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464s Biochemical SocietyTransactions ( 1 995) 23 Protein catabolism in metabolic acidosis: inhibition of glycolysis by low p H suggests a role for glucose Table 1. Protein content and glycolysis 1n L6 Each value represents the mean z S.E.M., n=6 (except for protein for which n=3) ALAN BEVINGTON and JOHN WALLS Department of Nephrology, Leicester General Hospital, Leicester LE5 4PW, U.K. In vivo metabolic acidosis is a potent stimulus for protein catabolism in skeletal muscle [l]. It has recently been shown that this occurs through the ATP-dependent pathway involving ubiquitin and proteasomes [l]. This response involves a co-ordinated increase in the expression of ubiquitin, of subunits of the proteasome [l] and of branched-chain keto-acid dehydrogenase, the enzyme thought to regulate branched chain amino-acid catabolism in skeletal muscle. The net effect is degradation of protein, accompanied by increased oxidation of the amino acids liberated [21. There is at present no explanation for how acidosis triggers this complex response, and no evidence that low pH directly activates the ATP-dependent pathway of protein degradation. However, it is known that low pH inhibits glycolysis, possibly through inhibition of 6-phosphofructo-lkinase [3], that low pH can induce expression of the so-called glucose response proteins, a group of stress-response proteins originally demonstrated in cells subjected to glucose starvation (41, and that abnormalities in glucose metabolism and protein catabolism correlate in skeletal muscle of rats in catabolic states [5]. The aim of the present study is to examine in vitro, using cultured L6 rat myoblasts, the possible link between impaired glycolysis and increased protein catabolism. Confluent cultures of L6 were incubated f o r up to 72h in Eagle's Minimum Essential Medium with 2mM glutamine and 10% (vol/vol) dialysed foetal bovine serum at 37OC under humidified 95% air/5% COz. The pH of the medium was adjusted by addition of NaHC03 or HC1, with extra NaCl added at low pH to maintain a constant Na concentration. Fresh culture medium was added at least every 24h, and glucose consumpt.ion and lactate production were measured in the medium as an index of glycolysis. Statistical significance of changes was assessed by Two-way Analysis of Variance and Duncan's Multiple Range Test. Low protein content and increased protein degradation have been reported in cultured BC3H1,myoblasts [61 after only 48h at low pH. In view of the ubiquitous nature of the proteins involved in the ATP-dependent pathway of protein degradation [71, it would be expected that protein wasting in response to acid would also be ubiquitous. However, even after 72h (Table l), L6 myoblasts showed no evidence of acid-induced protein wasting, even though a known catabolic stimulus (incubation without serum for 24h), caused a clear decrease in protein content (7.2~0.1 pg proteinlpg DNA versus 7.9~0.2 in control cells in medium with 10% serum, p<O.O2). In contrast, both glucose consumption and lactate production were impaired after only 3h at low pH (Table l), and this effect was still demonstrable after at least 48h. Therefore if impaired glycolysis is linked to I D, this increased protein catabolism & coupling seems to fail in L6 cells. It is well established that, in rapidly pH of medium 7.18*+0.03 7.29*+0.03 7.44~0.03 Protein (pglpg DNA) 9.0+0.5 8.8~0.9 9.3~0.8 Glucose (nmollpg protl3h) 2.9*+0.2 3.1~0.2 3.4~0.3 Lactate (nmo1/pg protl3h) 5.3*+0.2 5.5*+0.2 6.0+0.3 92+8 91+7 G 1ucose conversion to lactate ( % ) 92+5 *p<0.05 compared with value at pH 7.44 proliferating cultured cells, glycolysis is inappropriately stimulated, providing a supply of pyruvate in excess of that which can be oxidised by the mitochondria [El, with consequent conversion of the excess pyruvate to lactate. This "aerobic glycolysis" occurs in L6 (Table 1 1 , as there is nearquantitative conversion of glucose to lactate. Oxidation of glucose seems therefore to be only a minor contributor to energy metabolism in L6, s o that impairment of glycolysis by low pH is unlikely to have a marked impact on the supply of substrate to the mitochondria. A possible explanation for the acidinduced protein catabolism and increased amino acid oxidation is that impairment of glycolysis by low pH restricts the pyruvate supply to mitochondria, leading to catabolism of amino acids from protein as an alternative metabolic fuel. This leads to the prediction that acid-induced protein degradation is not ubiquitous, but should be confined to cells having a significant dependence on glucose oxidation. It also predicts that low cytosolic pH will not trigger protein degradation when other factors are stimulating glycolysis. This may explain why the profound intramuscular acidification observed during exercise (in which glycolysis is known to increase [3]) does not usually trigger protein catabolism. 1. Mitch, W.E., Medina, R., Grieber, S., May, R.C., England, B.K., Price, S.R., Bailey, J.L. & Goldberg, A.L. (1994) J. Clin. Invest. 93, 2127-2133 2. May, R.C., Masud, T., Logue, B., Bailey, J. & England, B.K. (1992) Min. Elect. Metab. 18, 245-249 3. Spriet, L.L. (1991) Can. J. Physiol. Pharmacol. 69, 298-304 4. Whelan, S.A. & Hightower, L.E. (1985) J. Cell. Physiol. 125, 251-258 5. May, R.C. , Clark, A.S., Goheer, M.A. & Mitch, W.E. (1985) Kid. Int. 28, 490-497 6. England, B.K., Chast-ain, J.L. & Mitch, W.E. (1991) Am. J. Physiol. 260, C277-C282 7. Rivett, A.J. (1989) Biochem. J. 263, 625633 8. Hue, L. & Rousseau, G.G. (1993) Adv. Enzyme Regul. 3 3 , 97-110