Metabolic fluxes in the liver of rats bearing the Walker-256 tumour: influence of the circulating levels of substrates and fatty acids

Studies on fatty acid and amino acid metabolism in the liver of Walker-256 tumour-bearing rats have revealed several changes. Comparisons, however, have been based on experiments performed with non-physiological, frequently unrealistic, substrate concentrations. The aim of the present work was to examine the influence of physiological substrate concentrations on gluconeogenesis, ketogenesis and related parameters. Isolated livers were perfused and substrates were infused at concentrations that were reported to occur in healthy and tumour-bearing rats. Ketogenesis and the mitochondrial NADH/NAD+ ratio were smaller in the tumour-bearing condition at low (0.2 mM) and high (0.8 mM) oleate concentrations. In the absence of oleate, gluconeogenesis from alanine (0.7 mM) and gluconeogenesis plus the associated changes in oxygen uptake due to lactate/pyruvate (2/0.2 and 6/0.3 mM) were smaller in livers of tumour-bearing rats. However, the response of gluconeogenesis from lactate/pyruvate in livers of tumour-bearing rats to 0.8 mM oleate was more pronounced so that a trend towards normalization was apparent at high substrate and oleate concentrations. Gluconeogenesis from 0.7 mM alanine was not significantly changed by oleate in the tumour-bearing state; in the control condition, stimulation occurred at 0.2 mM oleate and inhibition at 0.8 mM oleate. This diminution almost equalized the hepatic alanine-dependent gluconeogenesis of both control and tumour-bearing rats. Ureogenesis was smaller in the tumour-bearing state and was not affected by oleate. It was concluded that the high concentrations of fatty acids and lactate/pyruvate, which predominate in rats bearing the Walker-256 tumour, could be effective in normalizing the gluconeogenic response of livers from tumour-bearing rats.     

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.     

Calcium transport in bovine sperm mitochondria: effect of substrates and phosphate.

Calcium uptake into filipin-treated bovine spermatozoa is completely inhibited by the uncoupler CCCP or by ruthenium red. Both Pi and mitochondrial substrates are required to obtain the maximal rate of calcium uptake into the sperm mitochondria. Bicarbonate and other anions such as lactate, acetate or beta-hydroxybutyrate do not support a high rate of calcium uptake. There are significant differences among various mitochondrial substrates in supporting calcium uptake. The best substrates are durohydroquinone, alpha-glycerophosphate and lactate. Pyruvate is a relatively poor substrate, and its rate can be greatly enhanced by malate or succinate but not by oxalacetate or lactate. This stimulation is blocked by the dicarboxylate translocase inhibitor, butylmalonate and can be mimiced by the non-metabolized substrate D-malate. The Ka for pyruvate was found to be 17 microM and 67 microM in the presence and absence of L-malate, respectively. The Ka for L-malate is 0.12 mM. It is suggested that in addition to the known pyruvate/lactate translocase there is a second translocase for pyruvate which is malate/succinate-dependent and does not transport lactate. In the presence of succinate, glutamate stimulates calcium uptake 3-fold, and this effect is not inhibited by rotenone. In the presence of glutamate plus malate or oxalacetate there is only an additive effect. It is suggested that glutamate stimulates succinate transport and/or oxidation in bovine sperm mitochondria. The alpha-hydroxybutyrate is almost as good as lactate in supporting calcium uptake. Since the alpha-keto product is not further metabolized in the citric acid cycle, it is suggested that lactate can supply the mitochondrial needs for NADH from its oxidation to pyruvate by the sperm lactate dehydrogenase x. Thus, when there is sufficient lactate in the sperm mitochondria, pyruvate need not be further metabolized in the citric acid cycle in order to supply more NADH.     

