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 inhibition of gluconeogenesis in suckling newborn rats.

Inhibition of gluconeogenesis with 3-mercaptopicolinate in suckling newborn rats caused a fall in blood [glucose], but no change in their high plasma [free fatty acid] and blood [ketone bodies]. Active gluconeogenesis seems to be more important than sparing of glucose by high concentrations of fat-derived substrates for the maintenance of normal blood [glucose] in suckling newborn rats.     

In vivo induction of 4-enoyl-CoA reductase by clofibrate in liver mitochondria and its effect on pent-4-enoate metabolism

Feeding of clofibrate to male rats leads to a 4–7 fold increase in the activity of the 4-enoyl-CoA reductase in the liver. Concomitantly the inhibition of fatty acid oxidation by pent-4-enoate is abolished, and an increased glucose formation in the presence of pent-4-enoate is observed. It is suggested that pent-4-enoate is converted to propionyl-CoA via the reaction sequence pent-4-enoyl-CoA→pent-2,4-dienoyl-CoA→pent-2-enoyl-CoA→propionyl-CoA + acetyl-CoA.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Carbohydrate metabolism

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pent-2-enoic acid, pentanoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on glycolysis, glucose oxidation and gluconeogenesis in some rat tissues were determined. 2. None of the compounds at low concentrations inhibited glycolysis by particle-free supernatant fractions from rat liver, skeletal muscle and intestinal mucosa, though there was inhibition by cyclopropanecarboxylic acid and cyclobutanecarboxylic acid at 3mm concentration. 3. Pent-4-enoic inhibited the oxidation of [1-(14)C]palmitate by rat liver slices, but did not increase the oxidation of [U-(14)C]glucose. 4. Pent-4-enoic acid (0.01mm) strongly inhibited gluconeogenesis by rat kidney slices from pyruvate or succinate, but none of the other compounds inhibited significantly at low concentrations. 5. There was also some inhibition of gluconeogenesis in kidney slices from rats injected with pent-4-enoic acid. 6. The mechanism of the hypoglycaemic effect of pent-4-enoic acid is discussed; it is suggested that there is an inhibition of fatty acid and ketone-body oxidation and of gluconeogenesis so that glucose reserves become exhausted, leading to hypoglycaemia. 7. The mechanism of the hypoglycaemic action of pent-4-enoic acid appears to be similar to that of hypoglycin.     

Effects of pent-4-enoate on cellular redox state, glycolysis and fatty acid oxidation in isolated perfused rat heart

The metabolic effects of pent-4-enoate were studied in beating and potassium-arrested perfused rat hearts. The addition of 0.8mm-pent-4-enoate to the fluid used to perfuse a potassium-arrested heart resulted in a 70% increase in the O(2) consumption and a 66% decrease in the glycolytic flux as measured in terms of the de-tritiation of [3-(3)H]glucose, although the proportion of the O(2) consumption attributable to glucose oxidation decreased from an initial 30% to 10%. The pent-4-enoate-induced increase in O(2) consumption was only 15% in the beating heart. In the potassium-arrested heart, pent-4-enoate stimulated palmitate oxidation by more than 100% when measured in terms of the production of (14)CO(2) from [1-(14)C]palmitate, but in the beating heart palmitate oxidation was inhibited. Perfusion of the heart with pent-4-enoate had no effect on the proportion of pyruvate dehydrogenase found in the active form, in spite of large changes in the CoASH and acetyl-CoA concentrations and changes in their concentration ratios. The effects of pent-4-enoate on the cellular redox state were dependent on the ATP consumption of the heart. In the beating heart, pent-4-enoate caused a rapid mitochondrial NAD(+) reduction that subsequently faded out, so that the final state was more oxidized than the initial state. The arrested heart, however, remained in a more reduced state than initially, even after the partial re-oxidation that followed the initial rapid NAD(+) reduction. The ability of pent-4-enoate to increase or decrease fatty acid oxidation can be explained on the basis of the differential effects of pent-4-enoate on the concentration of citric acid-cycle intermediates under conditions of high or low ATP consumption of the myocardial cell. The proportion of the fatty acids in the fuel consumed by the heart is probably primarily determined by the regulatory mechanisms of glycolysis. When pent-4-enoate causes an increase in the citric acid-cycle intermediates, feedback inhibition of glycolysis results in an increase in the oxidation of fatty acids.     

