Interactions in vivo between oxidation of non-esterified fatty acids and gluconeogenesis in the newborn rat.

Metabolic interactions between fatty acid oxidation and gluconeogenesis were investigated in vivo in 16h-old newborn rats under various nutritional states. As the newborn rat has no white adipose tissue, starvation from birth induces a low rate of hepatic fatty acid oxidation. Hepatic gluconeogenesis in inhibited in the starved newborn rat when compared with the suckling rat, which receives fatty acids through the milk, at the steps catalysed by pyruvate carboxylase and glyceraldehyde 3-phosphate dehydrogenase. These inhibitions are rapidly reversed by triacylglycerol feeding. Inhibition of fatty acid oxidation by pent-4-enoate in the suckling animal mimics the effect of starvation on the pattern of hepatic gluconeogenic metabolites. It is concluded that, in the newborn rat in vivo, hepatic fatty acids oxidation can increase the gluconeogenic flux by providing the acetyl-CoA necessary for the reaction catalysed by pyruvate carboxylase and the reducing equivalents (NADH) to displace the reversible reaction catalysed by glyceraldehyde 3-phosphate dehydrogenase in the direction of gluconeogenesis.     

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.     

The role of calcium in the stimulation of gluconeogenesis by catecholamines

In hepatocytes isolated from fasted normal rats and incubated without albumin or gelatin, norepinephrine stimulated gluconeogenesis from fructose or dihydroxyacetone only in the absence of added calcium and from sorbitol or glycerol only in the presence of added calcium. The effects of calcium, norepinephrine, or calcium in combination with norepinephrine on the concentration of intermediary metabolites were therefore studied in hepatocytes metabolizing fructose or sorbitol as the representative oxidized or reduced substrate, respectively. With fructose as the substrate, addition of calcium increased the concentrations of lactate, pyruvate, glyceraldehyde 3-phosphate, and β-hydroxybutyrate, but decreased the concentrations of phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate, glucose 6-phosphate, malate, citrate, and α-oxoglutarate. With sorbitol as the substrate, calcium increased the concentrations of pyruvate, malate, β-hydroxybutyrate, and glucose. With either substrate, calcium caused a decrease in the lactate/ pyruvate ratio and an increase in the β-hydroxybutyrate/acetoacetate ratio, indicating the stimulation of transfer of reducing equivalents from cytosol to mitochondria. With sorbitol as the substrate, and with calcium present, norepinephrine promoted further electron transfer from cytosolic to mitochondrial NAD. Enhanced cytosolic calcium concentrations, when cells are exposed to catecholamines in the presence of medium calcium, stimulate the mitochondrial α-glycerophosphate dehydrogenase and thus the transfer of electrons between cell compartments.     

Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP-glucose-6-phosphate dehydrogenase, NADP-6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase, NADP-malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl-CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with ¹⁴C-labelled carbon precursors and the incorporation of ¹⁴C into starch and fatty acids was determined. ¹⁴C from glucose-6-phosphate (G-6-P), fructose, glucose, fructose-6-phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness- and ATP-dependent manner. The rate of starch synthesis was highest from G-6-P, although fructose gave rates which were 70% of those from G-6-P. Glucose-1-phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G-6-P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G-6-P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.     

The route of ethanol formation in Zymomonas mobilis

1. Enzymic evidence supporting the operation of the Entner-Doudoroff pathway in the anaerobic conversion of glucose into ethanol and carbon dioxide by Zymomonas mobilis is presented. 2. Cell extracts catalysed the formation of equimolar amounts of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate. Evidence that 3-deoxy-2-oxo-6-phosphogluconate is an intermediate in this conversion was obtained. 3. Cell extracts of the organism contained the following enzymes: glucose 6-phosphate dehydrogenase (active with NAD and NADP), ethanol dehydrogenase (active with NAD), glyceraldehyde 3-phosphate dehydrogenase (active with NAD), hexokinase, gluconokinase, glucose dehydrogenase and pyruvate decarboxylase. Extracts also catalysed the overall conversion of glycerate 3-phosphate into pyruvate in the presence of ADP. 4. Gluconate dehydrogenase, fructose 1,6-diphosphate aldolase and NAD-NADP transhydrogenase were not detected. 5. It is suggested that NAD is the physiological electron carrier in the balanced oxidation-reduction involved in ethanol formation.     