Changes in mitochondrial and microsomal lipid peroxidation and fatty acid profiles in adrenal glands, testes, and livers from alpha-tocopherol-deficient rats

Studies were done to evaluate the effects of alpha-tocopherol deficiency in rats on the fatty acid composition and sensitivity to lipid peroxidation (LP) of mitochondria and microsomes from adrenal glands, testes, and livers. In control (alpha-tocopherol-sufficient) animals, adrenal concentrations of alpha-tocopherol were approximately 10 times greater than those in livers and testes. Dietary deficiency of alpha-tocopherol for 8 weeks decreased adrenal and hepatic concentrations by 80-90% and testicular concentrations by approximately 60-70%. Incubation of testicular or hepatic mitochondria and microsomes from control rats with FeSO(4) (1.0 mM) caused a time-dependent stimulation of LP as indicated by the formation of thiobarbituric acid reactive substances (TBARS); the rate of TBARS production increased in preparations from alpha-tocopherol-deficient animals. TBARS formation was not demonstrable in adrenal mitochondria or microsomes from alpha-tocopherol sufficient rats, but reached high levels in alpha-tocopherol-deficient preparations. The fatty acid composition of mitochondria and microsomes was tissue-dependent. In particular, arachidonic acid comprised approximately 40% of the total fatty acids in adrenal membranes, but only 20-25% in testes and livers. alpha-Tocopherol deficiency increased oleic acid concentrations in adrenal and hepatic mitochondria and microsomes but not in testes. In all three tissues, linoleic acid concentrations decreased by approximately 50%, but arachidonic acid levels were unaffected by alpha-tocopherol deficiency. The results indicate a close relationship between tissue sensitivity to LP in vitro and alpha-tocopherol concentrations. Nonetheless, any oxidative stress in vivo caused by alpha-tocopherol deficiency seems to spare arachidonic acid in mitochondria and microsomes but decreases linoleic acid concentrations. It is possible that because of the important physiological functions of arachidonic acid, metabolic adaptations serve to maintain membrane content during periods of oxidative stress.     

Studies on the inhibition of hepatic lipogenesis by N6,O2′-dibutyryl adenosine 3′,5′-monophosphate

Fatty acid synthesis by isolated liver cells is dependent upon the availability of lactate and pyruvate. A lag in fatty acid synthesis is explained by time being required for lactate and pyruvate to accumulate to maximum concentrations in the incubation medium. The initial rate of fatty acid synthesis is not linear with cell concentration, being disproportionately greater at higher cell concentrations because optimal lactate and pyruvate concentrations are established in the medium more rapidly. The accumulation of lactate and pyruvate is inhibited markedly by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. This accounts in part for the inhibition of fatty acid synthesis caused by this cyclic nucleotide. Other sites of action are apparent, however, because exogenous lactate plus pyruvate only partially relieves the inhibition. The profile of metabolic intermediates suggests that N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate inhibits the conversion of glycogen to pyruvate and lactate by decreasing the effectiveness of phosphofructokinase and pyruvate kinase.     

The effects of specific lipogenic substrates and metabolic inhibitors on de Novo fatty acid synthesis in isolated hepatocytes from chow-fed female rats

