Biochimica et Biophysica Acta, 1051 (1990) 215-220
215
Elsevier BBAMCR 12617
Ca2+-fatty acid interaction in the control of hepatic gluconeogenesis Consuelo Gonzfilez-Manch6n
*, J u a n M e n a y a
* *, M a t i l d e S. A y u s o
and Roberto Parrilla Endocrine Physiology Unit, Centro de lnvestigaciones Biolbgicas (C. S. 1.C.), Madrid (Spain)
(Received 20 March 1989) (Revised manuscript received 12 September 1989)
Key words: Calciumion-fatty acid interaction; Gluconeogenesis; Fatty acid; (Rat liver)
Calcium depletion induced by perfusing livers with calcium-free buffer did not alter the rates of basal glucose production from pyruvate or from increasing concentrations of lactate. However, calcium deficiency selectively prevented the fatty acid-induced stimulation of gluconeogenesis from lactate. This effect is not related to the higher NAD redox potential consistently observed in Ca2+-deficient livers. On the other hand, octanoate was capable of inducing dose-dependent changes in the [pyruvate]0.5 in calcium-depleted livers pedused with lactate, ruling out that low cellular calcium content could perturb the mitochondrial transport of pyruvate. The observation that the effect of calcium deficiency can be overcome by supraphysiological concentrations of pyruvate supports the proposal that stimulation of the maximal capacity of the gluconeogenic pathway by fatty acid relies largely on the tricarboxylic acid cycle activity, restricted in calcium deficiency conditions.
Introduction Extracellular requirement for Ca/+ or the participation of intracellular Ca 2÷ fluxes on the hormonal stimulation of gluconeogenesis has been reported by numerous authors [1-3]. However, the reports on the degree of dependency of hepatic gluconeogenesis on extracellular Ca 2÷ are controversial [4-9]. As far as we know, there is no information available on possible Ca2+-fatty acid interactions in the control of gluconeogenesis, despite the possible involvement of both Ca 2÷ and fatty acid on hormonal responses [3,10,11]. Mutual interactions of Ca 2+ and fatty acids are well documented in the literature: (1) fatty acids have been reported to perturb Ca 2÷ homeostasis [12-15]; (2) similarly, Ca 2+ has been reported to alter fatty acid metabolism [16-18]. On the basis of the above considerations, we found it worthwhile to carry out a study in perfused rat liver, under controlled conditions of substrates and oxygen supply, on
the calcium dependency of the fatty acid stimulatory action of gluconeogenesis. Octanoate was chosen not only for the technical simplicity of its administration, but also because it offers important advantages; it is not substantially converted into triacylglycerols [19] and does not interact with fructose-biphosphatase [20]. Its intramitochondrial activation [21] also allows to exclude acylCoA-carnitine transfer across the mitochondrial membrane as an eventual site of interaction. Our results indicate that normal hepatic Ca 2÷ loading is not essential to maintain normal basal gluconeogenic responses to pyruvate or to rapidly increasing concentrations of lactate. In contrast, a normal Ca 2+ homeostasis is an obligatory requirement for fatty acid to increase the maximal velocity of gluconeogenesis from lactate but not from supraphysiological concentrations of pyruvate.
Materials and Methods L i v e r perfusion
Present addresses: * The Saik Institute, La Jolla, San Diego, CA 92138-9216, U.S.A. ** Department of Biochemistry, St. George's Hospital Medical School, London, U.K. Correspondence: R. Parriila, Centro de Investigaciones Biol6gicas, Velfizquez 144, 28006 Madrid, Spain.
