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Transcript
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to completely inhibit glucose production was species-dependent; rat (OSrnM), dog (2.5 mM), rabbit ( 5 mM) (M. Watford, unpublished work).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), Vol. 7 26 754 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