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.     

Energy sources for glutamate neurotransmission in the retina: absence of the aspartate/glutamate carrier produces reliance on glycolysis in glia

The mitochondrial transporter, the aspartate/glutamate carrier (AGC), is a necessary component of the malate/aspartate cycle, which promotes the transfer into mitochondria of reducing equivalents generated in the cytosol during glycolysis. Without transfer of cytosolic reducing equivalents into mitochondria, neither glucose nor lactate can be completely oxidized. In the present study, immunohistochemistry was used to demonstrate the absence of AGC from retinal glia (Müller cells), but its presence in neurons and photoreceptor cells. To determine the influence of the absence of AGC on sources of ATP for glutamate neurotransmission, neurotransmission was estimated in both light- and dark-adapted retinas by measuring flux through the glutamate/glutamine cycle and the effect of light on ATP-generating reactions. Neurotransmission was 80% faster in the dark as expected, because photoreceptors become depolarized in the dark and this depolarization induces release of excitatory glutamate neurotransmitter. Oxidation of [U-14C]glucose, [1-14C]lactate, and [1-14C]pyruvate in light- and dark-adapted excised retinas was estimated by collecting 14CO2. Neither glucose nor lactate oxidation that require participation of the malate/aspartate shuttle increased in the dark, but pyruvate oxidation that does not require the malate/aspartate shuttle increased to 36% in the dark. Aerobic glycolysis was estimated by measuring the rate of lactate appearance. Glycolysis was 37% faster in the dark. It appears that in the retina, ATP consumed during glutamatergic neurotransmission is replenished by ATP generated glycolytically within the retinal Müller cells and that oxidation of glucose within the Müller cells does not occur or occurs only slowly.     

Aminooxyacetate inhibits gluconeogenesis by isolated chicken hepatocytes

Although the pathway for glucose synthesis from lactate in avian liver is not thought to involve transamination steps, inhibitors of transamination (aminooxyacetate and L-2-amino-4-methoxy-trans-3-butenoic acid) block lactate gluconeogenesis by isolated chicken hepatocytes. Inhibition of glucose synthesis from lactate by aminooxyacetate is accompanied by a large increase in the lactate-to-pyruvate ratio. Oleate largely relieves inhibition by aminooxyacetate and lowers the lactate-to-pyruvate ratio. In parallel studies with rat hepatocytes, oleate did not overcome aminooxyacetate inhibition of glucose synthesis. The ratios of lactate used to glucose formed were greater than 2 with both rat and chicken hepatocytes, were increased by aminooxyacetate, and were restored toward 2 by oleate. Thus, in the absence of oleate, lactate is oxidized to provide the energy needed to meet the metabolic demand of chicken hepatocytes. Excess cytosolic reducing equivalents generated by the oxidation of lactate to pyruvate are transferred from the cytosol to the mitosol by the malate-aspartate shuttle. Aminooxyacetate inhibits the shuttle and, consequently, glucose synthesis for want of pyruvate.     

Control of reversible intracellular transfer of reducing potential.

Isolated rat liver mitochondria were incubated in the presence of a reconstituted malate-aspartate shuttle under carboxylating conditions in the presence of glutamate, octanoyl-carnitine and pyruvate, or a preset lactate/pyruvate ratio. The respiration and attendant energy state were varied with soluble F1-ATPase. Under these conditions reducing equivalents are exported due to pyruvate carboxylation. This was shown by lactate production from pyruvate and by a substantial increase in the lactate/pyruvate ratio. This led to a competition between malate export and energy-driven malate cycling via the malate-aspartate shuttle, resulting in a lowered redox segregation of the NAD systems between the mitochondrial and extramitochondrial spaces. If pyruvate carboxylation was blocked, this egress of reducing equivalents was also blocked, leading to an elevated value of redox segregation, delta G(redox) (in kJ) = -5.7 log(NAD+/NADHout)/(NAD+/NADHin) being then equal to approximately one-half of the membrane potential, in accordance with electrogenic glutamate/aspartate exchange. Reconstitution of malate-pyruvate cycling led to a further kinetic decrease in the original malate-aspartate shuttle-driven value of delta G(redox). Therefore, the value of segregation of reducing potential between mitochondria and cytosol caused by glutamate/aspartate exchange can be diminished kinetically by processes exporting reducing equivalents from mitochondria, such as pyruvate carboxylation and pyruvate cycling.     