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.     

Potentiation by ammonia of the metabolic effects of pent-4-enoate in isolated rat hepatocytes.

The metabolic effects of pent-4-enoate were studied in isolated rat hepatocytes; 1 mM-pent-4-enoate did not significantly inhibit gluconeogenesis from lactate, alanine and glycerol, but significantly decreased glucose synthesis from pyruvate. The addition of 1 mM-NH4Cl led to a drastic inhibition of glucose synthesis from all these substrates. In hepatocytes incubated with 10 mM-alanine and 1 mM-oleate, pent-4-enoate at 0.05-1 mM slightly inhibited glucose synthesis and ketogenesis. The addition of ammonia resulted in a dramatic potentiation of the metabolic effects of pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of ATP and acetyl-CoA were also decreased by the addition of ammonia, as were lactate/pyruvate ratio and beta-hydroxybutyrate/acetoacetate ratio. These data suggest that ammonia seriously interferes with the cellular metabolism of pent-4-enoate and leads to a dramatic potentiation of its effects.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of the free acids and their carnitine esters on coenzyme A-dependent oxidations in rat liver mitochondria

1. The synthesis of pent-4-enoyl-l-carnitine, cyclopropanecarbonyl-l-carnitine and cyclobutanecarbonyl-l-carnitine is described. 2. Pent-4-enoate strongly inhibits palmitoyl-l-carnitine oxidation in coupled but not in uncoupled mitochondria. Pent-4-enoyl-l-carnitine strongly inhibits palmitoyl-l-carnitine oxidation in uncoupled mitochondria. Prior intramitochondrial formation of pent-4-enoyl-CoA is therefore necessary for inhibition. 3. There was a small self-limiting pulse of oxidation of pent-4-enoyl-l-carnitine during which the ability to inhibit the oxidation of subsequently added palmitoyl-l-carnitine developed. 4. Pent-4-enoate and pent-4-enoyl-l-carnitine are equally effective inhibitors of the oxidation of all even-chain acylcarnitines of chain length C(4)-C(16). Pent-4-enoyl-l-carnitine also inhibits the oxidation of pyruvate and of 2-oxoglutarate. 5. Pent-4-enoate strongly inhibits the oxidation of palmitate but not that of octanoate. This is presumably due to competition between octanoate and pent-4-enoate for medium-chain acyl-CoA ligase. 6. There was less inhibition of the oxidation of pyruvate by pent-4-enoyl-l-carnitine, and of palmitoyl-l-carnitine by cyclopropanecarbonyl-l-carnitine, after pre-incubation with 10mm-arsenate. This suggests that these inhibitions were caused either by depletion of free CoA or by increase of acyl-CoA concentrations, since arsenate deacylates intramitochondrial acyl-CoA. There was little effect on the inhibition of palmitoyl-l-carnitine oxidation by pent-4-enoyl-l-carnitine. 7. Penta-2,4-dienoate strongly inhibited palmitoyl-l-carnitine oxidation in coupled mitochondria; acrylate only inhibited slightly. 8. Pent-4-enoate (0.1mm) caused a rapid and almost complete decrease in free CoA and a large increase in acid-soluble acyl-CoA when incubated with coupled mitochondria. Cyclopropanecarboxylate caused a similar decrease in CoA, with an equivalent rise in acid-soluble acyl-CoA concentrations. n-Pentanoate caused extensive lowering of CoA and a large increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. Octanoate caused a 50% lowering of CoA and an increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. 9. Cyclopropanecarboxylate and n-pentanoate were less potent inhibitors of palmitate oxidation than was pent-4-enoate. 10. It is concluded that pent-4-enoate causes a specific inhibition of beta-oxidation after the formation intramitochondrially of its metabolites.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Fatty acid oxidation