The effects of glucose (10 mm), glycerol (3 mm), and lactate/pyruvate (10 mm) on the incorporation of ³H from ³H₂O into fatty acids were studied in isolated hepatocytes prepared from chow-fed female rats. Lactate/pyruvate markedly increased lipogenic rates, while glucose and glycerol did not significantly affect rates of lipogenesis. In cells incubated with lactate/pyruvate plus glycerol, the increase in ³H incorporation was greater than observed with lactate/pyruvate alone. In hepatocytes isolated from 24-h starved rats, lactate/pyruvate again increased de novo fatty acid synthesis to a greater extent than either glucose or glycerol. Glycerol significantly increased lipogenesis compared to the endogenous rates and when incubated with lactate/pyruvate produced an increase above lactate/pyruvate alone. (?)-Hydroxycitrate, a potent inhibitor of ATP-citrate lyase (EC 4.1.3.8), and agaric acid, an inhibitor of tricarboxylate anion translocation, were studied in hepatocytes to determine their effects on lipogenesis by measuring ³H₂O, [1-¹⁴C]acetate, and [2-¹⁴C]lactate incorporation into fatty acids. ³H incorporation into fatty acids was markedly inhibited by both inhibitors with agaric acid (60 μm) producing the greater inhibition. (?)-Hydroxycitrate (2 mm) increased acetate incorporation into fatty acids from [1-¹⁴C]acetate and agaric acid produced a strong inhibitory effect. Combined effects of (?)-hydroxycitrate and agaric acid on lipogenesis from [1-¹⁴C]acetate showed an inhibitory response to a lesser extent than with agaric acid alone. With substrate concentrations of acetate present, there was no significant increase in rates of lipogenesis from [1-¹⁴C]acetate and the increase previously observed with (?)-hydroxycitrate alone was minimized. Agaric acid significantly inhibited fatty acid synthesis from acetate in the presence of exogenous substrate, but the effect was decreased in comparison to rates with only endogenous substrate present. With [2-¹⁴C]lactate as the lipogenic precursor, agaric acid and (?)-hydroxycitrate strongly inhibited fatty acid synthesis. However, agaric acid despite its lower concentration (60 μm vs 2 mm) was twice as effective as (?)-hydroxycitrate. A similar pattern was observed when substrate concentrations of lactate/pyruvate (10 mm) were added to the incubations. When (?)-hydroxycitrate and agaric acid were simultaneously incubated in the presence of endogenous substrate, there was an additive effect of the inhibitors on decreasing fatty acid synthesis. Results are discussed in relation to the origin of substrate for hepatic lipogenesis and whether specific metabolites increase lipogenic rates.     

Effectors of fatty acid synthesis in hepatoma tissue culture cells

An investigation was undertaken to better understand the process of fatty acid synthesis in hepatoma tissue culture (HTC) cells. By comparing the findings to the normal liver some of the differences between normal and cancer tissue were defined. Incubation of the HTC cells in a buffered salt-defatted albumin medium showed that fatty acid synthesis was dependent upon the addition of substrate. The order of stimulation was glucose + pyruvate ～- glucose + alanine ～- glucose + lactate ～- pyruvate > glucose > alanine ? no additions. Fatty acid synthesis in HTC cells was decreased by oleate. In these respects HTC cells are similar to the liver; however, in contrast to the normal liver, N⁶, O²-dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl-cAMP) did not inhibit glycolysis or fatty acid synthesis. The cytoplasmic redox potential, as reflected by the lactate to pyruvate ratio, was found to be elevated compared to normal liver but unchanged by the addition of dibutyryl cAMP. Since higher rates of fatty acid synthesis are associated with lower lactate-to-pyruvate ratios in normal liver, it was expected that by decreasing the lactate-to-pyruvate ratio in HTC cells the rate of fatty acid synthesis would increase. One way to lower the lactate to pyruvate ratio is to increase the activity of the malate-aspartate shuttle. Stimulators of the hepatic malate-aspartate shuttle in normal liver (ammonium ion, glutamine, and lysine) had mixed effects on the redox state and fatty acid synthesis in HTC cells. Both ammonium ion and glutamine decreased the redox potential and increased the rate of fatty acid synthesis. Lysine was without effect on either process. Since NH₄Cl and glutamine stimulate the movement of reducing equivalents into the mitochondria and decrease the redox potential, then the stimulation of fatty acid synthesis by NH₄Cl and glutamine may be due to an increase in the movement of reducing equivalents into the mitochondria. However, if the shuttle were rate determining for fatty acid synthesis the rate from added lactate would be the same as from glucose alone but would be lower than from pyruvate which does not require the movement of reducing equivalents. This was not the case. Lactate and pyruvate gave comparable rates which were higher than glucose alone. Other possible sites of stimulation were investigated. The possibility that NH₄⁺ and glutamine stimulated fatty acid synthesis by activating pyruvate dehydrogenase was excluded by finding that dichloroacetate, an activator of pyruvate dehydrogenase, did not stimulate fatty acid synthesis when glucose was added. Stimulation by NH₄⁺ and glutamine at steps beyond pyruvate dehydrogenase was ruled out by the observation that NH₄⁺ caused no stimulation from added pyruvate. NH₄⁺ and glutamine did not alter the pentose phosphate pathway as determined by ¹⁴CO₂ production from [1-¹⁴C]- or [6-¹⁴C]glucose. Ammonium ion and glutamine increased glucose consumption and increased lactate and pyruvate accumulation. The increased glycolysis in HTC cells appears to be the explanation for the stimulation of fatty acid synthesis by NH₄⁺ and glutamine, even though glycolysis is much more rapid than fatty acid synthesis in these cells. The following observations support this conclusion. First, the percentage increase in glycolysis caused by NH₄⁺ or glutamine is closely matched by the percentage increase in fatty acid synthesis. Second, the malate-aspartate shuttle, the pentose phosphate pathway, and the steps past pyruvate are not limiting in the absence of NH₄⁺ or glutamine.     