The influence of ammonium and calcium lons on gluconeogenesis in isolated rat hepatocytes and their response to glucagon and epinephrine

The use of n-butylmalonate as an inhibitor of malate transport from mitochondria and of aminooxyacetate as an inhibitor of glutamate-aspartate transaminase indicated that rat liver hepatocytes employ the aspartate shuttle for gluconeogenesis from lactate which supplies reducing equivalents to the cytosolic NAD system. In contrast, malate is transported from mitochondria to cytosol for gluconeogenesis from pyruvate. This conclusion is corroborated by the finding that the addition of ammonium ions enhances gluconeogenesis from lactate but inhibits glucose formation from pyruvate. In hepatocytes, glucagon and epinephrine have relatively little effect on glucose synthesis from lactate. Ammonium ions permit both of these hormones to exert their usual stimulation of gluconeogenesis from lactate.Calcium ions (1.3 mm) enhance gluconeogenesis from lactate and from lactatepyruvate mixtures (10:1). The stimulatory effects of Ca²⁺ and NH₄⁺ are additive and, when lactate is the substrate, the rates of gluconeogenesis achieved are so high as to preclude further stimulation by glucagon.     

Nitrogen-13 flux from L-[13N]glutamate in the isolated rabbit heart: effect of substrates and transaminase inhibition

Kinetic and biochemical parameters of nitrogen-13 flux from L-[13N]glutamate in myocardium were examined. Tissue radioactivity kinetics and chemical analyses were determined after bolus injection of L-[13N]glutamate into isolated arterially perfused interventricular septa under various metabolic states, which included addition of lactate, pyruvate, aminooxyacetate (a transaminase inhibitor), or a combination of aminooxyacetate and pyruvate to the standard perfusate containing insulin and glucose. Chemical analysis of tissue and effluent at 6 min allowed determination of the composition of the slow third kinetic component of the time-activity curves. 13N-labeled aspartate, alanine and glutamate accounted for more than 80% of the tissue nitrogen-13 under the experimental conditions used. Specific activities for these amino acids were constant, but not identical to each other, from 6 through 15 min after administration of L-[13N]glutamate. Little labeled ammonia (1.9%) and glutamine (4.7%) were produced, indicating limited accessibility of exogenous glutamate to catabolic mitochondrial glutamate dehydrogenase and glutamine synthetase, under control conditions. Lactate and pyruvate additions did not affect tissue amino acid specific activities. Aminooxyacetate suppressed formation of 13N-labeled alanine and aspartate and increased production of L-[13N]glutamine and [13N]ammonia. Formation of [13N]ammonia was, however, substantially decreased when aminooxyacetate was used in the presence of exogenous pyruvate. The data support a model for glutamate compartmentation in myocardium not affected by increasing the velocity of enzymatic reactions through increased substrate (i.e., lactate or pyruvate) concentrations but which can be altered by competitive inhibition of transaminases (via aminooxyacetate) making exogenous glutamate more available to other compartments.     

Inhibition of gluconeogenesis and lactate formation from pyruvate by N6, O2'-dibutyryl adenosine 3':5'-monophosphate.

N6,O2-Dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) inhibits gluconeogenesis and lactate formation but increases ketogenesis by isolated liver cells incubated with high concentrations of pyruvate. The inhibitory effects can not be explained on the basis of an inhibition of the pyruvate dehydrogenase complex nor by a change in the NAD+ oxidation-reduction potential of the mitochondrial compartment. Both oleate and 3-hydroxybutyrate substantially increase the rates of gluconeogenesis and lactate formation from pyruvate but do not overcome the inhibition caused by Bt2cAMP. A decreased effectiveness of pyruvate kinase is proposed to account for the inhibition of both gluconeogenesis and lactate formation by Bt2cAMP. This enzyme catalyzes a step required in the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasm and participates in the formation of glucose and lactate from pyruvate by the overall reaction: 2 pyruvate- + 2 NADHmito + 4 ATP4- + 4 H2O leads to 1/2 glucose + lactate- + 2 NAD+ mito + 4 ADP3- + 4 HPO4(2)- + H+. Inhibition of pyruvate kinase promotes gluconeogenesis with most substrates but inhibits gluconeogenesis from pyruvate for want of cytoplasmic reducing equivalents.     