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pentanoic acid, pent-2-enoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on the oxidation of saturated fatty acids by rat liver mitochondria were determined. 2. The formation of (14)CO(2) from [1-(14)C]palmitate was strongly inhibited by 0.01mm-pent-4-enoic acid. 3. The inhibition of oxygen uptake was less than that of (14)CO(2) formation, presumably because fumarate was used as a sparker. 4. The oxidation of [1-(14)C]-butyrate, -octanoate or -laurate was not strongly inhibited by 0.01mm-pent-4-enoic acid. 5. The other four non-hypoglycaemic compounds did not inhibit the oxidation of any saturated fatty acid when tested at 0.01mm concentration, though they all inhibited strongly at 10mm. 6. The oxidation of [1-(14)C]-myristate and -stearate, but not of [1-(14)C]decanoate, was strongly inhibited by 0.01mm-pent-4-enoic acid. 7. The oxidation of [1-(14)C]palmitate was about 50% carnitine-dependent under the experimental conditions used. 8. The percentage inhibition of [1-(14)C]palmitate oxidation by pent-4-enoic acid was the same whether carnitine was present or not. 9. Acetoacetate formation from saturated fatty acids was inhibited by 0.1mm-cyclopropanecarboxylic acid to a greater extent than their oxidation. 10. The other compounds tested inhibited acetoacetate formation from saturated fatty acids proportionately to the inhibition of oxidation. 11. Possible mechanisms for the inhibition of long-chain fatty acid oxidation by pent-4-enoic acid are discussed. 12. There was a correlation between the ability to inhibit long-chain fatty acid oxidation and hypoglycaemic activity in this series of compounds.     

Effect of pent-4-enoic acid, propionic acid and other short-chain fatty acids on citrulline synthesis in rat liver mitochondria.

19 The effect of pent-4-enoic acid, propionic acid and several other short-chain fatty acids on citrulline synthesis in rat liver mitochondria was studied. 2.Pent-4-enoate at 1 mM inhibited mitochondrial citulline synthesis by about 80-90%. It is concluded that pent-4-enoate inhibits citrulline synthesis by interfering with some aspect of mitochondrial energy metabolism. This results in impairment of mitochondrial ornithine uptake or depletion of mitochondrial ATP, which, in turn, impairs carbamoyl phosphate synthesis or both. Evidence in support of this conclusion includes: pent-4-enoate has no effect on citrulline synthesis supported by succinate or exogenous ATP; pent-4-enoate lowers the medium plus mitochondrial ATP concentration; finally, when glutamate is the oxidizable substrate, pent-4-enoate decreases the carbamoyl phosphate concentration in mitochondria incubated without ornithine to minimize citrulline synthesis and impairs the mitochondrial uptake of ornithine, but it has neither effect when succinate is the oxidizable substrate. 4. Propionate, butyrate and crotonate also inhibit mitochondrial citrulline synthesis, but much less than pent-4-enoate. 5. Acetate, pentanoate, pent-2-enoate, hexanoate, octanoate, isovalerate, tiglylate and alpha-methylbutyrate have little or no effect on mitochondrial citrulline synthesis.     

The effects of inhibition of gluconeogenesis on ketogenesis in starved and diabetic rats.

Experiments were performed in which the effects of inhibiting gluconeogenesis on ketone-body formation were examined in vivo in starved and severely streptozotocin-diabetic rats. The infusion of 3-mercaptopicolinate, an inhibitor of gluconeogenesis (DiTullio et al., 1974), caused decreases in blood [glucose] and increases in blood [lactate] and [pyruvate] in both normal and ketoacidotic rats. Patterns of liver gluconeogenic intermediates after 3-mercaptopicolinate infusion suggested inhibition at the level of phosphoenolpyruvate carboxykinase. This was confirmed by measurement of hepatic oxaloacetate concentrations which were increased 5-fold after 3-mercaptopicolinate administration. The infusion of 3-mercaptopicolinate caused a decrease in total ketone-body concentrations of 30% in starved rats and 73% in the diabetic animals. Blood glycerol and hepatic triglyceride concentrations remained unchanged. The decreases in ketone-body concentrations were associated with increases in the calculated hepatic cytosolic and mitochondrial [NADH]/[NAD+] ratios. The decrease in ketogenesis seen after inhibition of gluconeogenesis may have resulted from an inhibition of hepatic fatty acid oxidation by the more reduced mitochondrial redox state. It was concluded that gluconeogenesis may stimulate ketogenesis by as much as 30% in severe diabetic ketoacidosis.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of their coenzyme A esters on enzymes of fatty acid oxidation