Influence of ethanol on the metabolism of perfused normal, fatty and cirrhotic rat livers

1. The influence of ethanol on the metabolism of perfused livers from normal rats and rats in various stages of development of dietary cirrhosis was studied. A choline-deficient, low-protein and high-fat diet was used. Results were obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in fatty and cirrhotic livers than in normal livers. Ethanol had no effect on the oxygen consumption of any of the various livers. After addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely in normal livers. Only a slight decrease in the carbon dioxide production occurred in fatty and cirrhotic livers. 3. With every type of liver glucose was released from the liver into the perfusion medium during the initial control period. This release continued after the addition of ethanol to the perfusion medium in experiments with normal and fatty livers, whereas with cirrhotic livers a marked uptake of glucose from the medium was found. A simultaneous release of the glycolytic end products lactate and pyruvate into the medium occurred. 4. The production of ketone bodies was equal in normal and early fatty livers (6 weeks on the fat diet). It was smaller in late fatty livers (3–4 months on the fatty diet) and in cirrhotic livers. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 11 to 67 with normal livers, from 12 to 16 with early fatty livers, from 13 to 26 with late fatty livers and from 21 to 55 with cirrhotic livers when the livers were perfused with a medium containing ethanol. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 1·2 to 8·4 in normal livers, from 2·0 to 2·8 in early fatty livers, from 1·2 to 2·4 in late fatty livers and from 2·1 to 4·0 in cirrhotic livers when ethanol was added to the medium. 6. The effects of ethanol on liver metabolism during the development of dietary cirrhosis are discussed and related to human fatty liver and cirrhosis during chronic ethanol consumption.     

Zymosan-induced changes in glucose release and fatty acid oxidation in the perfused rat liver

The aim of the present study was to investigate the actions of zymosan on glucose release and fatty acid oxidation in perfused rat livers and to determine if Kupffer cells and Ca2+ ions are implicated in these actions. Zymosan caused stimulation of glycogenolysis in livers from fed rats. In livers from fasted rats zymosan caused gradual inhibition of glucose production and oxygen consumption from lactate plus pyruvate. Ketogenesis, oxygen consumption, and [14C-]-CO2 production were inhibited by zymosan when the [1-14C]-palmitate was supplied exogenously. However, ketogenesis and oxygen consumption from endogenous sources were not inhibited. An interference with substrate-uptake by the liver may be the cause of the changes in gluconeogenesis and oxidation of fatty acids from exogenous sources. The pretreatment of the rats with gadolinium chloride and the removal of Ca2+ ions did not suppress the effects of zymosan on glucose release, a finding that argues against the participation of Kupffer cells or Ca2+ ions in the liver responses. The hepatic metabolic changes caused by zymosan could play a role in the systemic metabolic alterations reported to occur after in vivo zymosan administration.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Evidence that the flux control coefficient of the respiratory chain is high during gluconeogenesis from lactate in hepatocytes from starved rats. Implications for the hormonal control of gluconeogenesis and action of hypoglycaemic agents.

