View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 23 1, number 1, 19-24 FEB 05732 April 1988 Evidence that stimulation of gluconeogenesis by fatty acid is mediated through thermodynamic mechanisms M.N. Berry, R.B. Gregory, A.R. Grivell, D.C. Henly, J.W. Phillips, P.G. Wallace and G.R. Welch* Department of Medical Biochemistry, School of Medicine, The Flinders University of South Australia, Bedford Park, SA -5042, Australia and *Department of Biological Sciences, University of New Orleans, Lakefront, New Orleans, LA 70148, USA Received 1 February 1988 We have studied the stimulatory effects of palmitate on the rate of glucose synthesis from lactate in isolated hepatocytes. Control of the metabolic flow was achieved by modulating the activity of enolase using graded concentrations of fluoride. Unexpectedly, palmitate stimulated gluconeogenesis even when enolase was rate-limiting. This stimulation was also observed when the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase were modulated using graded concentrations of quinolinate and aminooxyacetate, respectively. Linear force-flow relationships were found between the rate ofgluconeogenesis and indicators of cellular energy status (i.e. mitochondrial membrane and redox potentials and cellular phosphorylation potential). These findings suggest that the fatty acid stimulation of glucose syn- thesis is in part mediated through thermodynamic mechanisms. Gluconeogenesis; Metabolic regulation; Linear force-flow relationship; Irreversible thermodynamics; Electrochemical potential; Isolated hepatocyte 1. INTRODUCTION creasing the activity of glyceraldehyde-3-P dehy- drogenase [5,6]. Other suggested modes of action In a recent study [l] we demonstrated that the of fatty acid include the depression of pyruvate pathway of gluconeogenesis from lactate can be oxidation and consequent promotion of pyruvate under thermodynamic control. We here report carboxylation through inhibition of pyruvate de- work that suggests that the stimulatory effects of hydrogenase [3,4], the stimulation of phospho- fatty acid on gluconeogenesis may operate through enolpyruvate carboxykinase and phosphoglycerate thermodynamic-dependent mechanisms. The abili- kinase activities due to enhancement of ATP syn- ty of fatty acids to stimulate glucose synthesis has thesis [S], and the suppression of a futile cycle be- been known for more than 20 years but their mode tween fructose 6-P and fructose 1,6-P by a citrate- of action still remains uncertain [2-S]. A widely dependent inhibition of phosphofructokinase held idea is that the stimulation is a consequence of [5,7]. All these suggested mechanisms can be activation of pyruvate carboxylase by acetyl CoA categorized as kinetic. That is to say they entail an arising from fatty acid oxidation 14-61. In another increase in the catalytic activity of putative view it is the reducing equivalents derived from this regulatory enzymes of the gluconeogenic pathway, oxidation that promote glucose synthesis by in- either by increasing the availability of substrate or alternatively the level of allosteric effecters. Because evidence for the operation of these pro: Correspondence address: M.N. Berry, Department of Medical Biochemistry, School of Medicine, The Flinders University of cesses in vivo is meagre we have explored the South Australia, Bedford Park, SA 5042, Australia possibility that the fatty acid stimulation of Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/88/$3.50 0 1988 Federation of European Biochemical Societies 19 Volume 231, number 1 FEBS LETTERS April 1988 glucose synthesis may be mediated through ther- Fig.1 illustrates the inhibitory effect of fluoride modynamic rather than kinetic mechanisms. on glucose formation. The degree of depression of gluconeogenesis and accumulation of phospho- enolpyruvate, reflecting inhibition of enolase ac- 2. MATERIALS AND METHODS tivity, increased as a function of fluoride Collagenase and enzymes for metabolite determination were concentration. Fluoride also depressed palmitate- from Boehringer-Mannheim (FRG) as was bovine serum stimulated gluconeogenesis, but for any given con- albumin (fraction V); defatted according to [9]. Palmitate, centration of the inhibitor the rate of glucose syn- rotenone and aminooxyacetic acid were obtained from Sigma (USA). Quinolinic acid was from Matheson Coleman and Bell thesis was always greater in the presence of pal- Inc. (USA) and sodium fluoride from Ajax Chemicals Limited. mitate than in its absence. Nevertheless, palmitate [r4C]Methyitriphenylphosphonium iodide was purchased from had no significant effect on the steady-state con- Amersham (England). Palmitate (8 mM) was prepared in centrations of the