Bioehimica et Bioph.vsica Acta. 1092(1991) 277-283 O 1991 ElsevierScience Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100153Z
277
BBAMCR 12921
Relationship between the stimulation of citric acid cycle oxidation and the stimulation of fatty acid esterification and inhibition of ketogenesis by lactate in isolated rat hepatocytes Debra C. Henly and Michael N. Berry Department of Medical Biochemistr); Flinders University of South A ustralia. Bedford Park. (Australia)
(Received20 September1990)
Key words: Triacylglycerol;Ketonebody synthesis; Citric ;lcid cycle; Fatty acid esterification; (Rat hepatocyte) Isolated hepatocytes from fasted rats were used to study the effects of lactate on palmitate metabolism. Lactate was found to stimulate fatty acid esterification and citric acid cycle oxidation and to inhibit ketone body synthesis. These effects of lactate were largely maintained when gluconeogenesis was inhibited with either quinolinate or pergluorosuccinate, but were overcome by ot-cyano-4-hydroxycinnamate. However, the responses of hepatocytes to lactate could be restored in the presence of a-cyano-4-hydroxycinnamate by the further addition of propionate. The stimulation of triacylglyceroi synthesis by lactate was not associated with an increase in the concentration of glycerol 3-phosphate. Rather, there was a correlation between flux through the citric acid cycle and the rate of triacylglyceroi synthesis. In all instances reduction of ketone body formation in the presence of lactate was accompanied by a stimulation of citric acid cycle oxidation.
Introduction The 'ketone bodies' acetoacetate and 3-hydroxybutyrate, synthesised in the liver from acetyl-CoA, a product of fatty acid /3-oxidation, serve an important role during periods of prolonged starvation, where they act as a fuel source for peripheral tissues, in particular the brain [1]. The pioneering work of Edson using liver slices [2,3], subsequently confirmed by others in both the perfused liver [4,5] and isolated hepatocytes [6-8], has demonstrated that gluconeogenic precursors such as lactate, fructose or glycerol inhibit ketone body formation from fatty acid (however, see Ref. 9). The mechanism of this antiketogenic action is still controversial, but two main hypotheses have been proposed. The inhibition of ketone body synthesis has been suggested to be the consequence of an acceleration of esterifica-
Abbreviations: CHC, a-cyano-4-hydroxycinnamate; Jo, total oxygen consumption; J/~, oxygen consumption associated with g-oxidation; Jr, oxygenconsumptionassociated with citric acid cycleoxidation. Correspondence: D.C. Henly, Department of Medical Biochemistry, Flinders University of South Australia, G.P.O. Box 2100, Adelaide. South Australia, 5001, Australia.
tion at the expense of fl-oxidation [4,6,10,11]. However, although it has been demonstrated that triacylglycerol synthesis is elevated upon addition of a gluconeogenic precursor, it has not been shown that this limits floxidation, as there is some evidence that, at least in isolated hepatocytes, fatty acid uptake is also accelerated [9] and may compensate for the increased triacyiglycerol synthesis. A second hypothesis proposes that ghiconeogenic precursors are antiketogenic because they increase the citric acid cycle oxidation of acetyl-CoA [7,8,12-14] possibly by stimulating the demand for ATP [7] or by raising the steady state concentration of oxaloacetate [14-16]. However, it has proved difficult to measure the level of this intermediate accurately as over 90% is bound [17]. In any case, the significance of the measurements is dubious as there is evidence for channelling of oxaloacetate between malate dehydrogenase and citrate synthetase [18]. Precise measurement of citric acid cycle activity has also proved difficult, most workers relying upon the generation of ~4CO2 from radiolabelled fatty acid, a method which has been criticised as it grossly underestimates the total flux through the cycle [19]. In the light of this continuing uncertainty we have investigated the mechanism of the antiketogenic effect of lactate in isolated hepatocytes from fasted rats. We
278 have previously developed a method to calculate the oxygen consumption associated with B-oxidation and citric acid cycle oxidation [7] and have used this to determine whether the activity of either pathway is affected by the addition of lactate. In addition, inhibitors of a number of steps of the gluconeogenic pathway have been used to determine whether the stimulation of citric acid cycle oxidation or triacylglycerol synthesis, and the concomitant inhibition of ketone body synthesis, are maintained when glucose synthesis is significantly inhibited. These studies have enabled us to identify clearly the site of action of lactate on the ketogenic pathway and have led to an alternative idea to explain the stimulatory effects of lactate on the esterification pathway.