Livers from fasted male Wistar rats (180-200 g) were perfused with Krebs-Ringer bicarbonate buffer in a flow-through system. Technical details were similar to those previously described [22,23]. When needed, the flow rate was adjusted so that the outflow pO 2 never went below 80 mmHg. Hepatic calcium depletion was
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
216 g ~ wet wt.; P < 0.01). Addition of C a 2+ t o the perfusion fluid of Ca2+-depleted livers resulted in an immediate restoration of the normal expected rates of substrate respiration and gluconeogenesis, indicating that the treatment did not produce any permanent damage to the organ. The kinetics of octanoate oxidation in Ca 2 Ldeficient livers, perfused with no other substrates, were not perturbed by the degree of calcium depletion attained under our experimental conditions. Almost all the oct a n o a t e - i n d u c e d oxygen c o n s u m p t i o n could be accounted for by ketone production (results not shown). This observation implies that normal Ca 2+ homeostasis is not required for fatty acid oxidation to proceed at least up to acetyl CoA.
routinely achieved by perfusion of the organs for 50 min with Ca2+-free buffer before the experiments were started. Control livers were perfused for a similar period with Ca2+-containing buffer. Metabolite analysis Perfusate samples were analyzed immediately after their collection. Metabolites were assayed, spectrophotometrically or fluorimetrically, by enzymatic procedures previously described [24]. Hepatic calcium content was determined by atomic absorption spectrometry in extracts prepared from freeze-clamped biopsies. Rates of pyruvate decarboxylation were determined by measuring CO 2 production from [134C]pyruvate. The protocol was similar as previously described [25]. Materials Most of the reagents were obtained from Sigma (St. Louis, MO, U.S.A.). The enzymes were purchased from Boehringer Mannheim (F.R.G.). [134C]pyruvate, spec. act. 25.9 m C i / m m o l , was obtained from Amersham
Effect of hepatic' Ca : + depletion on the octanoate action on gluconeogenesis from lactate Ca 2+ depletion had no detectable effects on the gluconeogenic response to progressively increasing concentrations of lactate, up to 7 m M (Fig. 1). In contrast, the stimulatory action of octanoate was fully prevented (Fig. 1, right panel). The kinetic changes induced by octanoate [26] (Fig. 1, left panel): reciprocal relationship of VmaX and octanoate availability, and the displacement of the saturation curve to the right as a function of the fatty acid concentration, were also observable in Ca2+-depleted livers (Fig. 1, right panel).
(U.K.). Results
Perfusion of livers with Ca 2+-free buffer for 50 min resulted in a decrease of more than 60% in the total calcium content (from 0.62 + 0.09 to 0.21 + 0.04 ~mol -
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[LACTATE] (raM) Fig. 1. Effects of extracellular Ca 2+ and variable concentrations of octanoate on rates of glucose production from increasing concentrations of lactate. Livers from 48-h-starved rats were perfused for 50 min (right panel) with Ca2+-free buffer to induce Ca 2+ depletion. Control livers (left panel) were perfused for a similar period of time with Ca2+-containing buffer before the experiments were started. When steady-state rates of oxygen consumption were attained in the presence of the indicated concentrations of octanoate, linearly increasing (0.14 p, m o l - m i n -I ) concentrations of sodium lactate were infused up to a concentration of 7 mM. Each plot is the mean of, at least, seven experiments. The results were highly reproducible; for the sake of clarity error bars and intermediate points were omitted.
217 which is a s s u m e d to be a close a p p r o a c h to w h a t its c o n c e n t r a t i o n might be at the o u t e r m i t o c h o n d r i a l m e m b r a n e . F r o m this figure, it is evident that in C a 2÷deficient livers, o c t a n o a t e a v a i l a b i l i t y d i d not p r o d u c e a n y significant changes in VmaX of glucose p r o d u c t i o n . In C a 2 + - d e p l e t e d livers, the smallest c o n c e n t r a t i o n of o c t a n o a t e tested (25/~M), which d i d n o t alter the N A D redox potential, resulted in a significant increase in the c o n c e n t r a t i o n of p y r u v a t e giving h a l f - m a x i m a l gluconeogenic rates (Fig. 3). T h e [pyruvate]0.5 a p p r o a c h e d c o n t r o l values as the c o n c e n t r a t i o n of o c t a n o a t e increased (Fig. 3).