1. Increasing concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a mild respiratory-chain inhibitor [Halestrap (1987) Biochim. Biophys. Acta 927, 280-290], caused progressive inhibition of glucose production from lactate + pyruvate by hepatocytes from starved rats incubated in the presence or absence of oleate and gluconeogenic hormones. 2. No significant changes in tissue ATP content were observed, but there were concomitant decreases in ketone-body output and cytochrome c reduction and increases in NADH fluorescence and the ratios of [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate]. 3. The inhibition by DCMU of palmitoylcarnitine oxidation by isolated liver mitochondria was used to calculate a flux control coefficient of the respiratory chain towards gluconeogenesis. In the presence of 1 mM-oleate, the calculated values were 0.61, 0.39 and 0.25 in the absence of hormone and in the presence of glucagon or phenylephrine respectively, consistent with activation of the respiratory chain in situ as previously suggested [Quinlan & Halestrap (1986) Biochem. J. 236, 789-800]. 4. Cytoplasmic oxaloacetate concentrations were shown to decrease under these conditions, implying inhibition of pyruvate carboxylase. 5. Inhibition of gluconeogenesis from fructose and dihydroxyacetone was also observed with DCMU and was accompanied by an increased output of lactate + pyruvate, suggesting that activation of pyruvate kinase was occurring. With the latter substrate, measurements of tissue ADP and ATP contents showed that DCMU caused a small fall in [ATP]/[ADP] ratio. 6. Two inhibitors of fatty acid oxidation, pent-4-enoate and 2-tetradecylglycidate, were shown to abolish and to decrease respectively the effects of hormones, but not valinomycin, on gluconeogenesis from lactate + pyruvate, without changing tissue ATP content. 7. It is concluded that the hormonal increase in mitochondrial matrix volume stimulates fatty acid oxidation and respiratory-chain activity, allowing stimulation of pyruvate carboxylation and thus gluconeogenesis to occur without major changes in [ATP]/[ADP] or [NADH]/[NAD+] ratios. 8. The high flux control coefficient of the respiratory chain towards gluconeogenesis may account for the hypoglycaemic effect of mild respiratory-chain inhibitors.     

Pathway of carbon flow during fatty acid synthesis from lactate and pyruvate in rat adipose tissue

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.     

The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue

The metabolism of lactate, pyruvate and glucose was studied in epididymal adipose tissue of starved, normally fed and starved-re-fed rats. Lactate conversion into fatty acid occurred at an appreciable rate only in the adipocyte of starved-re-fed animals. NNN'N'-Tetramethyl-p-phenylenediamine, an agent that transports reducing power from the cytoplasm to the mitochondria, caused large increments of fatty acid synthesis from lactate and a smaller one from glucose but a decrease in that from pyruvate. Glucose (1.0mm) increased fatty acid synthesis from lactate 4.3-fold but only 1.67-fold from pyruvate in adipocytes from normally fed animals. 2-Deoxyglucose decreased fatty acid synthesis from lactate to a greater degree (threefold) compared to that from pyruvate in adipocytes from starved-re-fed animals. l-Glycerol 3-phosphate contents were approximately equal in epididymal fat-pads, incubated in the presence of lactate or pyruvate, from normally fed animals, whereas the addition of 1mm-glucose resulted in a tenfold increase in l-glycerol 3-phosphate content only in the presence of lactate. The l-glycerol 3-phosphate content was tenfold higher in adipose tissue from starved-re-fed animals incubated in the presence of lactate than in the presence of pyruvate. 2-Deoxyglucose caused these values to be slightly lowered in the presence of lactate. We suggest that lactate metabolism is limited by the rate of NADH removal from the cytoplasm. In the starved-re-fed state, this occurs by reduction of dihydroxyacetone phosphate formed from glycogen to produce l-glycerol 3-phosphate, thus permitting lactate conversion into fatty acid. When glucose is the substrate, and rates of transport are not limiting, the rate of removal of cytoplasmic NADH limits glucose conversion into fatty acid.     

