6). However, little if any of this exogenous pyruvate or PEP can have been reduced to lactate, for if the latter reaction had occurred to a significant extent, the lactate:glucose molar ratio would have risen above the observed value of 2:1 (Table 1). The fate of externally supplied pyruvate in anoxic articular cartilage remains unclear.
A likely mode of action common to both O2 and the other oxidants would be the removal of a reductant from the cells. Whereas MEB or DCIP might function intracellularly, FECY does not penetrate the plasma membrane (38, 39), so it must act at the cell surface, perhaps via a transmembrane oxidoreductase. Under conditions of sustained abundant supply of MEB, DCIP, or FECY, the quantities of these compounds reduced were of the same order as the quantity of additional lactate formed, relative to that in anoxia-only controls (Figure 3). In terms of reductant, decolorization of MEB, DCIP, or FECY at the maximum rates observed in anoxic cartilage (see Results) would consume ∼40–70 microequivalents of electrons/gm dry weight cartilage in 4.5 hours. This flux greatly exceeds the flow of reductant to O2 in aerobic cartilage, which is typically ∼9–18 microequivalents of electrons/gm dry weight cartilage in 4.5 hours (calculated from Table 1 in ref. 1). In short-term studies at least, lipid breakdown needs to be considered as a possible source of the abundant reductant going to MEB, DCIP, and FECY, and also perhaps to externally supplied keto-acids. Lipid droplets are commonly observed in electron microscopy studies of articular chondrocytes (12), and “neutral fats” have been demonstrated with a specific staining procedure (7). Beta-hydroxyacyl dehydrogenase (β-hydroxybutyrate dehydrogenase), a key enzyme in β-oxidation of fatty acids, has been detected cytochemically in articular cartilage (7, 8).
Modulation of the intracellular oxidation-reduction status by oxidants other than O2 has been demonstrated directly in other cell types, usually by measuring the ratio of intracellular pyruvate:lactate concentrations (which reflects the ratio of NADH:NAD+ concentrations in the cytosol). As the following examples show, a change toward a more oxidized intracellular milieu is often beneficial. Thus, in human erythrocytes, FECY decreased the cytosolic NADH:NAD+ ratio (i.e., a shift toward a more oxidized status) and promoted the pentose phosphate pathway and lactate production (40). Brief treatment with other mild oxidants (diamide, H2O2) also promoted erythrocyte glycolysis (41). FECY restored ATP synthesis in erythrocytes when this was partially inhibited by iodoacetate (38). In isolated hepatocytes, hypoxic injury (assessed by loss of ability to exclude trypan blue) was correlated with an increased cytosolic NADH:NAD+ ratio (42). Oxaloacetate, acetoacetate, MEB, or DCIP reduced this ratio and protected the hepatocytes against damage during hypoxia, whereas substrates that favored NADH production (including sorbitol, glycerol, and ethanol) made the injury more severe (42).
Over a longer term, tissue culture studies demonstrate many instances of oxidants improving the growth of cells, even in air-saturated media. Reducible keto-acids, especially glyoxylic, pyruvic, α-ketoglutaratic, and oxaloacetic acid, increased division and colony formation by a variety of cell types in aerobic, serum-free medium (43–45), while the corresponding reduced (hydroxy) forms of the acids were ineffective. Attachment and growth of human melanoma cells in a low-serum medium was enhanced by addition of FECY or pyruvate (46, 47). FECY, or the auto-oxidizable compounds naphthoquinone sulfonate or indigo tetrasulfonate, stimulated growth of HeLa cells in aerobic medium (48, 49). The stimulatory effects of exogenous oxidants on cartilage metabolism are thus consistent with those found in other systems.
For normal function, articular cartilage appears to require exogenous oxidants to stimulate glycolysis, a pathway which evidence from other studies (15–17) strongly suggests is the principal source of ATP for metabolic processes in this tissue, including (for example) the synthesis of extracellular matrix. Under physiologic conditions, O2 acts as this oxidant, but its role can be adequately assumed by other agents. How these oxidants influence glycolysis remains unclear.