Effect of prolonged ethanol ingestion on hepatic lipogenesis and related enzyme activities

1. Hepatic lipogenesis in vivo and the activities of enzymes associated with fatty acid synthesis in the liver were studied in rats fed for 21 days on liquid diets containing ethanol. 2. The ethanol-fed rats developed a moderate hepatic triacylglycerol accumulation during this period. When carbohydrate was replaced by ethanol in the diet, the rate of fatty acid synthesis was slower in the ethanol-fed rats on low-, medium- and high-fat diets than in the appropriate controls. However, when the fat/carbohydrate ratio was kept the same in the ethanol-fed and control rats, ethanol had no influence on the rate of fatty acid synthesis. 3. Glucose 6-phosphate dehydrogenase activity was lower in the ethanol-fed group. ;Malic' enzyme activity did not change during the ethanol treatment when the fat/carbohydrate ratio was kept unchanged. 4. The ATP citrate lyase activity was lower in the ethanol-fed rats on all diets, whereas acetyl-CoA synthetase activity was independent of the composition of the control diet, but was lower in the ethanol-fed rats, in which the concentration of the active form of pyruvate dehydrogenase was also lower. 5. It is concluded that hepatic fatty acid synthesis does not play any major role in ethanol-induced triacylglycerol accumulation. Careful design of the diets is necessary to reveal the specific effects of ethanol on the enzymes associated with lipogenesis.     

Effect of some glycolytic intermediates and palmitoyl-CoA on alpha-glycerophosphate dehydrogenase in mitochondria isolated from liver of triiodothyronine-treated rats

alpha-Glycerophosphate dehydrogenase (EC 1.1.99.5) in mitochondria from liver of the triiodothyronine-treated rats is competitively inhibited by phosphoenolpyruvate, glyceraldehyde 3-phosphate and 3-phosphoglycerate, the apparent Ki values for phosphoenolpyruvate being 0.76 mM at pH 7.0, 1.7 mM at pH 7.4 and 3.5 mM at pH 7.7. The apparent Ki values for glyceraldehyde 3-phosphate and 3-phosphoglycerate are also pH-dependent. Other glycolytic intermediates, such as 2-phosphoglycerate, 2,3-diphosphoglycerate, pyruvate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate did not alter significantly alpha-glycerophosphate dehydrogenase activity. Palmitoyl-CoA is a competitive inhibitor of this enzyme, with Ki value of about 30 micron.     

Enzymatic Studies on the Conversion of Erythritol Fermentation to d-Mannitol Fermentation in n-Alkane-grown Yeast Candida zeylanoides

In the polyol fermentation by Candida zeylanoides KY6166, which occurred preferentially by keeping the pH of medium at acidic side (below 4.0), phosphate ion played a precise role in the conversion of erythritol fermentation to d-mannitol fermentation. Enzymatic studies on the conversion mechanism provided the following evidences.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphate ion.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphateion. From these results, the alteration from erythritol fermentation to mannitol fermentation by phosphate ion was explained as the result of the change in the level of erythrose 4-phosphate and fructose 6-phosphate which was caused by the inhibition of transketolase.     

Inhibition of lipogenesis by halothane in isolated rat liver cells.

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.     

Reversal of glycolysis in yeast.