Evidence for the oxidation of glycolytic NADH by the malate-aspartate shuttle in Ehrlich ascites tumor cells.

Actively glycolyzing Ehrlich-Lettré ascites tumor cells have been tested for their ability to reoxidize into the mitochondria reduced nicotinamide adenine dinucleotide by the malate-aspartate shuttle. The aspartate transaminase inhibitor aminooxyacetate has been used as a tool for evaluating it. Measurements of the free cytosolic $\text{(NADH)}\text{(}\text{NAD}{}^{\mathrm{+}}\text{)}$ ratio indicate that it increases gradually in the inhibitor-treated cells up to a value about ninefold higher than in the controls after 30 min of glycolytic activity. Fructose 1, 6-diphosphate and dihydroxyacetone phosphate reach a steady-state level after 5 min of incubation in the untreated cells, whereas they accumulate in large amounts in the inhibited cells. Correspondingly, a decrease in 3-phosphoglycerate concentration is observed. On the other hand, the rate of glucose utilization is affected slightly only during long observation times. From these results it may be established that in Ehrlich ascites cells reducing equivalents generated in the cytosol during aerobic glycolysis strongly influence the NAD redox state when their intramitochondrial translocation is prevented by the inactivation of the malate-aspartate shuttle.     

Nitrogen-13 flux from L-[13N]glutamate in the isolated rabbit heart: effect of substrates and transminase inhibition

Kinetic and biochemical parameters of nitrogen-13 flux from L-[¹³N]-glutamate in myocardium were examined. Tissue radioactivity kinetics and chemical analyses were determined after bolus injection of L-[¹³N]glutamate into isolated arterially perfused interventricular septa under various metabolic states, which included addition of lactate, pyruvate, aminooxyacetate (a transminase inhibitor), or a combination of aminooxyacetate and pyruvate to the standard perfusate containing insulin and glucose. Chemical analysis of tissue and effluent at 6 min allowed determination of the composition of the slow third kind kinetic component of the time-activity curves. ¹³N-labeled aspartate, alanine and glutamate accounted for more than 80% of the tissue nitrogen-13 under the experimental conditions used. Specific activities for these amino acids were constant, but not identical to each other, from 6 through 15 min after administration of L-[¹³N]glutamate. Little labeled ammonia (1.9%) and glutamine (4.7%) were produced, indicating limited accessibility of exogenous glutamate to catabolic mitochondrial glutamate dehydrogenase and glutamine synthetase, under control conditions. Lactate and pyruvate additions did not affect tissue amino acid specific activities. Aminooxyacetate suppressed formation of ¹³N-labeled alanine and aspartate and increased production of L-[¹³N]glutamine and [¹³N]ammonia. Formation of [¹³N]ammonia was, however, substantially decreased when aminooxyacetate was used in the presence of exogenous pyruvate. The data support a model for glutamate compartmentation in myocardium not affected by increasing the velocity of enzymatic reactions through increased substrate (i.e., lactate or pyruvate) concentrations but which can be altered by competitive inhibition of transaminases (via aminooxyacetate) making exogenous glutamate more available to other compartments.     

Anaerobic energy metabolism in isolated adductor muscle of the sea musselMytilus edulis L.

Summary Energy metabolism during anaerobiosis was investigated in the isolated posterior adductor muscle of the sea mussel. Metabolism appeared to be similar to that observed in the intact organism. Glycogen and aspartate are simultaneously utilized and levels of alanine, succinate, strombine and octopine increase. The sum of the adenylates remains constant, whereas phosphoarginine is dephosphorylated. The influence of iodoacetate, aminooxyacetate and hadacidin, inhibitors of glycolysis, transamination and purine nucleotide cycle, respectively, on the utilization of substrates and the interconversion of metabolites has been studied. The results suggest that the purine nucleotide cycle is not involved in the inverse correlation of changes in levels of aspartate and alanine, but that this exclusively depends on transamination reactions. Pyruvate (required for alanine formation) arises about equally from glycolysis and aspartate decarboxylation. When the utilization of aspartate is blocked by aminooxyacetate, glycolytically formed pyruvate is metabolized by reductive condensation with glycine and arginine to yield strombine and octopine. Under this condition phosphoarginine is dephosphorylated at a faster rate in order to maintain the energy status of the cell.Abbreviations Ac acetate - AEC Atkinson energy charge - Ala alanine - Asp aspartate - Glu glutamate - Lac lactate - Mal malate - Oct octopine - PA phosphoarginine - Prop propionate - Pyr pyruvate - Str strombine - Suc succinate     

