Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μm palmitate, palmitoyl-CoA, or palmitoyl-l-carnitine. At palmitoyl-CoA concentrations higher than 30 μm oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO₂. At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of ¹⁴CO₂ and ¹⁴C-labeled perchloric acid-soluble products is higher from [1-¹⁴C]palmitate than that from [U-¹⁴C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of ¹⁴CO₂ production and ¹⁴C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring ¹⁴C-fatty acid oxidation than that of ¹⁴CO₂ production alone.     

Insulin-like effects of a physiologic concentration of carnitine on cardiac metabolism

Pharmacologic (millimolar) levels of carnitine have been reported to increase myocardial glucose oxidation, but whether physiologically relevant concentrations of carnitine affect cardiac metabolism is not known. We employed the isolated, perfused rat heart to compare the effects of physiologic levels of carnitine (50 M) and insulin (75 mU/l [0.5 nM]) on the following metabolic processes: (1) glycolysis (release of ³H₂O from 5-³H-glucose); (2) oxidation of glucose and pyruvate (production of ¹⁴CO₂ from U-¹⁴C-glucose, 1-¹⁴C-glucose, 3,4-¹⁴C-glucose, 1-¹⁴C-pyruvate, and 2-¹⁴C-pyruvate); and (3) oxidation of palmitate (release of ³H₂O from 9,10-³H-palmitate). We found that addition of carnitine (50 M) to a perfusate containing both glucose (10 mM) and palmitate (0.5 mM) stimulated glycolytic flux by 20%, nearly doubled the rate of glucose oxidation, and inhibited palmitate oxidation by 20%. These actions of carnitine were uniformly similar to those of insulin. When carnitine and insulin were administered together, their effects on the oxidation of glucose and palmitate, but not on glycolysis, were additive. When pyruvate (1 mM) was substituted for glucose, neither carnitine nor insulin influenced the rate of oxidation of pyruvate or palmitate. In combination, however, carnitine and insulin sharply suppressed pyruvate oxidation (75%) and doubled the rate of palmitate oxidation. None of the responses to carnitine or insulin was affected by varying the isotopic labeling of glucose or pyruvate. The results show that carnitine, at normal blood levels, exerts insulin-like effects on myocardial fuel utilization. They also suggest that plasma carnitine in vivo may interact with insulin both additively and permissively on the metabolism of carbohydrates and fatty acids     

INTERACTION OF FATTY ACID AND GLUCOSE OXIDATION BY CULTURED HEART CELLS

Chick embryo heart cells in tissue culture actively oxidize [1-¹⁴C]palmitate to ¹⁴CO₂. Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30–70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in ¹⁴CO₂ production from [1-¹⁴C]palmitate. Conversely, palmitate had a similar sparing effect on [¹⁴C]glucose oxidation to ¹⁴CO₂. Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.     

Glycine,Serine, and Leucine Metabolism in Different Regions of Rat Central Nervous System

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-¹⁴C]glycine, [2-¹⁴C]glycine, L-[3-¹⁴C]serine and L-[U-¹⁴C]leucine. We showed that the [1-¹⁴C]glycine oxidation to CO₂ in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-¹⁴C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-¹⁴C]leucine oxidation was lower than the [1-¹⁴C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO₂ in all structure studied. In adult rats brains, the highest oxidation of [1-¹⁴C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-¹⁴C]leucine was significantly higher than that from glycine in all regions and ages studied.     

Lipopolysaccharide (LPS) increases thein vivo oxidation of branched-chain amino acids in the rat: A cytokine-mediated effect

Septic rats (as induced by cecal puncture and ligation) showed an increased rate ofin vivo leucine oxidation as measured from the formation of¹⁴CO₂ from an intravenously injected [1-¹⁴C]leucine tracer dose. Acute lipopolysaccharide (LPS) administration (1 mg/kg) to rats caused a similar effect on the rate ofin vivo leucine oxidation. Additionally, both tumour necrosis factor- (TNF) and interleukin-1- (IL-1), in an acute dose of 100 g/kg, also increased the rate of the oxidation of the amino acid, although only IL-1 caused a similar increase to that observed following LPS. The observed increased leucine oxidation was related to lower leucine concentrations both in LPS- and cytokine-treated rats. Important decreases were also observed in the other branched-chain amino acids (valine and isoleucine) in the LPS- and IL-1-treated animals. Isolated incubated muscles from TNF- and IL-1-treated rats did not show any changes in the rate of leucine utilization, thus suggesting that the mechanism by which the cytokines stimulate whole-body leucine oxidation is not based on an increase in the activity of the enzymatic machinery responsible for leucine oxidation. Additionally, glucocorticoids do not seem to mediate the enhancedin vivo oxidation of the amino acid since, although they are increased by both LPS and cytokines, treatment of the animals with RU486 (a glucocorticoid antagonist) was not able to suppress the effects of the cytokine onin vivo leucine oxidation.     