components of the enolase- isotonic saline containing 9% (w/v) bovine serum albumin. Water-insoluble compounds were dissolved in dimethylsul- catalysed reaction (fig.l), tending to discount any phoxide. direct action of the fatty acid on that step. We also Isolated liver cells from male hooded Wistar rats (250-280 g observed than when the rate of glucose synthesis in body wt), starved for 24 h to deplete liver glycogen, were the presence of each concentration of fluoride was prepared and incubated at 37°C for 35 min in a bicarbonate- expressed as a percentage of the rat observed in the buffered medium as described [lo, 111. Unless stated otherwise, the added substrates were a mixture of lactate (10 mM) and absence of inhibitor, rather than in absolute units, pyruvate (1 mM). the degree of inhibition of gluconeogenesis for any Metabolites were measured by standard enzymic techniques given fluoride concentration was the same whether as in [12] on neutralized perchloric acid extracts of the in- or not palmitate was present (fig.2). This observa- cubated cells and medium [ 111. The level of 2-phosphoglycerate was derived from measurement of 3-phosphoglycerate on the tion implies that the amount of enolase-inhibitor basis of studies which consistently showed a constant relation- complex formed by exposure of the hepatocytes to ship between the concentrations of these metabolites. The a particular concentration of fluoride was unaf- metabolite assays were performed in a Cobas FARA centrifugal fected by the presence of palmitate. analyser (Roche Diagnostics, Switzerland) and the data These findings are not readily explicable in terms transferred to a PDP 1 l/73 computer (DEC, USA) for subse- quent processing. All fitted, straight-line relationships were of current concepts that account for metabolic calculated by least-squares linear regression analysis of un- regulation entirely on the basis of kinetic mechan- transformed data. Each plot is a representative example from isms [ 16,18,19]. For many years a cardinal element at least three related experiments. of these concepts has been the importance of a Mitochondrial membrane, redox and cellular phosphoryla- ‘rate-limiting step’ for control of flux through a tion potentials were determined as previously described [ 1,131. Values for cytoplasmic redox-potential (&c) defined as the half- metabolic pathway. It has been argued that, for a cell reaction of free [NAD+]/freemADH] (Es = E,’ + given pathway, the enzyme with the lowest (RT/nF)ln[pyruvate]/[lactate]) were obtained by measurement catalytic activity will in large measure determine of the concentrations of pyruvate and lactate on the assumption the overall rate of flux [la]. In the experiments of an E,’ at 37°C of -215 mV [14]. described here, however, an artifactual rate- limiting step was created by exposing hepatocytes to fluoride, thereby reducing the activity of enolase 3. RESULTS AND DISCUSSION (which is normally not rate-limiting) below that of In this study of the effects of palmitate on the physiologically limiting regulatory enzyme ac- gluconeogenesis the rate of the metabolic flow tivity. Evidence for the rate-limiting nature of the from lactate to glucose has been modulated using enolase reaction was provided by the incremental graded concentrations of fluoride, an inhibitor of accumulation of phosphoenolpyruvate in response enolase [ 151 which is not regarded as a rate-limiting to increasing fluoride concentration. Paradoxical- enzyme of the pathway under physiological condi- ly, paImitate though stimulating glucose synthesis, tions [16]. Enolase was chosen for this study had no effect on this accumulation. We concluded because it is believed to catalyse a ‘near- from this that palmitate was not exerting a specific equilibrium’ reaction [16,17], and is not known to stimulatory effect on the enolase reaction, a con- be affected by fatty acid at concentrations which clusion in keeping with current views concerning stimulate gluconeogenesis. the mechanisms whereby gluconeogenesis is stimu- 20 Volume 23 1, number 1 FEBS LETTERS April 1988 1.5 % 2.5 100 2.0 g 15 60 . % I 3 60 . 2 . 1: z 2 ” 40 . I 20 0 L 0 2 4 6 8 10 Fluoride concentration hM1 Fig. 1. Inhibition of gluconeogenesis from lactate by fluoride. Fig.2. Percentage inhibition of ghrconeogenesis from lactate by Hepatocytes were incubated for 35 min at 37OC with 10 mM lac- fluoride. The rates of glucose synthesis shown in fig.1 are plot- tate and 1 mM pyruvate in the absence ( l) or presence (A) of ted as percentages with the uninhibited rate of glucose forma- 2 mM palmitate. Fluoride concentrations in the range l-10 mM tion being taken as 100%. Conditions and symbols are as for were included