When a buffered, aerobic suspension of ethanol-grown cells of Saccharomyces cerevisiae is treated with ethanol, a rapid flux of metabolism is observed from endogenous phosphoenolpyruvate to hexose monophosphates. Intracellular concentrations of phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate record a monotonic drop, while those of triose phosphates and fructose 1,6-diphosphate fall after an early rise; fructose 6-phosphate, mannose 6-phosphate, and glucose 6-phosphate levels rise to a plateau. Prior growth on glucose extinguishes fructose 1,6-diphosphatase activity and completely arrests the rise of the hexose monophosphates. By using mutants blocked at a number of glycolytic steps it has been concluded that the metabolic flow takes place along the Embden-Meyerhof pathway in the reverse direction bypassing pyruvate kinase and fructose 6-phosphate kinase. Ethanol acts as a trigger by supplying NADH at the glyceraldehyde 3-phosphate dehydrogenase step. The rate of the reversal in the span phosphoenolpyruvate to fructose 1,6-diphosphate approaches 40 μ mol of 3-carbon units per minute per gram of wet cells. The in vivo activity of fructose 1,6-diphosphatase is nearly a quarter of this rate.     

A new 3-hydroxybutyrate fermenting anaerobe,Ilyobacter polytropus,gen. nov. sp. nov., possessing various fermentation pathways

From marine anoxic mud, a new strictly anaerobic, Gram-negative, non-sporeforming bacterium was isolated with 3-hydroxybutyrate as substrate. 3-Hydroxybutyrate and crotonate were fermented to acetate and butyrate. Glycerol was fermented to 1,3-propanediol and 3-hydroxypropionate. Acetate and formate were the only products of pyruvate or citrate fermentation. Glucose and fructose were fermented to acetate, formate and ethanol. Malate and fumarate were fermented to acetate, formate and propionate. Neither sulfate, sulfur, nor nitrate was reduced. The DNA base ratio was 32.2±0.5 mol% guanine plus cytosine. Strain CuHbu1 is described as type strain of a new genus and species, Ilyobacter polytropus gen. nov. sp. nov., in the family Bacteroidaceae.     

Acute effects of ethanol on the perfused rat liver. Studies on lipid and carbohydrate metabolism, substrate cycling and perfusate amino acids

1. Livers from fed rats were perfused in situ with whole rat blood containing glucose labelled uniformly with (14)C and specifically with (3)H at positions 2, 3 or 6. 2. When ethanol was infused at a concentration of 24mumol/ml of blood the rate of utilization was 2.8mumol/min per g of liver. 3. Ethanol infusion raised perfusate glucose concentrations and caused a 2.5-fold increase in hepatic glucose output. 4. Final blood lactate concentrations were decreased in ethanol-infused livers, but the mean uptake of lactate from erythrocyte glycolysis was unaffected. 5. Production of ketone bodies (3-hydroxybutyrate+3-oxobutyrate) and the ratio [3-hydroxybutyrate]/[3-oxobutyrate] were raised by ethanol. 6. Formation of (3)H(2)O from specifically (3)H-labelled glucoses increased in the order [6-(3)H]<[3-(3)H]<[2-(3)H]. Production of (3)H(2)O from [2-(3)H]glucose was significantly greater than that from [3-(3)H]glucose in both control and ethanol-infused livers. Ethanol significantly decreased (3)H(2)O formation from all [(3)H]glucoses. 7. Liver glycogen content was unaffected by ethanol infusion. 8. Production of very-low-density lipoprotein triacylglycerols was inhibited by ethanol and there was a small increase in liver triacylglycerols. Very-low-density-lipoprotein secretion was negatively correlated with the ratio [3-hydroxybutyrate]/[3-oxobutyrate]. Perfusate fatty acid concentrations and molar composition were unaffected by perfusion with ethanol. 9. Ethanol decreased the incorporation of [U-(14)C]glucose into fatty acids and cholesterol. 10. The concentration of total plasma amino acids was unchanged by ethanol, but the concentrations of alanine and glycine were decreased and ([glutamate]+[glutamine]) was raised. 11. It is proposed that the observed effects of ethanol on carbohydrate metabolism are due to an increased conversion of lactate into glucose, possibly by inhibition of pyruvate dehydrogenase. The increase in gluconeogenesis is accompanied by diminished substrate cycling at glucose-glucose 6-phosphate and at fructose 6-phosphate-fructose 1,6-bisphosphate.     

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.     