Measuring plasma fatty acid oxidation with intravenous bolus injection of 3H- and 14C-fatty acid

Accurate measures of plasma FA oxidation can improve our understanding of diseases characterized by impaired FA oxidation. We describe and compare the 24 h time-courses of FA oxidation using bolus injections of [1-¹⁴C]palmitate versus [9,10-³H]palmitate under postabsorptive, postprandial, and walking conditions. Fifty-one men and 95 premenopausal women participated in one condition (postabsorptive, postprandial, or walking), one tracer (¹⁴C- or ³H-labeled), and an acetate or palmitate study. Groups were matched for sex, age, and body mass index (BMI). At 24 h, cumulative [³H]acetate recovery as ³H₂O was 80 ± 6%, 78 ± 2%, and 81 ± 6% in the postabsorptive, postprandial, and walking conditions, respectively (not significant). Model-predicted maximum [1-¹⁴C]acetate recovery as expired ¹⁴CO₂ was 59 ± 12%, 52 ± 8%, and 65 ± 10% in the postabsorptive, postprandial, and walking condition, respectively (one way ANOVA, P = 0.12). When corrected with the corresponding acetate recovery factors, 24 h time-courses of FFA oxidation were similar between [1-¹⁴C]palmitate and [9,10-³H]palmitate in all three conditions. In contrast to previous meal ingestion studies, an acetate-hydrogen recovery factor was needed to achieve comparable oxidation rates using an intravenous bolus of [³H]palmitate. In conclusion, intravenous boluses of [9,10-³H]palmitate versus [1-¹⁴C]palmitate gave similar estimates of 24 h cumulative FFA oxidation in age-, sex- and BMI-matched individuals.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

Comparison of linoleate,palmitate and acetate metabolism in rat ventral prostate

Rat ventral prostate incorporated (1-¹⁴C)acetate, (1-¹⁴C)palmitate and (1-¹⁴C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI+LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+LPE. Acetate showed the highest oxidation to CO₂ whereas no differences were observed in the radioactivity incorporated into CO₂ from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO₂ oxidation.Abbreviations PL total phospholipids - PC choline glycerophospholipids - PE ethanolamine glycerophospholipids - PI+LPE inositol glycerophospholipids plus lysoethanolamine glycerophospholipids - PS serine glycerophospholipids - SM sphingomyelin     

Comparison of oxidative metabolism in starved, fat-fed and carbohydrate-fed rats

1. Rats were starved for 48hr. or fed for 1 week on a high-fat or a high-carbohydrate diet. The effects of these dietary alterations on the rate of production of ¹⁴CO₂ from trace amounts of [U-¹⁴C]glucose, [1-¹⁴C]palmitate or [1-¹⁴C]acetate administered intravenously were studied. 2. The oxidation of [¹⁴C]glucose was most rapid in the carbohydrate-fed condition and was decreased significantly and to the same extent after starvation and after feeding with fat. 3. Under all dietary regimes studied the maximum rate of elimination of ¹⁴CO₂ from [¹⁴C]palmitate occurred within a few minutes after injection, but considerably more was oxidized after starvation and feeding with fat than after feeding with carbohydrate. 4. Alterations in diet had no effect on the oxidation and high recovery of administered [¹⁴C]acetate as ¹⁴CO₂. 5. Graphical analysis showed the presence of several exponential components in the ¹⁴CO₂-elimination curves. 6. In all studies a marked similarity in oxidative pattern was noted between the starved and the fat-fed rat.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Effect of α-Ketoisocaproate and Leucine on the in Vivo Oxidation of Glutamate and Glutamine in the Rat Brain

Leucine and -ketoisocaproate (-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or -KIC on the oxidation of L-[U-¹⁴C]glutamate and L-[U-¹⁴C]glutamine in the brain were determined in the non-anesthetized rat. ¹⁴CO₂ generated by the metabolic oxidation of [^l4C]glutamate and [¹⁴C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and -KIC exhibited differential effects on ¹⁴CO₂ generation from radioactive glutamate or glutamine. Infusion of 0.5 mM -KIC increased [^l4C]glutamate oxidation approximately 2-fold; higher concentrations of -KIC did not further stimulate [¹⁴C]glutamate oxidation. The enhanced oxidation of [¹⁴C]glutamate may be attributed to the function of -KIC as a nitrogen acceptor from [¹⁴C]glutamate yielding [¹⁴C]-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [¹⁴C-]glutamine oxidation was not stimulated as much as [¹⁴C-]glutamate oxidation and only increased at 10 mM -KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [¹⁴C]glutamate or [¹⁴C]glutamine. In maple syrup urine disease elevated -KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.     