Functional inhibition of cytosolic and mitochondrial aspartate aminotransferase by l-2-amino-4-methoxy-trans-3-butenoic acid in isolated rat hepatocytes and mitochondria

The transaminase inhibitor l-2-amino-4-methoxy-trans-3-butenoic acid (AMB) decreased aspartate aminotransferase activity by approximately two-thirds in isolated rat liver mitohondria incubated with succinate, ammonia, and ornithine. Aspartate production by the mitochondria was unaffected over the 30-min incubation period, indicating that mitochondrial aspartate aminotransferase activity is normally far in excess of that required for maximal rates of aspartate production. In rat hepatocytes incubated with lactate, ammonia, and ornithine the inhibition of both the cytosolic and mitochondrial isozymes of aspartate aminotransferase by AMB was partially blocked by the presence of ammonia and ornithine. When pyruvate was substituted for lactate as a carbon source with isolated hepatocytes, the presence of ammonia and ornithine blocked the inhibition by AMB of the mitochondrial but not the cytosolic isozyme of aspartate aminotransferase. Urea formation by cells incubated with lactate, ammonia, and ornithine was unaffected by AMB unless the cells were preincubated with the inhibitor prior to the addition of substrates. However, urea formation by cells incubated in the presence of pyruvate, ammonia, and ornithine was inhibited strongly by AMB even without preincubation. The results suggest that the stimulation of ureogenesis from ammonia and ornithine by pyruvate involves the cytosolic isozyme of aspartate aminotransferase. In contrast, the stimulation of ureogenesis elicited by lactate primarily involved mitochondrial aspartate aminotransferase.     

The effect of fatty acids and starvation on the metabolism of gluconeogenic precursors by isolated sheep liver cells.

Isolated liver cells prepared from fed sheep synthesize glucose from propionate at twice the rate observed with cells from starved animals. Addition of palmitate or palmitate + carnitine to incubations of liver cells from starved animals inhibited the rate of glucose synthesis with lactate as a precursor, but had little effect when propionate and pyruvate were substrates. Liver cells from fed and starved sheep synthesized lactate and pyruvate when incubated with propionate. Fatty acids inhibited this formation of lactate and pyruvate from propionate. It is proposed that the different responses of gluconeogenic precursors to fatty acids can be explained by the effect of reducing equivalents on the transport of carbon atoms across the mitochondrial membrane.     

Evidence for the occurrence of the malate-citrate shuttle in intact Ehrlich ascites tumor cells

A possible activity of the malate-citrate shuttle has been investigated in Ehrlich ascites cells by testing the effects of 1,2,3-benzenetricarboxylic acid, an inhibitor of the malate-citrate exchange, and (?)-hydroxycitrate, an inhibitor of the citrate cleavage enzyme, on the glucose-dependent oxidation-reduction rates of pyridine nucleotides and cytochrome b as well as on ATP levels of glycolyzing cells. Moreover, to quantitate such an activity, the effects of these two inhibitors have been compared with those induced under the same experimental conditions by aminooxyacetate, an inhibitor of the malate-aspartate shuttle which is known to operate in this strain of ascites tumor. Both benzenetricarboxylic acid and hydroxycitrate are able to increase the reduction of pyridine nucleotides, which follows glucose addition to whole cells, to about the same extent. A much more pronounced effect is elicited by aminooxyacetate under the same condition. When n-butylmalonate is added to slow down the flux of glycolytic reducing equivalents to the respiratory chain via the malate-aspartate shuttle, benzenetricar-boxylic acid or hydroxycitrate promotes an ATP-driven reversal of electron transfer. Indeed, the glucose-induced reduction of cytochrome b becomes sensitive to oligomycin and the ATP level is raised significantly with respect to the value of uninhibited cells. It is concluded that the malate-citrate shuttle operates in Ehrlich ascites cells, although with a substantially lower activity with respect to the malate-aspartate shuttle.     