1. Pent-4-enoyl-CoA and its metabolites penta-2,4-dienoyl-CoA and acryloyl-CoA, as well as n-pentanoyl-CoA, cyclopropanecarbonyl-CoA and cyclobutanecarbonyl-CoA, were examined as substrates or inhibitors of purified enzymes of beta-oxidation in an investigation to locate the site of inhibition of fatty acid oxidation by pent-4-enoate. 2. The reactions of various acyl-CoA derivatives with l-carnitine and of various acyl-l-carnitine derivatives with CoA, catalysed by carnitine acetyltransferase, were investigated and V(max.) and K(m) values were determined. Pent-4-enoyl-CoA and n-pentanoyl-CoA were good substrates, whereas cyclobutanecarbonyl-CoA, cyclopropanecarbonyl-CoA and acryloyl-CoA reacted more slowly. A very slow rate with penta-2,4-dienoyl-CoA was detected. Pent-4-enoyl-l-carnitine, n-pentanoyl-l-carnitine and cyclobutanecarbonyl-l-carnitine were good substrates and cyclopropanecarbonyl-l-carnitine reacted more slowly. 3. Pent-4-enoyl-CoA and n-pentanoyl-CoA were substrates for butyryl-CoA dehydrogenase and for octanoyl-CoA dehydrogenase, and both compounds were equally effective competitive inhibitors of these enzymes with butyryl-CoA or palmitoyl-CoA respectively as substrates. V(max.), K(m) and K(i) values were determined. 4. None of the acyl-CoA derivatives inhibited enoyl-CoA hydratase or 3-hydroxybutyryl-CoA dehydrogenase. Penta-2,4-dienoyl-CoA was a substrate for enoyl-CoA hydratase when the reaction was coupled to that catalysed by 3-hydroxybutyryl-CoA dehydrogenase. 5. In a reconstituted sequence with purified enzymes crotonoyl-CoA was largely converted into acetyl-CoA, and pent-2-enoyl-CoA into acetyl-CoA and propionyl-CoA. Penta-2,4-dienoyl-CoA was slowly converted into acetyl-CoA and acryloyl-CoA. 6. Penta-2,4-dienoyl-CoA, a unique metabolite of pent-4-enoate, was the only compound that specifically inhibited an enzyme of the beta-oxidation sequence, 3-oxoacyl-CoA thiolase. The formation of penta-2,4-dienoyl-CoA could explain the strong inhibition of fatty acid oxidation in intact mitochondria by pent-4-enoate.     

Isolated rat heart mitochondria are able to metabolize pent-4-enoate to tricarboxylic acid-cycle intermediates.

The metabolism of four short-chain odd-number-carbon fatty acids, pentanoate, pent-4-enoate, propionate and acrylate, was studied in isolated rat heart mitochondria incubated in [14C]bicarbonate buffer. Under these conditions pentanoate was metabolized with a concomitant accumulation of malate and incorporation of 14CO2 into non-volatile compounds. The metabolism of propionate to tricarboxylic acid-cycle intermediates required the addition of ATP and oligomycin. After addition of a small amount of rotenone to the incubation medium, pent-4-enoate was metabolized with an increase in malate from less than 3 nmol/mg of protein to 34.0 +/- 1.5 nmol/mg in 40 min, during which time the amount of 14CO2 fixed in acid-stable compounds increased from 1.56 +/- 0.30 to 41.1 +/- 2.6 nmol/mg of protein. Acrylate was not metabolized under any of the conditions tested. The results show that cardiac mitochondria must have an enzyme system that is capable of reducing the double bond of either pent-4-enoate or its metabolities. That the metabolism of pent-4-enoate occurs through a reductive step and energy-dependent carboxylation is evident from the requirement for NAD+ reduction by partial inhibition of the mitochondrial respiratory chain and the presence of ATP and CO2. The results do not enable us to say whether the compound reduced is pent-4-enoyl-CoA or acryloyl-CoA.     