In vitro substrate utilization for lipid synthesis in liver explants from hyperthyroid chickens

• 1.1. Indian River male broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 18, 24 and 30% protein + 0 or 1 mg triiodothyronine (T₃)/kg of diet to study energetic costs of lipogenesis and the use of various substrates for in vitro lipogenesis.
• 2.2. De novo lipid and CO₂ production were determined in the presence of [1-¹⁴C]pyruvate, [2-¹⁴q]pyruvate, [3-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine.
• 3.3. Oxygen consumption was determined in mitochondrial preparations to estimate the energetic costs in expiants synthesizing lipid.
• 4.4. Radiolabeled CO₂ derived from [1-¹⁴C]pyruvate was used as an estimate of coenzyme A availability in liver expiants. Lipids derived from [2-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine estimate relative substrate efficiency.
• 5.5. Labeled CO₂ production from [1-¹⁴C]pyruvate was greatest in that group fed a 12% protein diet and least in the group fed a 30% protein diet.
• 6.6. In addition, T₃ increased CO₂ production from [1-¹⁴C]pyruvate.
• 7.7. The production of ¹⁴CO₂ from the second carbon of pyruvate or acetate was increased by T₃.
• 8.8. The low-protein diet (12% protein) increased (P <0.05) lipogenesis.
• 9.9. Adding T₃ to the diets decreased carbon flux into lipid from all substrates, but increased CO₂ production from all substrates without changing stage 3 and 4 respiration rates in mitochondrial preparations.
• 10.10. These observations imply that coenzyme A availability may have regulated de novo lipogenesis in the present study.
• 11.11. It was also concluded that previously noted effects of T₃ on intermediary metabolism may involve metabolic pathways that do not involve changes in mitochondrial function.

     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

Nutrient absorption,storage and remobilization in octopus vulgaris

Average absorption and conversion to ¹⁴CO₂for free leucine included in a meal were 96% and 30% after 24 h. The values for glucose were 98% and 48% and for palmitate 46% and 12.5%. Muscle was the major repository of leucine (38% of the total ingested) and glucose (44%), but the digestive gland contained most of the palmitate (20%).

During normal feeding ¹⁴CO₂ production from octopuses given leucine dropped to low stable levels after only 2 days, those given glucose required 4 to 5, but palmitate apparently did not enter a stable reserve. Fasting increased the release of ¹⁴CO₂ from octopuses given palmitate and leucine, but glucose animals showed little change. A 5‐fold increase in ¹⁴CO₂ production during forced exercise after fasting by octopuses given glucose may indicate increased carbohydrate catabolism. Reduced ¹⁴CO₂production in exercise after other substrates is consistent with this, suggesting muscle carbohydrate reserves may be exclusively for locomotion.

A comparison of nutrient uptake, catabolism and growth suggests that lipids are the limiting nutrient for octopuses on a crab diet.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Effects of dietary protein contents and habitual endurance exercise on supplemental leucine oxidation in mice

The effects of dietary protein contents and regular exercise on the oxidation of supplemented leucine were examined. In the short-term study, male BALB/cCrSlc mice were fed diets containing 0, 10, 20, 35, and 60% protein: energy ratios for 1 week. In the long-term study, exercised and sedentary mice were fed diets containing 20, 35, and 60% protein ratios for 9 weeks. After the feeding periods, the mice were a bolus administered oral supplements of l-[1-¹³C] leucine. Expired gas was analyzed, and oxidized leucine was expressed as a relative ¹³CO₂/¹²CO₂ ratio. In the short-term study, the peak ¹³CO₂/¹²CO₂ ratio significantly increased with diet protein concentrations. Moreover, the long-term study also showed that the peak ¹³CO₂/¹²CO₂ ratio was significantly increased by high protein diets in both exercised and sedentary mice. Our results indicate that supplemental leucine oxidation is associated with consumption of a high-protein diet, irrespective of exercise status.

Abbreviations: AUC: area under the curve; EX: exercise; RQ: respiratory quotient; SED: sedentary; VO₂/W: oxygen uptake per body weight     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.