Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria

We report here initial studies on phosphoenolpyruvate metabolism in coupled mitochondria isolated from Jerusalem artichoke tubers. It was found that:

(1)

phosphoenolpyruvate can be metabolized by Jerusalem artichoke mitochondria by virtue of the presence of the mitochondrial pyruvate kinase, shown both immunologically and functionally, located in the inner mitochondrial compartments and distinct from the cytosolic pyruvate kinase as shown by the different pH and inhibition profiles.

(2)

Jerusalem artichoke mitochondria can take up externally added phosphoenolpyruvate in a proton compensated manner, in a carrier-mediated process which was investigated by measuring fluorimetrically the oxidation of intramitochondrial pyridine nucleotide which occurs as a result of phosphoenolpyruvate uptake and alternative oxidase activation.

(3)

The addition of phosphoenolpyruvate causes pyruvate and ATP production, as monitored via HPLC, with their efflux into the extramitochondrial phase investigated fluorimetrically. Such an efflux occurs via the putative phosphoenolpyruvate/pyruvate and phosphoenolpyruvate/ATP antiporters, which differ from each other and from the pyruvate and the adenine nucleotide carriers, in the light of the different sensitivity to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of both pyruvate and ATP. The appearance of citrate and oxaloacetate outside mitochondria was also found as a result of phosphoenolpyruvate addition.

     

Relationship of phosphoenolpyruvate transport, acyl coenzyme A inhibition of adenine nucleotide translocase and calcium ion efflux in guinea pig heart mitochondria.

The transport of phosphoenolpyruvate by the adenine nucleotide translocase system of heart mitochondria may be directly involved in the mechanism of phosphoenolpyruvate-induced calcium ion efflux. In contrast to liver mitochondria, the transport of phosphoenolpyruvate via the tricarboxylate carrier system is low or absent in heart mitochondria. The translocation of phosphoenolpyruvate which catalyzed adenine nucleotide and calcium efflux from heart mitochondria was inhibited by palmitoyl-CoA as well as atractylate and ATP. These results suggest that phosphoenolpyruvate, which is preferentially transported on the tricarboxylate carrier of liver mitochondria, is transported primarily via the adenine nucleotide translocase system in heart mitochondria. As a result of its inward transport, phosphoenolpyruvate is able to catalyze calcium ion as well as adenine nucleotide efflux from the mitochondrial matrix. Although not yet proven, either or both phosphoenolpyruvate and long chain acyl-CoA esters may act as natural physiological effectors in the regulation and distribution of intracellular calcium.     

Jerusalem artichoke mitochondria can export reducing equivalents in the form of malate as a result of D-lactate uptake and metabolism

We found that as a result of d-lactate uptake and metabolism by Jerusalem artichoke mitochondria, reducing equivalents were exported from the mitochondrial matrix to the exterior in the form of malate. The rate of malate efflux, as measured photometrically using NADP+ and malic enzyme, depended on the rate of transport across the mitochondrial membrane. It showed saturation characteristics (K(m) = 5 mM; V(max) = 9 nmol/min mg of mitochondrial protein) and was inhibited by non-penetrant compounds. We conclude that reducing equivalent export from mitochondria is due to the occurrence of a putative d-lactate/malate antiporter which differs from other mitochondrial carriers, as shown by the different inhibitor sensitivity.     

The effect of phosphoenolypyruvate on calcium transport by mitochondria

Phosphoenolpyruvate was found to inhibit net uptake of Ca²⁺ by rat heart and liver mitochondria. The main action of phosphoenolpyruvate is to increase the rate of efflux of mitochondrial Ca²⁺. The effect of phosphoenolpyruvate on mitochondrial Ca²⁺ transport is antagonized by ATP and by atractylate and is observed when mitochondria are respiring in the presence of NAD-linked subtrates such as glutamate and pyruvate plus malate. In liver mitochondria phosphoenolpyruvate is also effective in the presence of succinate but not when rotenone is added. Glycolytic intermdiates other than phosphoenolpyruvate had little effect on mitochondrial Ca²⁺ transport.     

Stimulation of tumor-cell respiration by inhibitors of pyruvate kinase.