Enzymatic analysis of the pathways of glucose catabolism and gluconeogenesis in Pseudomonas citronellolis

Extracts of Pseudomonas citronellolis cells grown on glucose or gluconate possessed all the enzymes of the Entner-Doudoroff pathway. Gluconokinase and either or both 6-phosphogluconate dehydratase and KDPG aldolase were induced by growth on these substrates. Glucose and gluconate dehydrogenases and 6-phosphofructokinase were not detected. Thus catabolism of glucose proceeds via an inducible Entner-Doudoroff pathway. Metabolism of glyceraldehyde 3-phosphate apparently proceeded via glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase. These same enzymes plus triose phosphate isomerase were present in lactate-grown cells indicating that synthesis of triose phosphates from gluconeogenic substrates also occurs via this pathway. Extracts of lactate grown-cells possessed fructose diphosphatase and phosphohexoisomerase but apparently lacked fructose diphosphate aldolase thus indicating either the presence of an aldolase with unusual properties or requirements or an alternative pathway for the conversion of triose phosphate to fructose disphosphate. Cells contained two species of glyceraldehyde 3-phosphate dehydrogenase, one an NAD-dependent enzyme which predominated when the organism was grown on glycolytic substrates and the other, an NADP-dependent enzyme which predominated when the organism was grown on gluconeogenic substrates.     

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.     

Interrelation of aerobic glycolysis and lipogenesis in isolated perfused liver of well-fed rats

The rates of glycolysis and lipogenesis in isolated perfused liver of well-fed rats were studied. When liver was allowed to synthesize [14C]glycogen prior to perfusion, no more than 9% of the degraded [14C]glycogen was recovered in lactate and 6% in lipid. Addition of glucose, fructose and sorbitol enhanced concomitantly the formation of lactate and pyruvate and the rate of release of triglyceride and free fatty acid. Glucose was less efficient than fructose or sorbitol. The incorporation of 14C from these 14C-labelled substrates into lactate, pyruvate and lipids confirmed their role as carbon sources. Incorporation of 14C into the glycerol moiety of neutral lipid exceeded that found in the fatty acids, suggesting that these substrates contributed largely to the esterification of fatty acids. The total rate of de novo fatty acid synthesis was correlated with the formation of lactate and pyruvate. It is concluded that increased rates of aerobic glycolysis are related to increased rates of lipogenesis.     

Interrelationship of islet metabolism, adenosine triphosphate content and insulin release

The oxidation of some exogenous substrates and their effects on ATP content and insulin release in mouse pancreatic islets were measured. The ATP concentration of islets incubated without exogenous substrate shows a gradual decrease, which can be prevented by glucose or mannose (20mm) or leucine (2.5mm); d-glyceraldehyde (5mm) is as effective as glucose (5mm); fructose or N-acetylglucosamine (20mm), pyruvate (10mm) and dl-3-hydroxybutyrate (2mm) are less effective; galactose (20mm), acetate (10mm), octanoate (2mm) and succinate (10mm) have no ATP-maintaining ability. Islets oxidize glucose, mannose, glyceraldehyde, leucine and, less readily, N-acetylglucosamine and glucosamine; galactose, however, is poorly metabolized. Mannoheptulose inhibits the oxidation of glucose but not of glyceraldehyde. Insulin release, measured over a 2h incubation, is stimulated by glucose, mannose, leucine, glyceraldehyde or glucosamine but not by fructose or N-acetylglucosamine. The latter, however, potentiates the effects of glucose or glyceraldehyde (5mm) or leucine (2.5mm) on release; the potentiating effects are inhibited by mannoheptulose, which also blocks glucose-, but not glyceraldehyde- or leucine-stimulated release. In the presence of glucose (20mm), metabolic inhibitors depress insulin release and islet ATP content in parallel. However, rates of insulin release and ATP content measured after incubation with various combinations of exogenous substrates do not appear to be correlated. Sulphonylureas stimulate insulin release but decrease islet ATP concentrations. These results provide further evidence of a close association between the metabolic activity of exogenous substrates and their ability to initiate insulin release. Glucoreceptor models are formulated in the light of these observations and discussed.     