palmitate ar palmitate and lactate for 35 rain. As has been frequently observed [4,7,8,14], addition of lactate caused an inhibition of ketone body synthesis from palmitate (Table I). In the presence of lactate there was a stimulation of oxygen consumption above that measured in incubations with palmitate alone which could be entirely accounted for by an increase in Jc There was also a large increase in the rate of triacylglycerol synthesis in the presence of lactate, but despite this stimulation of esterification, B-oxidation was unaffected. Instead, cellular palmitate uptake was increased in a compensatory manner so that extra palmitate was esterified without the diversion of fatty acid from the B-oxidative pathway (Table I). Glycerol 3-phosphate concentrations were also unchanged by the addition of lactate (Table II).
Materials and Methods
Materials, Collagenase and enzymes for metabolite determination were from Boehringer-Mannheim (F.R.G.) as was bovine serum albumin (Fraction V) defatted according to Chen [20]. Palmitate, lactate, pyruvate and a-cyano-4-hydroxycinnamate were from the Sigma Cbemical Company (U.S.A.). Perfluorosuccinate was obtained from Pfaltz and Bauer (U.S.A.) while quinolinate was from Matheson, Coleman and Bell (U.S.A.). Other reagents were of the highest grade commercially available. Palmitate (8 mM) was prepared in isotonic saline containing 9% bovine serum albumin. Methods. Isolated liver cells (110-120 mg wet weight) from male Hooded Wistar rats (260-300 g body weight), starved for 24 h to deplete liver glycogen, were prepared by standard techniques [21,22]. Hepatocytes were incubated at 37"C for 35 rain on a Gilson differential respirometer in 2 ml bicarbonate-buffered saline medium [23,24] containing 2.25% (w/v) albumin with a gas phase of 95% 02, 5% CO 2. Oxygen consumption in the presence of CO 2 was measured by a manometric method [251 and the proportion of total oxygen consumption (J,,) attributable to B-oxidation (Jr0 and citric acid cycle oxidation (J,.) were calculated as outlined previously [71. Metabolites in neutralised perchloric acid extracts were measured by standard enzymic techniques as in Bergmeyer [261 in a Cobas FARA centrifugal analyser (Roche Diagnostics, Switzerland) and the data were transferred to a PDP 11/73 (D.E.C., U.S.A.) for subsequent processing. [14C]Palmitate and [14C]triacylglycerol were extracted in isopropanol-heptane according to Borgstrom [27]. Results
Mechanism of antiketogenic action of lactate The antiketogenic action of the gluconeogenic precursor lactate was examined in a series of experiments in which cells from fasted rats were incubated with
Effect of inhibitors of gluconeogenesis ot-Cyano.4-hydroxycinnamate, a-Cyano-4-hydroxycinnamate (CHC), an inhibitor of the pyruvate transporter [28], prevents the entry of pyruvate into the mitochondria and would therefore be expected to inhibit the accumulation of oxaloacetate upon addition of lactate. CHC caused a small inhibition of Jo, J~ and Jc when included in incubations containing only palmitate and reduced the rate of triacylglycerol synthesis, but the inhibitor did not significantly affect the rate of ketogenesis (Table I). In contrast, the addition of CHC to incubations containing palmitate and lactate reduced Jo to a rate that was not significantly different to that measured in incubations of hepatocytes with palmitate alone. This inhibition was the result of a reduction in J, to a rate approaching that measured in the presence of palmitate alone, J~ was unaffected. Ketone body synthesis was not significantly different to that measured in the presence of paimitate alone, and triacylglycerol synthesis was also reduced to a rate that was only slightly greater than that measured in hepatocytes incubated with palmitate in the absence of lactate. Glycerol 3-phosphate concentrations were not significantly different to those measured in liver cells incubated with either palmitate or palmitate and lactate, though they were somewhat higher than in incubations containing palmitate and CHC (Table II). The citric acid cycle intermediates malate and citrate were reduced by the addition of CHC to incubations containing palmitate and lactate to concentrations that were not significantly different to those measured with palmitate alone. CHC did not alter the concentration of malate when included in incubations containing only palmitate. Citrate concentrations in the presence of the inhibitor proved to be too low for accurate measurement. To gain a more precise measure of the effects of CHC, cells incubated with palmitate and lactate were titrated with the inhibitor. As glucose synthesis was
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277
BBAMCR 12921