Competition between fatty acids and carbohydrate or ketone bodies as metabolic fuels for the isolated perfused heart

The ability of carbohydrate fuels (lactate, pyruvate, glucose) and the ketone bodies (acetoacetate, beta-hydroxybutyrate) to compete with fatty acids as fuels of respiration in the isolated Langendorf-perfused heart was studied. Oleate and octanoate were used as fatty acid fuels since oleate requires carnitine for entry into mitochondria, whereas octanoate does not. The two ketone bodies inhibited the oxidation of both oleate and octanoate implying an intramitochondrial site of action. Pyruvate, lactate, and lactate plus glucose inhibited oleate oxidation but not octanoate oxidation, indicating a mechanism of inhibition that involves the carnitine system. Pyruvate was a more potent inhibitor than lactate at equal concentrations, but the effect of lactate could be greatly increased by dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase. The physiological and mechanistic implications of these observations are discussed.     

The action of vasopressin and calcium on palmitate metabolism in hepatocytes and isolated mitochondria from rat liver

Vasopressin inhibits fatty acid oxidation and stimulates fatty acid esterification, glycogenolysis, and lactate production in hepatocytes from fed rats. In cells from fasted rats, the effect of the hormone on palmitate oxidation was absent, while gluconeogenesis was stimulated. The inhibitory action of vasopressin on palmitate oxidation was not due to the increased lactate production. Neither was it correlated to glycogen content or stimulation of glycogenolysis, which were restored earlier than the vasopressin effect on palmitate oxidation when previously fasted rats were refed a carbohydrate diet. The level of malonyl-CoA was moderately increased by vasopressin. Isolated mitochondria from rat liver were incubated in the presence of [U-14C]palmitate, ATP, CoA carnitine, glycerophosphate, ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, and varying amounts of calcium. The oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1 to 10 microM. Simultaneously, palmitate esterification was stimulated. This effect of calcium was observed also with mitochondria from fasted rats and with octanoate as well as palmitate as the substrate. Carnitine acylation was not affected by calcium. The possibility that the observed effects of calcium on mitochondrial fatty acid utilization is part of the mechanism of action of vasopressin on hepatocyte fatty acid metabolism is discussed.     

Effects of branched chain alpha-ketoacids on the metabolism of isolated rat liver cells. I. Regulation of branched chain alpha-ketoacid metabolism.

alpha-Ketoisocaproate (ketoleucine) is shown to be metabolized to ketone bodies rapidly by isolated rat liver cells. Acetoacetate is the major end product and maximum rates were observed with 2 mM substrate. Studies with 2-tetradecylglycidic acid (an inhibitor of long chain fatty acid oxidation) showed that ketogenesis from alpha-ketoisocaproate and from endogenous fatty acids were additive. With alpha-ketoisocaproate present as soole substrate at 2 mM, leucine production was less than 10% of alpha-ketoisocaproate uptake and only 30% of the acetyl coenzyme A generated was oxidized in the citric acid cycle. Metabolism of alpha-ketoisocaproate was inhibited by fatty acids, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and pyruvate. Oxidation of acetyl-CoA generated from alpha-ketoisocaproate was suppressed by oleate and by pyruvate, but was enhanced by lactate. Metabolism between the different branched chain alpha-ketoacids was mutually competitive. When alpha-ketoisocaproate (2 mM) was added in the presence of high pyruvate concentrations (4.4 mM), flux through pyruvate dehydrogenase was decreased, and the proportion of total pyruvate dehydrogenase in the active form (PDHa) also fell. With lactate as substrate, PDHa was only 25% of total activity and was little affected by addition of alpha-ketoisocaproate. These data suggest that enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis. However, acetyl-CoA generation from pyruvate is apparently insufficiently inhibited by alpha-ketoisocaproate to cause a diversion of acetyl-CoA formed during alpha-ketoisocaproate metabolism from ketone body formation to oxidation in the citric acid cycle. Measurements of the cell contents of CoASH, acetyl-CoA, acid-soluble acyl-CoA, and acid-insoluble fatty acyl-CoA indicated that when the branched chain alpha-ketoacids were added as sole substrate, their oxidation was limited at a step distal to the branched chain alpha-ketoacid dehydrogenase. Acid-soluble acyl-CoA derivatives were depleted after oleate addition in the presence of alpha-ketoisocaproate, suggesting an inhibition of the branched chain alpha-ketoacid dehydrogenase by the elevation of the mitochondrial NADH/NAD+ ratio observed during fatty acid oxidation. This effect was not observed in the presence of oleate and 2-tetradecylglycidic acid.     