Metabolic pathways involved in lipogenesis from lactate and acetate in bovine adipose tissue: Effects of metabolic inhibitors

Metabolic inhibitors were used in vitro in an attempt to elucidate the biochemical pathways by which lactate is converted to fatty acids by bovine adipose tissue. Subcutaneous adipose tissue samples were obtained by biopsy techniques from steers fed a high-energy ration. Kynurenate (α-2-diamino-γ-oxabenzenebutanoic acid) (5–10 mm), an inhibitor of acetyl-CoA carboxylase, and cerulenin (2,3-epoxy-4-oxo-7,10-dodecadienamide) (20–100 μg/ml), an inhibitor of the fatty acid synthetase enzyme complex, inhibited fatty acid synthesis from both acetate and lactate. The hydrogen acceptor, N-methylphenazonium methosulfate (10 μm) inhibited acetate but not lactate incorporation into fatty acids. α-Cyanohydroxycinnamate (5 mm) and phenylpyruvate (10 mm), which inhibit pyruvate entry into the mitochondria and pyruvate carboxylase, respectively, decreased lipogenesis from both acetate and lactate. The effects of phenylpyruvate on lipogenesis from acetate were greater in the presence of glucose plus insulin. Agaric acid (2-hydroxy-1,2,3-nonadecanetricarboxylic acid) (0.2 and 1.0 mm), which inhibits citrate efflux from the mitochondria also decreased lipogenesis from both acetate and lactate. Fluoroacetate (2.5 mm), an inhibitor of aconitate hydratase, had no effect on lipogenesis from acetate; but, in the presence of glucose or pyruvate, decreased lactate incorporation into fatty acids. n-Butylmalonate (5 mm), which blocks malate transport across the mitochondrial membrane, decreased lipogenesis from lactate but not acetate. Malate transport during lipogenesis is not associated with an operative malate:asparate shuttle in bovine adipose tissue, as indicated by the lack of effect of either 0.2 or 1.0 mm aminooxyacetate, a transaminase inhibitor, on lipogenesis from acetate or lactate. The results suggest a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine subcutaneous adipose tissue and that this pathway may be involved in lipogenesis from acetate as well as lactate.     

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 effect of 2,4-dinitrophenol on adipose-tissue metabolism

1. The effect of dinitrophenol on the metabolism of glucose labelled with (14)C and tritium by epididymal fat-pad segments from fed rats was studied. Dinitrophenol at concentrations of 0.1-0.3mm: (a) had little effect on glucose utilization; (b) depressed synthesis of fatty acids and greatly increased that of lactate; (c) increased the T/(14)C ratio in fatty acids synthesized from [U-(14)C,3-T]glucose and decreased that in fatty acids synthesized from [U-(14)C,4-T]glucose; (d) abolished randomization of (14)C from [6-(14)C]glucose in lactate. 2. Dinitrophenol stimulated oxidation of pyruvate and greatly inhibited the oxidation of lactate. It inhibited lipogenesis from pyruvate and lactate. 3. From the isotope data it was calculated that: (a) dinitrophenol stimulates oxidation via the tricarboxylic acid cycle three- to six-fold; (b) dinitrophenol depresses markedly the operation of the pentose cycle; (c) in the presence of dinitrophenol, NADPH formed in the pentose cycle provides all the hydrogen equivalents for fatty acid reduction, whereas, in its absence, NADPH provides 50-70% of the hydrogen equivalents; (d) in the presence of dinitrophenol, there is an excess of ATP produced in the cytoplasm, which flows into the mitochondria. A reverse flow operates in the absence of dinitrophenol. 4. A balance of formation and utilization of reduced nicotinamide nucleotides in the cytoplasm was established. With dinitrophenol there is some excess of NADH. There are indications that this excess may be transferred into mitochondria in the form of malate. 5. Our results are interpreted to indicate the absence from adipose tissue of the alpha-glycerophosphate shuttle for transferring reducing equivalents from the cytoplasm to mitochondria. 6. The effects of dinitrophenol are accounted for in terms of decreased ATP concentrations in the cells, leading to marked decrease in pyruvate carboxylation in the mitochondria and depression of fatty acid synthesis in the cytoplasm.