Relationship between the stimulation of citric acid cycle oxidation and the stimulation of fatty acid esterification and inhibition of ketogenesis by lactate in isolated rat hepatocytes Debra C. Henly and Michael N. Berry Department of Medical Biochemistr); Flinders University of South A ustralia. Bedford Park. (Australia)
(Received20 September1990)
Key words: Triacylglycerol;Ketonebody synthesis; Citric ;lcid cycle; Fatty acid esterification; (Rat hepatocyte) Isolated hepatocytes from fasted rats were used to study the effects of lactate on palmitate metabolism. Lactate was found to stimulate fatty acid esterification and citric acid cycle oxidation and to inhibit ketone body synthesis. These effects of lactate were largely maintained when gluconeogenesis was inhibited with either quinolinate or pergluorosuccinate, but were overcome by ot-cyano-4-hydroxycinnamate. However, the responses of hepatocytes to lactate could be restored in the presence of a-cyano-4-hydroxycinnamate by the further addition of propionate. The stimulation of triacylglyceroi synthesis by lactate was not associated with an increase in the concentration of glycerol 3-phosphate. Rather, there was a correlation between flux through the citric acid cycle and the rate of triacylglyceroi synthesis. In all instances reduction of ketone body formation in the presence of lactate was accompanied by a stimulation of citric acid cycle oxidation.
Introduction The 'ketone bodies' acetoacetate and 3-hydroxybutyrate, synthesised in the liver from acetyl-CoA, a product of fatty acid /3-oxidation, serve an important role during periods of prolonged starvation, where they act as a fuel source for peripheral tissues, in particular the brain [1]. The pioneering work of Edson using liver slices [2,3], subsequently confirmed by others in both the perfused liver [4,5] and isolated hepatocytes [6-8], has demonstrated that gluconeogenic precursors such as lactate, fructose or glycerol inhibit ketone body formation from fatty acid (however, see Ref. 9). The mechanism of this antiketogenic action is still controversial, but two main hypotheses have been proposed. The inhibition of ketone body synthesis has been suggested to be the consequence of an acceleration of esterifica-
Abbreviations: CHC, a-cyano-4-hydroxycinnamate; Jo, total oxygen consumption; J/~, oxygen consumption associated with g-oxidation; Jr, oxygenconsumptionassociated with citric acid cycleoxidation. Correspondence: D.C. Henly, Department of Medical Biochemistry, Flinders University of South Australia, G.P.O. Box 2100, Adelaide. South Australia, 5001, Australia.
tion at the expense of fl-oxidation [4,6,10,11]. However, although it has been demonstrated that triacylglycerol synthesis is elevated upon addition of a gluconeogenic precursor, it has not been shown that this limits floxidation, as there is some evidence that, at least in isolated hepatocytes, fatty acid uptake is also accelerated [9] and may compensate for the increased triacyiglycerol synthesis. A second hypothesis proposes that ghiconeogenic precursors are antiketogenic because they increase the citric acid cycle oxidation of acetyl-CoA [7,8,12-14] possibly by stimulating the demand for ATP [7] or by raising the steady state concentration of oxaloacetate [14-16]. However, it has proved difficult to measure the level of this intermediate accurately as over 90% is bound [17]. In any case, the significance of the measurements is dubious as there is evidence for channelling of oxaloacetate between malate dehydrogenase and citrate synthetase [18]. Precise measurement of citric acid cycle activity has also proved difficult, most workers relying upon the generation of ~4CO2 from radiolabelled fatty acid, a method which has been criticised as it grossly underestimates the total flux through the cycle [19]. In the light of this continuing uncertainty we have investigated the mechanism of the antiketogenic effect of lactate in isolated hepatocytes from fasted rats. We
278 have previously developed a method to calculate the oxygen consumption associated with B-oxidation and citric acid cycle oxidation [7] and have used this to determine whether the activity of either pathway is affected by the addition of lactate. In addition, inhibitors of a number of steps of the gluconeogenic pathway have been used to determine whether the stimulation of citric acid cycle oxidation or triacylglycerol synthesis, and the concomitant inhibition of ketone body synthesis, are maintained when glucose synthesis is significantly inhibited. These studies have enabled us to identify clearly the site of action of lactate on the ketogenic pathway and have led to an alternative idea to explain the stimulatory effects of lactate on the esterification pathway.
