Download The Formation of Pyruvate from Citric Acid

5 8 0 t h Meeting
Held at the University of Nottingham
on 3,4 and 5 January 7979
CO M M U N ICAT10 N
The Formation of Pyruvate from Citric Acid-Cycle Intermediates in
Kidney Cortex
MALCOLM WATFORD, PATRICK VINAY, G U Y LEMIEUX and
ANDRE GOUGOUX
Rerial Laboratory, H6tel- Dieii Hospirril rirrrl Deprirrnicwt of Merliciiw,
University of Montreal, Monrreul, Qiicdwc, Criiiarlri H Z W I T8
In studies to investigate the relationship of renal gluconeogenesis to ammonia production in isolated tubules from rat kidney cortex we observed the following: when
phosphoenolpyruvate carboxykinase [GTP-oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1 .I .32] is inhibited by 3-mercaptopicolinate, glucose production can
be conipletely suppressed without affecting glutamine oxidation (see Table I ) . This
implies the formation of pyruvate from citric acid-cycle intermediates via an alternative
pathway not involving phosphoenolpyruvate carboxykinase. This communication
compares such pathways in the kidney cortex of rats, dogs and rabbits.
) isolated kidney tubules from rats or from
The metabolism of glutamine ( 5 m ~ by
dogs is similar (Table I ) . In both species, glutamine utilized can be accounted for by
glucose (30%), amino acids (mainly glutamate and aspartate) ( 3 5 % ) and CO, ( 3 5 % ) .
The accumulation of amino acids is an artefact of the experimental conditions and would
not be a major end product of glutamine metabolism in vivo. The substrate for rabbit
tubules was a-oxoglutarate ( 5 mM) as neither glutamine nor glutamate gave satisfactory
rates of gluconeogenesis, possibly due to inhibition of rabbit renal glutamate dehydrogenase by ammonia (see Klahr, 1971). The a-oxoglutarate utilized by rabbit kidney
tubules (Table I ) was recovered as glucose (3573, amino acids (mainly glutamate,
malate (7%) and lactate (10%) with comparatively
glutamine and aspartate) (35
little substrate undergoing complete oxidation (1 5 %).
The addition of 3-mercaptopicolinate caused a decrease in gluconeogenesis in all
three species, however, the concentration of inhibitor necessary to completely inhibit
glucose production was species-dependent; rat (OSrnM), dog (2.5 mM), rabbit ( 5 mM)
(M. Watford, unpublished work).
Despite its effect on glucose production (Table 1) 3-mercaptopicolinate (0.5-2.5 mM)
did not suppress glutamine oxidation in isolated tubules from rats and dogs (Table I ) .
The glutamine utilized was accounted for by an accumulation of amino acids in the rat
and by amino acids, malate and lactate in the dog. In the rabbit 3-mercaptopicolinate
( 5 m ~ caused
)
increases in the concentrations of amino acids and of malate and conipletely suppressed both glucose production and substrate (a-oxoglutarate) oxidation,
with a decrease in lactate production.
These results imply that when phosphoenolpyruvate carboxykinase is inhibited, then
an alternative pathway for pyruvate formation from citric acid-cycle intermediates
exists in the kidney cortex of rats and dogs, but not of rabbits. Two such pathways are
x),
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26
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BIOCHEMICAL SOCIETY TRANSACTIONS
Table I . Metabolism of pliitamitze and a-oxoghrtarate by isolated kidney tirbirles
Isolated kidney tubules were prepared from fed animals and incubated at 37°C for
30min as previously described (Vinay et al., 19780). The results are expressed as pmol
or substrate utilized (-) or product formed (+)/30min per g wet wt. and are the means
for at least four separate experiments. The amount of glutamine undergoing complete
oxidation was calculated by three methods; from the carbon balance, from the ammonia
balance (see Vinay et al., 19786) and from the production of ‘‘C0, from [U-L4C]gl~~tamine after correction for dilution via the citric acid cycle as described by Vinay et al.
(19784. The amount of a-oxoglutarate oxidized was estimated by the carbon balance
only.
