Patricia G. Wallace

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: Further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.

Linear relationships between mitochondrial forces and cytoplasmic flows argue for the organized energy‐coupled nature of cellular metabolism

We have studied rates of formation of glucose, urea and lactate by isolated hepatocytes incubated with a variety of inhibitors of energy transduction. Linear relationships have been found between these metabolic rates and mitochondrial forces (membrane, redox and phosphorylation potentials). The findings are suggestive of extensive enzyme organization within these metabolic pathways.

Evidence that stimulation of gluconeogenesis by fatty acid is mediated through thermodynamic mechanisms

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 of gluconeogenesis 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 synthesis is in part mediated through thermodynamic mechanisms.

Energy-dependent regulation of the steady-state concentrations of the components of the lactate dehydrogenase reaction in liver

It has been known for some years that the components of the lactate dehydrogenase reaction in liver are maintained in a steady-state believed to be close to thermodynamic equilibrium [ 1,2]. The ratio, [lactate]/[pyruvate] has thus been considered to reflect the ‘redox state’ (the ratio of ‘free’ [NAD]/ [NADH]) within the cytoplasmic compartment of the hepatic cell [3]. Analogous investigations of mitochondrial dehydrogenase systems in liver have led to the conclusion that the mitochondrial redox state is -lOO-times more reduced than that of the cytoplasm [3]. It has been pointed out that because of these differences in redox state, the transfer of reducingequivalents from cytoplasmic to mitochondrial NAD(H) pools would be against the electrochemical potential gradient and therefore likely to be energydependent [4,5]. However, few relevant experimental observations using whole cell preparations have been described. The work presented here provides evidence for a direct involvement of energy in the maintenance of the steady-state [lactate]/[pyruvate] ratio in.isolated liver cells.

Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes

The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in isolated hepatocytes of fasted rats. Both lactate removal and glucose synthesis were inhibited by butylmalonate, aminooxyacetate or cycloserine confirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butylmalonate was considerably reduced, yet the transfer of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for butylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulatory effect on uptake of sorbitol, a substrate whose oxidation principally involves the alpha-glycerophosphate shuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potential (delta psi) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate oxidation was strongly inhibited when delta psi fell from 130 to 105 mV whereas O2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by delta psi. In the presence of added ammonium ions the removal of lactate was much less impaired by valinomycin, suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochondrial matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the butylmalonate-sensitive, energy-dependent, malate-aspartate shuttle.

Electrochemical Aspects of Metabolism

The opportunity to write a Chapter on this topic presents something of a challenge. Apart from the prescient suggestions of a few individuals, (1–3) the importance of electrochemical processes in intermediary metabolism has received little recognition until comparatively recently. Even now, the details of biological electrochemical mechanisms remain obscure and it is not feasible to write a comprehensive account without indulging in a substantial degree of speculation. We do not see this as necessarily undesirable. Abundant standard textbook and review articles are available for those who wish to familiarize themselves with conventional concepts concerning metabolic regulation. We see our task as providing an alternative and integrative point of view that, it is to be hoped, will lead to new experimental approaches. The time seems well overdue for a multidisciplinary approach to the understanding of metabolic regulation and we trust that this review may serve towards that end.

THE FATE OF GLUCOSE IN STRAINS S288C AND S173-6B OF THE YEAST SACCHAROMYCES CEREVISIAE

Intracellular metabolic flux has been investigated in two strains of Saccharomyces cerevisiae grown into stationary phase under both glucose‐repressed and glucose‐derepressed conditions. By employing a variety of simple methodologies (manometry, enzymatic analysis and colorimetric analysis) we have been able to identify and quantitate carbon flow from glucose without the need for isotopically labelled substrate. We can account for 88–98% (depending on strain and growth conditions) of the carbon products of glucose metabolism under both glycolytic and oxidative conditions as ethanol (27–40%), carbon dioxide (15–26%), acetate (2–3%), glycerol (5–11%), glycogen (5–13%) and trehalose (9–39%).©1997 John Wiley & Sons, Ltd.

Constraints in the Application of Control Analysis to the Study of Metabolism in Hepatocytes

In recent years it has been argued that quantitative methods are essential for providing new insights into the nature of the living state (Kacser, 1983). A number of mathematical approaches have evolved to meet these demands for quantitative methodologies (Heinrich & Rapoport, 1974; Savageau, 1976; Kacser & Burns, 1979), and of these, control analysis has perhaps attracted the greatest attention. Although its advent has been greeted enthusiastically by many theorists, who see it as an ideal way to quantify the regulatory role of the enzymes of a metabolic pathway, but this analytical method has not yet been widely embraced by experimentalists, because of the difficulties encountered in applying it to complex cellular systems. The work that we shall report in this chapter will illustrate some of these difficulties. The cell system we have used for our studies is the isolated hepatocyte preparation, and we shall present some representative examples from the many hundreds of experiments we have performed in this area.

Ethanol Oxidation in Isolated Hepatocytes

In isolated hepatocytes from fed and starved rats, rates of ethanol oxidation were 1.15 and 0.71 μmol x min−1 x (g wet wt)−1 respectively and were unchanged over the ethanol concentration range 8-96mM. The addition of the competitive inhibitors of alcohol dehydrogenase (ADH), pyrazole and particularly 4-methyl pyrazole (4-MP) abolished the oxidation of 8 mM ethanol and subsequently inhibited oxidation of 96 mM ethanol.

Biological significance of compartmentation of hepatic ethanol oxidation

Intact cell preparations, isolated from the livers of fasted rats and treated with rotenone or antimycin A, retain a limited ability to oxidize long chain fatty acids but not short chain species or other usual substrates. Cells prepared from livers of animals treated with clofibrate to induce peroxisomal proliferation have even greater long chain fatty acid oxidizing capacity in the presence of these inhibitors. We infer that the peroxisomes of intact cells are capable of catabolizing long chain fatty acids by a superoxide-generating pathway. In normal cells or those from clofibrate-treated rats, reducing equivalents generated within peroxisomes compete with those originating in the cytosol for mitochondrial disposition. Because of this, peroxisomal fatty acid metabolism inhibits ethanol oxidation. Peroxisomal oxidations appear to be coupled to conversation of free energy and mitochondrial-peroxisomal relationships are regulated by interaction of free-energy transducing processes. Hence, uncoupling agents release the inhibition of ethanol oxidation induced by long chain fatty acid. When mitochondrial metabolism is impaired, reducing equivalents may flow from mitochondria to peroxisomes for reaction with O2. Thus, there exists a two-way interaction between these organelles. The biological and pathological implications of these relationships for ethanol oxidation and overall energy metabolism are discussed.