palmitate ar palmitate and lactate for 35 rain. As has been frequently observed [4,7,8,14], addition of lactate caused an inhibition of ketone body synthesis from palmitate (Table I). In the presence of lactate there was a stimulation of oxygen consumption above that measured in incubations with palmitate alone which could be entirely accounted for by an increase in Jc There was also a large increase in the rate of triacylglycerol synthesis in the presence of lactate, but despite this stimulation of esterification, B-oxidation was unaffected. Instead, cellular palmitate uptake was increased in a compensatory manner so that extra palmitate was esterified without the diversion of fatty acid from the B-oxidative pathway (Table I). Glycerol 3-phosphate concentrations were also unchanged by the addition of lactate (Table II).
Materials and Methods
Materials, Collagenase and enzymes for metabolite determination were from Boehringer-Mannheim (F.R.G.) as was bovine serum albumin (Fraction V) defatted according to Chen [20]. Palmitate, lactate, pyruvate and a-cyano-4-hydroxycinnamate were from the Sigma Cbemical Company (U.S.A.). Perfluorosuccinate was obtained from Pfaltz and Bauer (U.S.A.) while quinolinate was from Matheson, Coleman and Bell (U.S.A.). Other reagents were of the highest grade commercially available. Palmitate (8 mM) was prepared in isotonic saline containing 9% bovine serum albumin. Methods. Isolated liver cells (110-120 mg wet weight) from male Hooded Wistar rats (260-300 g body weight), starved for 24 h to deplete liver glycogen, were prepared by standard techniques [21,22]. Hepatocytes were incubated at 37"C for 35 rain on a Gilson differential respirometer in 2 ml bicarbonate-buffered saline medium [23,24] containing 2.25% (w/v) albumin with a gas phase of 95% 02, 5% CO 2. Oxygen consumption in the presence of CO 2 was measured by a manometric method [251 and the proportion of total oxygen consumption (J,,) attributable to B-oxidation (Jr0 and citric acid cycle oxidation (J,.) were calculated as outlined previously [71. Metabolites in neutralised perchloric acid extracts were measured by standard enzymic techniques as in Bergmeyer [261 in a Cobas FARA centrifugal analyser (Roche Diagnostics, Switzerland) and the data were transferred to a PDP 11/73 (D.E.C., U.S.A.) for subsequent processing. [14C]Palmitate and [14C]triacylglycerol were extracted in isopropanol-heptane according to Borgstrom [27]. Results
Mechanism of antiketogenic action of lactate The antiketogenic action of the gluconeogenic precursor lactate was examined in a series of experiments in which cells from fasted rats were incubated with
Effect of inhibitors of gluconeogenesis ot-Cyano.4-hydroxycinnamate, a-Cyano-4-hydroxycinnamate (CHC), an inhibitor of the pyruvate transporter [28], prevents the entry of pyruvate into the mitochondria and would therefore be expected to inhibit the accumulation of oxaloacetate upon addition of lactate. CHC caused a small inhibition of Jo, J~ and Jc when included in incubations containing only palmitate and reduced the rate of triacylglycerol synthesis, but the inhibitor did not significantly affect the rate of ketogenesis (Table I). In contrast, the addition of CHC to incubations containing palmitate and lactate reduced Jo to a rate that was not significantly different to that measured in incubations of hepatocytes with palmitate alone. This inhibition was the result of a reduction in J, to a rate approaching that measured in the presence of palmitate alone, J~ was unaffected. Ketone body synthesis was not significantly different to that measured in the presence of paimitate alone, and triacylglycerol synthesis was also reduced to a rate that was only slightly greater than that measured in hepatocytes incubated with palmitate in the absence of lactate. Glycerol 3-phosphate concentrations were not significantly different to those measured in liver cells incubated with either palmitate or palmitate and lactate, though they were somewhat higher than in incubations containing palmitate and CHC (Table II). The citric acid cycle intermediates malate and citrate were reduced by the addition of CHC to incubations containing palmitate and lactate to concentrations that were not significantly different to those measured with palmitate alone. CHC did not alter the concentration of malate when included in incubations containing only palmitate. Citrate concentrations in the presence of the inhibitor proved to be too low for accurate measurement. To gain a more precise measure of the effects of CHC, cells incubated with palmitate and lactate were titrated with the inhibitor. As glucose synthesis was
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