Regulation of glucose formation from lactate and pyruvate in isolated tubules of chicken kidney.

1. Isolated kidney tubules from chicken have been used to study the actions of ethanol, ouabain and aminooxyacetate on glucose formation from lactate and pyruvate. 2. In kidney tubules from well-fed chickens the rate of glucose production from lactate was higher than from pyruvate. Ethanol (10 mM) and ouabain (0.1 mM) were found to increase glucose formation from pyruvate but not from lactate. 3. It is concluded that in the presence of ethanol the fluxes of pyruvate through pyruvate dehydrogenase are in favour of the pyruvate carboxylase reaction restricted. 4. Glucose formation from lactate is decreased by aminooxyacetate (0.1 mM) and ouabain (0.1 mM). 5. Aminooxyacetate inhibited glucose formation from lactate, although chicken phosphoenolpyruvate carboxykinase is located intramitochondrially. 6. The results indicate that the effect of aminooxyacetate like that of ouabain is caused by the restricted formation of pyruvate.     

The role of glucose,pyruvate and lactate in ATP production by rat spermatocytes and spermatids

The ATP content of pachytene spermatocytes and round spermatids, isolated from rat testes, was not maintained during incubation of the germ cells in the presence of glucose. Glucose was metabolized via glycolysis at a considerable rate, but the rate of oxidation of the resulting endogenous pyruvate in the mitochondria was too low to support fully ATP production. Exogenous pyruvate (0.25 mM) or exogenous l-lactate (3–6 mM), however, were effective energy substrates. The lactate dehydrogenase reaction in isolated germ cells favoured the rapid conversion of pyruvate to lactate, at the expense of reducing equivalents from mitochondrial NADH. Hence, to support ATP production by the germ cells via mitochondrial metabolism of endogenous pyruvate, a relatively high concentration of exogenous lactate may be essential. In the spermatogenic microenvironment in vivo, such high concentrations of lactate could result from the net production of lactate by Sertoli cells. The mitochondria of the isolated germ cells produced ATP probably at a close to maximal rate, and spermatogenesis therefore may be extremely sensitive to compounds which interfere with mitochondrial energy metabolism and respiratory control.     

Thyroid hormone and dehydroepiandrosterone permit gluconeogenic hormone responses in hepatocytes

The importance of the sn-glycerol- 3-phosphate (G-3-P) electron transfer shuttle in hormonal regulation of gluconeogenesis was examined in hepatocytes from rats with decreased mitochondrial G-3-P dehydrogenase activity (thyroidectomized) or increased G-3-P dehydrogenase activity [triiodothyronine (T(3)) or dehydroepiandrosterone (DHEA) treated]. Rates of glucose formation from 10 mM lactate, 10 mM pyruvate, or 2.5 mM dihydroxyacetone were somewhat less in hypothyroid cells than in cells from normal rats but gluconeogenic responses to calcium addition and to norepinephrine (NE), glucagon (G), or vasopressin (VP) were similar to the responses observed in cells from normal rats. However, with 2. 5 mM glycerol or 2.5 mM sorbitol, substrates that must be oxidized in the cytosol before conversion to glucose, basal gluconeogenesis was not appreciably altered by hypothyroidism but responses to calcium and to the calcium-mobilizing hormones were abolished. Injecting thyroidectomized rats with T(3) 2 days before preparing the hepatocytes greatly enhanced gluconeogenesis from glyc erol and restored the response to Ca(2+) and gluconeogenic hormones. Feeding dehydroepiandrosterone for 6 days depressed gluconeogenesis from lactate or pyruvate but substantially increased glucose production from glycerol in euthyroid cells and restored responses to Ca(2+) in hypothyroid cells metabolizing glycerol. Euthyroid cells metabolizing glycerol or sorbitol use the G-3-P and malate/aspartate shuttles to oxidize excess NADH generated in the cytosol. The transaminase inhibitor aminooxyacetate (AOA) decreased gluconeogenesis from glycerol 40%, but had little effect on responses to Ca(2+) and NE. However, in hypothyroid cells, with minimal G-3-P dehydrogenase, AOA decreased gluconeogenesis from glycerol more than 90%. Thus, the basal rate of gluconeogenesis from glycerol in the euthyroid cells is only partly dependent on electron transport from cytosol to mitochondria via the malate/aspartate shuttle and almost completely dependent in the hypothyroid state, and the hormone enhancement of the rate in euthyroid cells involves primarily the G-3-P cycle. These data are consistent with Ca(2+) being mobilized by gluconeogenic hormones and G-3-P dehydrogenase being activated by Ca(2+) so as to permit it to transfer reducing equivalents from the cytosol to the mitochondria.     