In a model system consisting of highly coupled rat liver mitochondria respiring in the presence of substrate, pyruvate kinase, phosphoenolpyruvate, ATP, hexokinase and glucose, the increase in the mitochondrial concentration results in a progressive decrease in the activity of pyruvate kinase. These results are in accord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with mitochondrial oxidative phosphorylation for the available ADP. The addition of adequate amounts of the amino acids, cysteine, alanine and phenylalanine, known as inhibitors of pyruvate kinase, to living Ehrlich ascites tumor cell suspensions results in a stimulation of the respiratory rate and in a decrease of the glycolytic rate of the cells. Concomitant with these changes, there is an accumulation of intracellular phosphoenolpyruvate and ADP, and a decrease in pyruvate and ATP. These results provide additional evidence for paying attention to pyruvate kinase as another key enzyme whose properties and activities may be major determinants for the control of glycolysis and the Crabtree and Pasteur effects of tumor cells.     

Relationship of the intra- and extramitochondrial adenine nucleotide ratios during synthesis of phosphoenolpyruvate using extramitochondrial ATP

Mitochondria prepared from the livers of guinea pig, chicken, and pigeon all actively synthesize phosphoenolpyruvate from oxalacetate and GTP, utilizing phosphoenolpyruvate carboxykinase. It was previously shown (Wilson, D. F., Erecińska, M., and Schramm, V. L. (1983). J. Biol. Chem. 258, 10464-10473) that phosphoenolpyruvate carboxykinase is freely reversible and that, in conjunction with nucleoside diphosphate kinase and malate dehydrogenase, which are also present in the mitochondria, it can be used to measure the intramitochondrial [ATPfree]/[ADPfree]. In this study, synthesis of phosphoenolpyruvate by guinea pig liver mitochondria was studied under conditions for which the only source of GTP was extramitochondrial ATP via adenine nucleotide translocase and nucleoside diphosphate kinase (the mitochondria were treated with rotenone, oligomycin, uncoupler, and fluorocitrate). When the extramitochondrial [ATP]/[ADP] was greater than the intramitochondrial [ATPfree]/[ADPfree] calculated from the phosphoenolpyruvate carboxykinase reaction, there was net synthesis of phosphoenolpyruvate, but when it was less, there was net disappearance of phosphoenolpyruvate. Thus, the intramitochondrial [ATPfree]/[ADPfree] was equal to the extramitochondrial value at the point of reversal of the phosphoenolpyruvate carboxykinase reaction. This equality of the intra- and extramitochondrial adenine nucleotide ratios occurred with a measured mitochondrial membrane potential of approximately -36 mV, whereas in the previous experiments, equality was observed for conditions in which the measured membrane potential was -111 to -125 mV. Thus, adenine nucleotide translocation was not dependent on the transmembrane electrical potential and must, therefore, have occurred by electroneutral exchange.     

Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement

In this work we looked at whether and how mitochondria isolated from Saccharomyces cerevisiae (SCM) oxidize d-lactate. We found that: (1). externally added d-lactate causes oxygen uptake by SCM with P/O ratio equal to 1.5; in the presence of antimycin A (AA), P/O ratio was 1.8, differently in the presence of the non-penetrant alpha-cyanocinnamate (alpha-CCN-) no P/O ratio could be measured. Consistently, mitochondrial electrical membrane potential (deltapsi) generation was found, due to externally added d-lactate in the presence of antimycin A, but not of alpha-CCN-. (2). SCM oxidize d-lactate in two different manners: (i). via inner membrane d-lactate dehydrogenase which leads to d-lactate oxidation without driving deltapsi generation and ATP synthesis and (ii). via the matrix d-lactate dehydrogenase, which drives deltapsi generation and ATP synthesis by using taken up d-lactate. (3). Pyruvate newly synthesised in the mitochondrial matrix is exported via the novel d-lactate/pyruvate antiporter. d-Lactate/pyruvate antiport proved to regulate the rate of pyruvate efflux in vitro. (4). The existence of the d-lactate/H+ symporter is also proposed as shown by mitochondrial swelling. The d-lactate carriers and d-lactate dehydrogenases could account for the removal of the toxic methylglyoxal from cytosol, as well as for the d-lactate-dependent gluconeogenesis.     

Transport and metabolism of D-lactate in Jerusalem artichoke mitochondria

We report here initial studies on D-lactate metabolism in Jerusalem artichoke. It was found that: 1) D-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-D-lactoyl-glutathione. 2) Externally added D-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) D-lactate was metabolized inside mitochondria by a flavoprotein, a putative D-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added D-lactate by means of a D-lactate/H(+) symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial D-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.     

The role of adenylate kinase in dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.