o
20O F> 150 13_ '-~ I00 I-
215
Elsevier BBAMCR 12617
Ca2+-fatty acid interaction in the control of hepatic gluconeogenesis Consuelo Gonzfilez-Manch6n
*, J u a n M e n a y a
* *, M a t i l d e S. A y u s o
and Roberto Parrilla Endocrine Physiology Unit, Centro de lnvestigaciones Biolbgicas (C. S. 1.C.), Madrid (Spain)
(Received 20 March 1989) (Revised manuscript received 12 September 1989)
Key words: Calciumion-fatty acid interaction; Gluconeogenesis; Fatty acid; (Rat liver)
Calcium depletion induced by perfusing livers with calcium-free buffer did not alter the rates of basal glucose production from pyruvate or from increasing concentrations of lactate. However, calcium deficiency selectively prevented the fatty acid-induced stimulation of gluconeogenesis from lactate. This effect is not related to the higher NAD redox potential consistently observed in Ca2+-deficient livers. On the other hand, octanoate was capable of inducing dose-dependent changes in the [pyruvate]0.5 in calcium-depleted livers pedused with lactate, ruling out that low cellular calcium content could perturb the mitochondrial transport of pyruvate. The observation that the effect of calcium deficiency can be overcome by supraphysiological concentrations of pyruvate supports the proposal that stimulation of the maximal capacity of the gluconeogenic pathway by fatty acid relies largely on the tricarboxylic acid cycle activity, restricted in calcium deficiency conditions.
Introduction Extracellular requirement for Ca/+ or the participation of intracellular Ca 2÷ fluxes on the hormonal stimulation of gluconeogenesis has been reported by numerous authors [1-3]. However, the reports on the degree of dependency of hepatic gluconeogenesis on extracellular Ca 2÷ are controversial [4-9]. As far as we know, there is no information available on possible Ca2+-fatty acid interactions in the control of gluconeogenesis, despite the possible involvement of both Ca 2÷ and fatty acid on hormonal responses [3,10,11]. Mutual interactions of Ca 2+ and fatty acids are well documented in the literature: (1) fatty acids have been reported to perturb Ca 2÷ homeostasis [12-15]; (2) similarly, Ca 2+ has been reported to alter fatty acid metabolism [16-18]. On the basis of the above considerations, we found it worthwhile to carry out a study in perfused rat liver, under controlled conditions of substrates and oxygen supply, on
the calcium dependency of the fatty acid stimulatory action of gluconeogenesis. Octanoate was chosen not only for the technical simplicity of its administration, but also because it offers important advantages; it is not substantially converted into triacylglycerols [19] and does not interact with fructose-biphosphatase [20]. Its intramitochondrial activation [21] also allows to exclude acylCoA-carnitine transfer across the mitochondrial membrane as an eventual site of interaction. Our results indicate that normal hepatic Ca 2÷ loading is not essential to maintain normal basal gluconeogenic responses to pyruvate or to rapidly increasing concentrations of lactate. In contrast, a normal Ca 2+ homeostasis is an obligatory requirement for fatty acid to increase the maximal velocity of gluconeogenesis from lactate but not from supraphysiological concentrations of pyruvate.
Materials and Methods L i v e r perfusion
Present addresses: * The Saik Institute, La Jolla, San Diego, CA 92138-9216, U.S.A. ** Department of Biochemistry, St. George's Hospital Medical School, London, U.K. Correspondence: R. Parriila, Centro de Investigaciones Biol6gicas, Velfizquez 144, 28006 Madrid, Spain.