Lipoic acid and diabetes: Effect of dihydrolipoic acid administration in diabetic rats and rabbits

Relative α-lipoic acid content of diabetic livers was considerably less than that of normal livers as determined by gas chromatography. It was not possible to detect any dihydrolipoic acid in the livers. Biochemical abnormalities such as hyperglycaemia, ketonemia, reduction in liver glycogen and impaired incorporation of [2-¹⁴C] -acetate into fatty acids in alloxan diabetic rats were brought to near normal levels by the oral or intraperitoneal administration of dihydrolipoic acid. The effect of α-lipoic acid was comparable to that of dihydrolipoic acid in reducing the blood sugar levels of diabetic rabbits during a glucose tolerance test. The results suggest that the mode of action of lipoic acid was through stimulation of pyruvate dehydrogenase.     

Cloning of yeast glycolysis genes by complementation

In hepatocytes isolated from fed rats, low concentrations of oxalate (50 to 100 μM) greatly enhance ketogenesis and decrease fatty acid synthesis. These metabolic changes are due to pyruvate carboxylase inhibition. Dichloroacetate, which can be catabolized into oxalate enhances ketogenesis only when cells are enriched with lactate and pyruvate and has no obvious effect on lipogenesis. The enhancement of ketogenesis, in both cases, is due to an imbalance between pyruvate dehydrogenase and pyruvate carboxylase but with oxalate, the primary event is oxaloacetate shortage and with dichloroacetate, mitochondrial acetyl CoA excess. This work demonstrates that the studied effects of dichloroacetate are not mediated by oxalate and that low concentrations of oxalate alter the lipid metabolism of hepatocytes.     

Uptake of glucose and release of fatty acids and glycerol by rat brown adipose tissue in vivo

The net in vivo uptake or release of free fatty acids glycerol, glucose, lactate, and pyruvate by the interscapular brown adipose tissue (IBAT) of barbital-anesthetized, cold-acclimated rats was determined from measurements of plasma arteriovenous concentration differences across IBAT and tissue blood flow. Measurements were made without stimulation of the tissue and also during submaximal and maximal stimulation by infused noradrenaline (NA), the physiological activator of BAT thermogenesis. There was no appreciable uptake of glucose or release of fatty acids and glycerol by the nonstimulated tissue. At both levels of stimulation there was significant uptake of glucose (1.7 and 2.0 mumol/min) and release of glycerol (0.9 and 1.2 mumol/min), but only at maximal stimulation was there significant release of fatty acids (1.9 mumol/min). Release of lactate and pyruvate accounted for 33% of the glucose taken up at submaximal stimulation and 88% at maximal stimulation. By calculation, the remainder of the glucose taken up was sufficient to have fueled about 12% of the thermogenesis at submaximal stimulation, but only about 2% at maximal stimulation. As estimated from the rate of glycerol release, the rate of triglyceride hydrolysis was sufficient at submaximal stimulation to fuel IBAT thermogenesis entirely with the resulting fatty acids, but it was not sufficient to do so at maximal stimulation when some of the fatty acid was exported. It is suggested that at maximal NA-induced thermogenesis a portion of lipolysis proceeded only to the level of mono- and di-glycerides with the result that glycerol release did not fully reflect the rate of fatty acid formation.(ABSTRACT TRUNCATED AT 250 WORDS)