To investigate the existence of dynamic adenine nucleotide (AdN) compartment in the mitochondrial intermembrane space, we used reconstituted systems consisting of (i) functional intact liver and heart mitochondria and (ii) pyruvate kinase plus phosphoenolpyruvate, both competing for ADP either formed in the intermembrane space by adenylate kinase or added directly into, or regenerated by ATPase within, the extramitochondrial space. It is shown that ADP formation in the mitochondrial intermembrane space is a prerequisite for a dominating oxidative phosphorylation in reconstituted systems, suggesting dynamic ADP compartmentation in that space.     

Transport and metabolism of d-lactate in Jerusalem artichoke mitochondria

We report here initial studies on d-lactate metabolism in Jerusalem artichoke. It was found that: 1) d-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-d-lactoyl-glutathione. 2) Externally added d-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) d-lactate was metabolized inside mitochondria by a flavoprotein, a putative d-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added d-lactate by means of a d-lactate/H⁺ symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial d-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.     

Pyruvate kinase in pig liver mitochondria

The existence of the pyruvate kinase (PK) in pig liver mitochondria was shown by monitoring photometrically the PK reaction in solubilised mitochondria with either phosphoenolpyruvate (PEP) or ADP used as a substrate. In distinction with the cytosolic isoenzyme, the mitochondrial PK showed a sigmoidal dependence on either PEP or ADP concentrations. The occurrence of the mitochondrial PK was confirmed by immunological analysis. Titration with digitonin showed that mPK is restricted to the matrix. PEP addition to mitochondria resulted in reduction of the intramitochondrial NAD(P)⁺ inhibited by either the non-penetrant thiol reagent mersalyl or by arsenite, an inhibitor of the pyruvate dehydrogenase complex. Citrate/oxaloacetate appearance outside mitochondria also occurred as result of PEP addition to PLM. Taken together these findings support a role for PEP itself in triggering fatty acid synthesis via its mitochondrial metabolism.     

The lack of direct coupling between ATP-ADP translocase and creatine phosphokinase in isolated rabbit heart mitochondria

The formation of creatine phosphate by isolated rabbit heart mitochondria in the presence of creatine, α-ketoglutarate, ATP, and inorganic phosphate was studied. Creatine phosphate formation was inhibited by oligomycin. This was most probably due to increased concentration of ADP favoring the reverse reaction (formation of creatine and ATP from phosphocreatine and ADP). The inhibitory effect of oligomycin disappeared in the presence of phosphoenolpyruvate and pyruvate kinase. The results do not indicate any direct coupling between mitochondrial creatine phosphokinase and ATP-ADP translocase as has been suggested for rat heart mitochondria.     

Direct measurement of energy fluxes from mitochondria into cytoplasm in permeabilized cardiac cells <Emphasis Type="Italic">in situ:</Emphasis> some evidence for mitochondrial interactosome

The aim of this study was to measure energy fluxes from mitochondria in isolated permeabilized cardiomyocytes. Respiration of permeabilized cardiomyocytes and mitochondrial membrane potential were measured in presence of MgATP, pyruvate kinase – phosphoenolpyruvate and creatine. ATP and phosphocreatine concentrations in medium surrounding cardiomyocytes were determined. While ATP concentration did not change in time, mitochondria effectively produced phosphocreatine (PCr) with PCr/O₂ ratio equal to 5.68 ± 0.14. Addition of heterodimeric tubulin to isolated mitochondria was found to increase apparent Km for exogenous ADP from 11 ± 2 μM to 330 ± 47 μM, but creatine again decreased it to 23 ± 6 μM. These results show directly that under physiological conditions the major energy carrier from mitochondria into cytoplasm is PCr, produced by mitochondrial creatine kinase (MtCK), which functional coupling to adenine nucleotide translocase is enhanced by selective limitation of permeability of mitochondrial outer membrane within supercomplex ATP Synthasome-MtCK-VDAC-tubulin, Mitochondrial Interactosome.     

In vitro 14C-leucine incorporation into nonsynaptic and synaptic rat brain mitochondria isolated by Ficoll gradients

The in vitro incorporation of 14C-leucine by nonsynaptic and synaptic rat brain mitochondria purified by means of discontinuous Ficoll gradients has been characterised. The incorporation was linear for the first 45 min for both populations. Synaptic mitochondria showed a higher rate of incorporation than the nonsynaptic mitochondria at high concentrations of leucine. The incorporation was more effective in the presence of Mg2+ and inhibited by dinitrophenol. The incorporation was sensitive to chloramphenicol and insensitive to cycloheximide. Bacterial contamination was in any case lower than 1,000 colonies per ml after the incubation period. The incorporation was carried out in the presence of either an external ATP-generating system consisting of ATP, phosphoenolpyruvate and pyruvate kinase or with mitochondria respiring with oxidisable substrates plus ADP (state III). The rates obtained for incorporation in this state III were higher for all the substrates assayed (succinate, pyruvate and glutamate) than in the presence of exogenous ATP. The highest rate obtained was found when glutamate was the respiratory substrate. No significant metabolic oxidation of leucine occurs in either synaptic or nonsynaptic mitochondria in the presence of exogenous ATP. Glutamate did not increase leucine uptake in any mitochondrial populations.     

