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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.
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J. & England, B.K. (1992) Min. Elect.
Metab. 18, 245-249
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&
Mitch, W.E. (1985) Kid. Int. 28, 490-497
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W.E. (1991) Am. J. Physiol. 260, C277-C282
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(1993) Adv.
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