Rabbit
5 nlMRat
Dog
5 mM-glutaniine
a-oxoglutarate
Substrate . . . 5 mM-gluiamine
3-Mercaptopicolinate (mM) . .. 0
0.5
0
2.5
0
5 .O
Metabolite changes
*--
Substrate
Glucose (as C, units)
NH3
Lactate
Malate
Amino acids
Mean C, recovery (%)
Substrate undergoing
complete oxidation:
from carbon balance
from NH, balance
from 14C02production
-95.4
+34.7
+172.5
+0.33
<0.01
+34.5
72.9
25.9
34.6
29.8
-76.7
+0.8
+123.5
+0.27
+3.13
+53.1
74.7
19.4
31.3
34.7
-47.3
-60.1
$13.6
f1.13
+69.7
+76.8
+2.1
+6.67
<0.01
+6.2
+18.9
f35.1
73.2
81.7
12.5
9.7
11.1
-53.75
f19.3
-
+5.5
-55.5
+1.0
-
+4.0
+17.1
85.4
+3.1
f32.2
+21.2
105.0
-7.85
-
10.9
7.0
7.0
-
-
-
-
Table 2. Distribution of ‘malic’ enzyme and oxalocicetate decarboxylnse in kidney cortex
The activities are expressed as pniol of pyruvate formed/min per g wet wt. at 37°C and
are the means for at least four determinations. ‘Malic’ enzyme was measured by the
method of Ochoa (1955) and oxalocetate decarboxylase by that of Wojtczak & Walajtys
(1974). The mitochondria1 activities were ‘solubilized’ by freezing and thawing four
times and the values shown have been corrected for cross-contamination of the fractions
using lactate dehydrogenase and glutaniate dehydrogenase as cytosolic and mitochondrial markers respectively.
Pyruvate formed (pmol/min per g)
,
‘Malic’ enzyme
Species Homogenate Cytosol Mitochondria
Rat
2.22
1.66
0.54
0.28
0.74
Dog
1.02
<0.10
Rabbit
Oxaloacetate decarboxylase
Homogenate Cytosol Mitochondria
3.10
0.80
2.20
1.43
0.29
0.58
1.98
0.56
I .57
known to exist, ‘malic’ enzyme ( E C I . I . I .40) and oxaloacetate decarboxylase (see Dean
& Bartley, 1973; Wojtczak & Walajtys, 1974).
‘Malic’ enzyme activity is present in the kidney cortex of rats and dogs but not rabbits
(Table 2), whereas oxaloacetate decarboxylase is present in all three species (Table 2).
Both enzymes are of sufficient activity to explain the maximum rates of pyruvate for1979
580th MEETING, NOTTINGHAM
755
niation (for oxidation via the citric acid cycle or for lactate production) observed with
isolated tubules and neither enzyme is inhbited by 3-mercaptopicolinate (5mM) (M.
Watford, unpublished work).
As neither complete oxidation nor conversion into lactate, of citric acid cycle intermediates, occurs in a species lacking renal ‘nialic’ enzyme (rabbit) we propose that
‘nialic’ enzyme can function to provide pyruvate when phosphoenolpyruvate carboxykinase is inhibited.
Implication of ‘malic’ enzyme activity also implies the generation of NADPH, whereas
the formation of lactate (in dog tubules) requires reducing equivalents in the form of
NADH. Oxaloacetate decarboxylase could also explain pyruvate formation and would
indirectly provide N A D H (via the malate dehydrogenase reaction). However it is
difficult to explain why this pathway is apparently inoperative in rabbit tubules. At the
moment the nietabolic processes involved in the possible utilization of NADPH and the
provision of N A D H cannot be ascertained. I t is of interest that Saggerson (1978) has
recently reported that lactate production from pyruvate in rat kidney tubules is only
partially sensitive to inhibition by 3-mercaptopicolinate. This study as well as our results
imply the transfer of mitochondria1 reducing equivalents to the cytosol during phosphoenol pyruvate carboxykinase inhi bit ion.
The 3-niercaptopicolinic acid was generously given by Dr. N. W. DiTullio, Smith, Kline and
French, Philadelphia, PA, U . S . A .
Dean, B. & Bartley, W. (1973) Eiocheni. J . 135, 667-672
Klahr, S . (1971) h i . J . Physiol221, 69-74
Ochoa, S . (1955) Methods Enzyniol. 1 , 739-753
Saggerson, E. D. (1978) Eiochem.J. 174, 131-142
Vinay, P., Lemieux, G. & Gougoux. A . (1978~1)Ccimid. J . Biochetn. 56, 305-314
Vinay, P., Lemieux, G., Gougoux, A. & Watford, M . (19786) Proc. Int. Congr.. A’ephro1og.v 7 f h ,
199-207
Vinay, P., Mapes, J. P. & Krebs, H. A. (1978~)Am. J . Physiol. 234, F123-FI29
Wojtczak, A . B. & Walajtys, E. (1974) Eiochim. Bioph)ts. Acta347, 168-182
Vol. 7