Studies on the relationship between glycolysis, lipogenesis, gluconeogenesis, and pyruvate kinase activity of rat and chicken hepatocytes.

Chicken hepatocytes synthesize glucose and fatty acids at rates which are faster than rat hepatocytes. The former also consume exogenous lactate and pyruvate at a much faster rate and, in contrast to rat hepatocytes, do not accumulate large quantities of lactate and pyruvate by aerobic glycolysis. α-Cyano-4-hydroxycinnamate, an inhibitor of pyruvate transport, causes lactate and pyruvate accumulation by chicken hepatocytes. Glucagon and N⁶,O²′-dibutyryl adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) convert pyruvate kinase (EC 2.7.1.40) of rat hepatocytes to a less active form. This effect explains, in part, inhibition of glycolysis, inhibition of lipogenesis, stimulation of gluconeogenesis, and inhibition of the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment by these compounds. In contrast, pyruvate kinase of chicken hepatocytes is refractory to inhibition by glucagon or dibutyryl cyclic AMP. Rat liver is known to have predominantly the type L isozyme of pyruvate kinase and chicken liver predominantly the type K. Thus, only the type L isozyme appears subject to interconversion between active and inactive forms by a cyclic AMP-dependent, phosphorylation-dephos-phorylation mechanism. This explains why the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment of chicken hepatocytes is insensitive to cyclic AMP. However, glucagon and dibutyryl cyclic AMP inhibit net glucose utilization, inhibit fatty acid synthesis, inhibit lactate and pyruvate accumulation in the presence of α-cyano-4-hydroxycinnamate, and stimulate gluconeogenesis from lactate and dihydroxyacetone by chicken hepatocytes. Thus, a site of action of cyclic AMP distinct from pyruvate kinase must exist in the glycolytic-gluconeogenic pathway of chicken liver.     

Methotrexate: studies on cellular metabolism. IV. Effect on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells

The effect of methotrexate (MTX) on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells was studied. MTX inhibited (100 per cent) malate dehydrogenase activity, but no effect was observed on that of GOT. MTX (0.5 mM) inhibited (100 per cent) the activity of reconstituted enzymatic system MDH-GOT, probably as a consequence of inhibition of malate dehydrogenase activity. MTX decreased pyruvate production (54 per cent), demonstrating its inhibitory action on the malate-aspartate shuttle. Blockage of the malate-aspartate shuttle by MTX accounts for the decrease in cellular energetic gain. The results obtained are consistent with the view that in HeLa cells, as well as in other tumour cells, the transport of reducing equivalents from cytoplasmic NADH into the respiratory chain of mitochondria is via the malate-aspartate shuttle.     

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.     

Energy metabolism of isolated rat thymus cells

The energy metabolism of rat thymus cells has been investigated using preparations of isolated cells obtained by mechanical treatment of whole organs. The addition of glycolytic substrates such as glucose, pyruvate and lactate stimulates the endogenous respiration of these cells by 50%. On the other hand, succinate, glutamate and malate do not produce any effect. Oligomycin (10 mug/ml) inhibits both endogenous and glucose stimulated respiration by about 40%; 2, 4-DNP (50 muM) increases by 100% glucose induced respiration. The results obtained by using mitochondrial and glycolytic inhibitors as well as aminoxyacetic acid (AOA) and following pyridine nucleotides redox changes, support the idea that in thymus cells glucose is able to induce a great enhancement of O2 consumption both by raising the level of endogenous pyruvate and feeding the mitochondrial respiratory chain with cytosolic reducing equivalents, through an active malate-aspartate shuttle. Thymus cells exhibit a high Pasteur effect (74%). Both AOA and 2,4 DNP are able to stimulate aerobic lactate accumulation by 200% and 100% respectively, indicating that either the redox or phosphate potential do influence the rate of aerobic glycolysis in isolated thymus cells. Similar experiments are also reported on other cells with well known biochemical characteristics.