Decreased hepatic fatty acid oxidation at weaning in the rat is not linked to a variation of malonyl-CoA concentration

In rats weaned on a high-carbohydrate diet, hepatic fatty acid oxidation capacity is decreased when compared to suckling rats. Previous studies (Benito et al., 1979) suggested that a malonyl-CoA-dependent mechanism could be at the origin of this decrease. Studies on isolated hepatocytes show that despite, respectively, a low and a high lipogenic rate in suckling and weaned rats, malonyl-CoA concentrations are similar in the two groups. This might be due to the lower ratio fatty acid synthetase/acetyl-CoA carboxylase (EC 6.4.1.2) activities during suckling than after weaning. Different rates of hepatic fatty acid oxidation despite similar malonyl-CoA concentrations can be explained by the 2.5-fold higher carnitine palmitoyltransferase I (EC 2.3.1.21) activity in suckling rats together with a 7-fold higher Ki for malonyl-CoA. This precludes a tight control of fatty acid oxidation by [malonyl-CoA] in suckling rats. Weaning on a high-fat carbohydrate-free diet abolishes the changes previously described for the kinetic characteristics of carnitine palmitoyltransferase I suggesting that nutritional modifications rather than a developmental stage are involved. Thus, during the suckling-weaning transition, a variation of [malonyl-CoA] is not responsible for the decrease in hepatic fatty acid oxidation. It involves, in addition, a decrease in carnitine palmitoyltransferase I activity and an increase of the sensitivity of this enzyme to malonyl-CoA.     

Likely individuality of the enzymes catalyzing the phosphorylation of choline and ethanolamine.

Dichloroacetate has effects upon hepatic metabolism which are profoundly different from its effects on heart, skeletal muscle, and adipose tissue metabolism. With hepatocytes prepared from meal-fed rats, dichloroacetate was found to activate pyruvate dehydrogenase, to increase the utilization of lactate and pyruvate without effecting an increase in the net utilization of glucose, to increase the rate of fatty acid synthesis, and to decrease slightly [1-¹⁴C]oleate oxidation to ¹⁴CO₂ without decreasing ketone body formation. With hepatocytes isolated from 48-h-starved rats, dichloroacetate was found to activate pyruvate dehydrogenase, to have no influence on net glucose utilization, to inhibit gluconeogenesis slightly with lactate as substrate, and to stimulate gluconeogenesis significantly with alanine as substrate. The stimulation of fatty acid synthesis by dichloroacetate suggests that the activity of pyruvate dehydrogenase can be rate determining for fatty acid synthesis in isolated liver cells. The minor effects of dichloroacetate on gluconeogenesis suggest that the regulation of pyruvate dehydrogenase is only of marginal importance in the control of gluconeogenesis.     

Effects of adrenaline on ketogenesis from long- and medium-chain fatty acids in starved rats.

1. Injection of adrenaline into 24 h-starved rats caused a 69% decrease in blood [ketone-body] (3-hydroxybutyrate plus acetoacetate), accompanied by a decreased [3-hydroxybutyrate]/[acetoacetate] ratio. Blood [glucose] and [lactate] increased, but [alanine] was unchanged. 2. Adrenaline also decreased [ketone-body] after intragastric feeding of both long- and medium-chain triacylglycerol. The latter decrease was observed after suppression of lipolysis with 5-methylpyrazole-3-carboxylic acid, indicating that the antiketogenic action of adrenaline was not dependent on the chain length of the precursor fatty acid. 3. The actions of adrenaline to decrease blood [ketone-body] and to increase blood [glucose] were not observed after administration of 3-mercaptopicolinate, an inhibitor of gluconeogenesis. This suggests that these effects of the hormone are related. 4. The possible clinical significance of the results is discussed with reference to the restricted ketosis often observed after surgical or orthopaedic injury.     

Protection of rats by clofibrate against the hypoglycaemic and toxic effects of hypoglycin and pent-4-enoate. An ultrastructural and biochemical study.

