Vol. 63, No. 3, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE REGULATIONOF CARBON FLOWTHROUGHPYRUVATE DEHYDROGENASE IN THE PERFUSEDGUINEA PIG LIVER1 Korechika Ogata2, Richard W. Hanson3 and Sidney Weinhouse Fels Research I n s t i t u t e and Department of Biochemistry Temple University Medical School, Philadelphia, Pa. 19140
Received February 18,1975 SUMMARY. The r e l a t i v e contributions of lactate, propionate and octanoate to the acetyl CoA pool for keto~ body formation in the perfused guinea pig l i v e r was investigated with Crlabeled substratesJm In the presence of octanoate, incorporation of 3-(14C)-lactate or 3-(l.C)-propionate into acetoacetate wa~,extremely low, amounting to only about 0.2% of the incorporation of l-(~C)-octanoate which accounted for 28% of the acetoacetate synthesized, when this labeled f a t t y acid was perfus@~ together with un-14 . labeled lactate or propionate. Incorporation Of 3-(l~C)-]actate and 3-(' C}propionate into CO2 was likewise very low, in the same @~periments amounting only to less than 0.7% and 7.0% respectively of the l-(i~C)-octanoate carbon oxidized to CO2 in the presence of unlabeled lactate or propionate. Despite the potential a v a i l a b i l i t y of acetyl CoA from lactate via pyruvate; and from propionate via succinate, oxalacetate, P-enolpyruvate and pyruvate, i t s formation was minimal. The results indicate that pyruvate dehydrogenase is strongly inhibited during ketogenesis, when under these conditions, there is v i r t u a l l y complete conversion of metabolic pyruvate to glucose. INTRODUCTION. During periods of enhanced gluconeogenesis, the l i v e r is provided with a variety of gluconeoQenic substrates such as lactate and alanine, and with f a t t y acids mobilized from depot steres.
During gluco-
neogenesis, pyruvate formed from lactate or alanine is diverted from decarboxylation via pyruvate dehydrogenase toward carboxylation via pyruvate carboxylase ( I ) .
Evidence from several experimental approaches suggests
that f a t t y acid oxidation plays a role in this process.
Walter et al. (2)
noted that octanoate markedly inhibited the oxidative decarboxylation of pyruvate by isolated rat l i v e r mitochondria; and Bremer (3) found that acylIThis research was supported by Public Health Service Grants AM 16009 from the National I n s t i t u t e of A r t h r i t i s and Metabolic Diseases, CA-I0916 and 12227 from the National Cancer InstitUte and BC-74 from the American Cancer Society. 2present address: Department of Biochemistry, School of Medicine, Nagoya University, Nagoya, Japan. 3Career Development Awardee of the U.S, Public Health Service, K04 AM 15365.
Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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carnitines i n h i b i t the mitochondrial conversion of pyruvate to acetoacetate. That this effect was due to an inhibition of pyruvate dehydrogenase was suggested by Jagow et al. (4) who demonstrated an inhibition by palmitate of the conversion of I-(14C) pyruvate to 14C02. Work with purified Dig heart pyruvate dehydrogenase has indicated that acetyl CoA (5), NADH (5) and ATP (6) can act as inhibitors of the enzyme. In a previous study from this laboratory (7), we noted that when 2-(14C) pyruvate was the substrate for gluconeogenesis by perfused rat l i v e r , octanoate markedly increased ketone body formation but almost t o t a l l y abolished the incorporation of label into ketone bodies.
These studies, together with
the results of the present report, indicate clearly that f a t t y acids effect i v e l y regulate the flow of pyruvate carbon during gluconeogenesis by inhibi t i n g i t s conversion to acetyl CoA, thereby providing substrate for pyruvate carboxylase with consequent enhanced gluconeogenesis. METHODS. The guinea pigs used in this study were of the Hartley strain and were fed Wayne Guinea Pig Chow supplemented with greens. fasted 48 hrs. before the experiments.
The animals were
The non-recycling l i v e r perfusion
technique has been described in detail in a previous publication (8).
During
the perfusion, we continuously monitored 02 consumption by determining d i f ferences between the concentrations of 02 in the fluid entering and leaving the l i v e r , using a Clark-type oxygen electrode. The perfusion fluid was Krebs-Ringer bicarbonate, pH 7.4, containing as substrates, when indicated, 2mM Dropionate and 0.2 mM sodium octanoate. In one series of experiments, 3-(14C)-lactate was perfused with unlabeled octanoate, and in another, l-(14C)-octanoate with unlabeled lactate.
The
same arrangement was employed for combinations of 3-(14C)-propionate and octanoate. The concentrations of glucose (9), acetoacetate (I0), and ~-hydroxybutyrate (I0) were determined on the effluent perfusion medium, and the radioactivity in (14C)-acetoacetate was estimated by a slight modification of
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the procedure of Reichard et al. ( I I ) .
Since the ~-hydroxybutyrate represents
less than 20 percent of the total ketone bodies produced by guinea pig l i v e r (12), only the specific a c t i v i t y of acetoacetate was determined.
The 14C02
output of the l i v e r was measured on a 5 ml sample of perfusion medium, collected from the cannula leaving the l i v e r , to minimize diffusion of gas.
The l i q u i d
was transferred to a 25 ml Erlenmeyer flask sealed with a plasma stopper equipped with a plastic hanging bucket (Kontes Glass, Vineland, N.J.).
The medium was
a c i d i f i e d by the introduction of 1 ml 2 N H2SO4 through the plasma stopper, and the 14C02 collected in hyamine hydroxide contained in the plastic bucket.
After
a 40 min period for diffusion and absorption of CO2, the bucket was removed, the contents dissolved in l i q u i d s c i n t i l l a t i o n f l u i d , and the r a d i o a c t i v i t y determined. MATERIALS. NAD+, NADH and B-hydroxybutyrate dehydrogenase were purchased from Boehringer Mannheim Corp., New York.
I octanoate
o