Livers from fasted male Wistar rats (180-200 g) were perfused with Krebs-Ringer bicarbonate buffer in a flow-through system. Technical details were similar to those previously described [22,23]. When needed, the flow rate was adjusted so that the outflow pO 2 never went below 80 mmHg. Hepatic calcium depletion was
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
216 g ~ wet wt.; P < 0.01). Addition of C a 2+ t o the perfusion fluid of Ca2+-depleted livers resulted in an immediate restoration of the normal expected rates of substrate respiration and gluconeogenesis, indicating that the treatment did not produce any permanent damage to the organ. The kinetics of octanoate oxidation in Ca 2 Ldeficient livers, perfused with no other substrates, were not perturbed by the degree of calcium depletion attained under our experimental conditions. Almost all the oct a n o a t e - i n d u c e d oxygen c o n s u m p t i o n could be accounted for by ketone production (results not shown). This observation implies that normal Ca 2+ homeostasis is not required for fatty acid oxidation to proceed at least up to acetyl CoA.
routinely achieved by perfusion of the organs for 50 min with Ca2+-free buffer before the experiments were started. Control livers were perfused for a similar period with Ca2+-containing buffer. Metabolite analysis Perfusate samples were analyzed immediately after their collection. Metabolites were assayed, spectrophotometrically or fluorimetrically, by enzymatic procedures previously described [24]. Hepatic calcium content was determined by atomic absorption spectrometry in extracts prepared from freeze-clamped biopsies. Rates of pyruvate decarboxylation were determined by measuring CO 2 production from [134C]pyruvate. The protocol was similar as previously described [25]. Materials Most of the reagents were obtained from Sigma (St. Louis, MO, U.S.A.). The enzymes were purchased from Boehringer Mannheim (F.R.G.). [134C]pyruvate, spec. act. 25.9 m C i / m m o l , was obtained from Amersham
Effect of hepatic' Ca : + depletion on the octanoate action on gluconeogenesis from lactate Ca 2+ depletion had no detectable effects on the gluconeogenic response to progressively increasing concentrations of lactate, up to 7 m M (Fig. 1). In contrast, the stimulatory action of octanoate was fully prevented (Fig. 1, right panel). The kinetic changes induced by octanoate [26] (Fig. 1, left panel): reciprocal relationship of VmaX and octanoate availability, and the displacement of the saturation curve to the right as a function of the fatty acid concentration, were also observable in Ca2+-depleted livers (Fig. 1, right panel).
(U.K.). Results
Perfusion of livers with Ca 2+-free buffer for 50 min resulted in a decrease of more than 60% in the total calcium content (from 0.62 + 0.09 to 0.21 + 0.04 ~mol -
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m
200
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[LACTATE] (raM) Fig. 1. Effects of extracellular Ca 2+ and variable concentrations of octanoate on rates of glucose production from increasing concentrations of lactate. Livers from 48-h-starved rats were perfused for 50 min (right panel) with Ca2+-free buffer to induce Ca 2+ depletion. Control livers (left panel) were perfused for a similar period of time with Ca2+-containing buffer before the experiments were started. When steady-state rates of oxygen consumption were attained in the presence of the indicated concentrations of octanoate, linearly increasing (0.14 p, m o l - m i n -I ) concentrations of sodium lactate were infused up to a concentration of 7 mM. Each plot is the mean of, at least, seven experiments. The results were highly reproducible; for the sake of clarity error bars and intermediate points were omitted.
217 which is a s s u m e d to be a close a p p r o a c h to w h a t its c o n c e n t r a t i o n might be at the o u t e r m i t o c h o n d r i a l m e m b r a n e . F r o m this figure, it is evident that in C a 2÷deficient livers, o c t a n o a t e a v a i l a b i l i t y d i d not p r o d u c e a n y significant changes in VmaX of glucose p r o d u c t i o n . In C a 2 + - d e p l e t e d livers, the smallest c o n c e n t r a t i o n of o c t a n o a t e tested (25/~M), which d i d n o t alter the N A D redox potential, resulted in a significant increase in the c o n c e n t r a t i o n of p y r u v a t e giving h a l f - m a x i m a l gluconeogenic rates (Fig. 3). T h e [pyruvate]0.5 a p p r o a c h e d c o n t r o l values as the c o n c e n t r a t i o n of o c t a n o a t e increased (Fig. 3).
o
20O F> 150 13_ '-~ I00 I-