An ultrastructural and biochemical study of the toxic and hypoglycaemic effects of hypoglycin and pent-4-enoate was made on the livers of normal and clofibrate-fed rats. Injection of hypoglycin to rats doubles (from 22% to 44%) the volume fraction of mitochondria and decreases (from 1.05% to 0.26%) the volume fraction of peroxisomes in hepatocytes. The fast-acting toxin pent-4-enoate causes few ultrastructural changes except for the accumulation of lipids. In male adult rats fed with 0.5% clofibrate in their diet for 1-2 months, the volume fraction occupied by peroxisomes and mitochondria in hepatocytes rose to 6.26% and 29.5% respectively. Clofibrate feeding apparently protected the animals against the toxic, hypoglycaemic and hypothermic effects of hypoglycin and of pent-4-enoate, and completely prevented the ultrastructural damage caused by hypoglycin. After hypoglycin administration, hepatic mitochondrial butyryl-CoA dehydrogenase activity was inhibited by more than 90% and, surprisingly, the activity of the peroxisomal enzymes studied was largely preserved. When hypoglycin was given to rats fed on a clofibrate-containing diet, the oxidation of decanoylcarnitine, which was incomplete after hypoglycin treatment alone, remained incomplete with uncoupled mitochondria, but became apparently complete with coupled mitochondria. In the latter condition, there was a slowing of the rate during the last quarter of the pulse of oxygen uptake. Further, butyryl-CoA dehydrogenase activity was much less affected by hypoglycin in clofibrate-fed animals. Pent-4-enoate does not inhibit beta-oxidation in coupled mitochondria from clofibrate-treated rats.     

Effects of 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate on fatty acid and glucose metabolism in perfused rat hearts determined using iodine-125 16-iodohexadecanoate

Long-chain fatty acid oxidation by the isolated perfused rat heart was assayed by external counting using [125I]16-iodohexadecanoic acid as substrate after administration of the hypoketonemic and hypoglycemic compound 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate to rats. Glucose metabolism was also assessed by measuring release of tritium from [2-T]glucose. The oxidation of long chain fatty acids was virtually suppressed in hearts from fed or starved rats given 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate while glucose utilization was increased 2-2.5 fold.     

Effect of L-alanine infusion on gluconeogenesis and ketogenesis in the rat in vivo.

1. In 48 h-starved 6-week-old rats the 14C incorporation in vivo into blood glucose from a constant-specific-radioactivity pool of circulating [14c]actateconfirmed that lactate is the preferred gluconeogenic substrate. 2. Increasing the blood [alanine] to that occurrring in the fed state increased 14C incorporation into blood glucose 2.3-fold from [14c]alanine and 1.7-fold from [14c]lactate. 3. When the blood [alanine] was increased to that in the fed state, the 14C incorporation into liver glycogen from circulating [14c]alanine or [14c]lactate increased 13.5- and 1.7-fold respectively. 4. The incorporation of 14C into blood acetoacetate and 3-hydroxybutyrate from a constant-specific-radioactivity pool of circulating [14c]oleate was virtually abolished by increasing the blood [alanine] to that existing in the fed state. However, the [acetoacetate] remained unchanged, whereas [3-hydroxybutyrate] decreased, although less rapidly than did its radiochemical concentration. 5. It is concluded that during starvation in 6-week-old rats, the blood [alanine] appears to influence ketogenesis for circulating unesterfied fatty acids and inversely affects gluconeogenesis from either lactate or alanine. A different pattern of gluconeogenesis may exist for alanine and lactate as evidenced by comparative 14C incorporation into liver glycogen and blood glucose.     

Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid (TDGA) on glucose and fatty acid oxidation in isolated rat soleus muscle

1. The effect of 2-tetradecylglycidic acid (TDGA), a potent, specific inhibitor of long-chain fatty acid oxidation, on fatty acid and glucose oxidation by isolated rat soleus muscle was studied. 2. TDGA inhibited [1-14C]palmitate oxidation by soleus muscle in a concentration-dependent manner. 3. TDGA inhibited the activity of soleus muscle mitochondrial carnitine palmitoyltransferase A (CPT-A). 4. Added palmitate (0.5 mM) significantly inhibited D-[U-14C]glucose oxidation and, under conditions where TDGA inhibited palmitate oxidation, the oxidation of D-[U-14C]glucose by isolated soleus muscle was significantly stimulated. 5. TDGA stimulation of glucose oxidation was reversed by octanoate, a medium-chain fatty acid whose oxidation is not inhibited by TDGA. 6. When nondiabetic rats were treated with TDGA (10 mg/kg p.o./day x 3 days), fasting plasma glucose was significantly lowered and the ability of isolated contralateral soleus muscles to oxidize palmitate was inhibited while glucose oxidation was significantly stimulated.