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

Characterization of phosphate efflux pathways in rat liver mitochondria.

ATP hydrolysis catalysed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, we have attempted to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited, Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). In conclusion, results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalysing Pi influx. Alternatively, a separate Pi efflux carrier may exist.     

Inhibition of carbon dioxide fixation by lead acetate in rat liver mitochondria.

These studies were undertaken to determine the mechanism by which intravenously administered lead salts inhibit hepatic gluconeogenesis. Within 1 h after the intravenous administration of lead acetate (10 mg), there is 97% inhibition of CO2 fixation in isolated rat liver mitochondria. This effect is concentration-dependent. The induction of phosphoenolpyruvate carboxykinase activity observed with starvation was also inhibited by intravenously administered lead acetate, but the activities of pyruvate kinase, glucose 6-phosphate dehydrogenase and pyruvate carboxylase were unaffected, as was the oxidation of palmitate and palmitoyl-CoA by mitochondria from Pb2+-treated animals. The addition of reduced glutathione to mitochondria from Pb2+-treated animals had no effect on the inhibited CO2 fixation. ATP concentrations in mitochondria from Pb2+-treated animals are decreased and the dose-response relationships for the effect of Pb2+ on CO2 fixation and ATP concentrations correspond. We conclude that the decrease in mitochondrial ATP in Pb2+-treated animals is probably responsible for the marked inhibition ov CO2 fixation, and hence the impairment of gluconeogenesis from alanine, lactate and pyruvate observed by others.     

Radioisotopic assay of femtomole quantities of total adenine nucleotides,ATP plus ADP,and AMP

AMP is converted to ATP by incubating overnight with pyruvate kinase, phosphoenolpyruvate and adenylate kinase in th prensence of endogenous ATP (ADP) as primer. In a subsequent incubation in the presence of pyruvate kinase, phosphoenolpyruvate, radioactive glucose and hexokinase. ATP and ADP are estimated together by coupling their recycling to the formation of glucose 6-phosphate. The latter is separated by precipitation using 76% (v/v) acetone for radioactivity measurement in the same Eppendorf tube. The sensitivity of these simple procedures matches or exceeds those of luciferase methods of nucleotide determination.     

Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP

Previous studies from this laboratory have shown that mitochondrial bound hexokinase is markedly elevated in highly glycolytic hepatoma cells (Parry, D. M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). A pore-forming protein, porin, within the outer membrane appears to comprise at least part of the receptor site (Nakashima, R.A., Mangan, P.S., Colombini, M., and Pedersen, P.L. (1986). Biochemistry 25, 1015-1021). In studies reported here experiments were carried out to assess the functional significance of mitochondrial bound tumor hexokinase. Two approaches were used to determine whether the bound enzyme has preferred access to mitochondrially generated ATP relative to cytosolic ATP. The first approach compared the time course of glucose 6-phosphate formation by AS-30D hepatoma mitochondria under conditions where ATP was regenerated endogenously via oxidative phosphorylation or exogenously by added pyruvate kinase and phosphoenolpyruvate. The second approach involved the measurement of the specific radioactivity of glucose 6-phosphate formed following the addition of [gamma-32P]ATP to either phosphorylating or nonphosphorylating AS-30D mitochondria. Both approaches provided results which show that the source of ATP for bound hexokinase is derived preferentially from the ATP synthase residing within the inner mitochondrial membrane compartment rather than from the medium (i.e. from the cytosolic compartment). These results provide the first direct demonstration that the exceptionally high level of hexokinase bound to mitochondria of highly glycolytic tumor cells has preferred access to mitochondrially generated ATP, a finding that may have rather profound metabolic significance for such tumors.     

Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: Implications for lipogenesis

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricar☐ylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or a means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate car☐ykinasein vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA car☐ylase, a carbon source via ATP:citrate lyase and NADPH via NADP: malate dehydrogenase or NADP: isocitrate dehydrogenase.