Pseudoketogenesis in the perfused rat heart

Ketogenesis is usually measured in vivo by dilution of tracers of (3R)-hydroxybutyrate or acetoacetate. We show that, in perfused working rat hearts, the specific activities of (3R)-hydroxybutyrate and acetoacetate are diluted by isotopic exchanges in the absence of net ketogenesis. We call this process pseudoketogenesis. When hearts are perfused with buffer containing 2.3 mM of [4-3H]- plus [3-14C]acetoacetate, the specific activities of [4-3H] and [3-14C]acetoacetate decrease while C-1 of acetoacetate becomes progressively labeled with 14C. This is explained by the reversibility of reactions catalyzed by mitochondrial 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. After activation of labeled acetoacetate, the specific activity of acetoacetyl-CoA is diluted by unlabeled acetoacetyl-CoA derived from endogenous fatty acids or glucose. Acetoacetyl-CoA thiolase partially exchanges 14C between C-1 and C-3 of acetoacetyl-CoA. Finally, 3-oxoacid-CoA transferase liberates weakly labeled acetoacetate which dilutes the specific activity of extracellular acetoacetate. An isotopic exchange in the reverse direction is observed when hearts are perfused with unlabeled acetoacetate plus [1-14C]-, [13-14C]-, or [15-14C]palmitate; here also, acetoacetate becomes labeled on C-1 and C-3. Computations of specific activities of (3R)-hydroxybutyrate, acetoacetate, and acetyl-CoA yield minimal rates of pseudoketogenesis ranging from 19 to 32% of the net uptake of (3R)-hydroxybutyrate plus acetoacetate by the heart.     

Contribution of omega-oxidation to fatty acid oxidation by liver of rat and monkey.

Contributions of omega-oxidation to overall fatty acid oxidation in slices from livers of ketotic alloxan diabetic rats and of fasted monkeys are estimated. Estimates are made from a comparison of the distribution of 14C in glucose formed by the slices from omega-14C-labeled compared to 2-14C-labeled fatty acids of even numbers of carbon atoms and from [1-14C]acetate compared to [2-14C]acetate. These estimates are based on the fact that 1) the dicarboxylic acid formed via omega-oxidation of a omega-14C-labeled fatty acid will yield [1-14C]acetate and [1-14C]succinate on subsequent beta-oxidation, if beta-oxidation is assumed to proceed to completion; 2) only [2-14C]acetate will be formed if the fatty acid is metabolized solely via beta-oxidation; and 3) 14C from [1-14C]acetate and [1-14C]succinate is incorporated into carbons 3 and 4 of glucose and 14C from [2-14C]acetate is incorporated into all six carbons of glucose. From the distributions found, the contribution of omega-oxidation to the initial oxidation of palmitate by liver slices is estimated to between 8% and 11%, and the oxidation of laurate between 17% and 21%. Distributions of 14C in glucose formed from 14C-labeled palmitate infused into fasted and diabetic rats do not permit quantitative estimation of the contribution of omega-oxidation to fatty acid oxidation in vivo. However, the distributions found also indicate that, of the fatty acid metabolized by the whole animal in the environment of glucose formation, at most, only a minor portion is initially oxidized via omega-oxidation. As such, omega-oxidation cannot contribute more than a small extent to the formation of glucose.     

Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6)

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.     

Acetoacetate is a cholesterogenic precursor for myelinating rat brain and spinal cord. Incorporation of label from [3-14C]acetoacetate, [14C]glucose and 3H2O

Rat pups, 3 weeks old, were injected i.p. with combinations of 3H2O and either [3-14C]acetoacetate or [14C]glucose. 3H/14C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [14C]glucose was distributed between fatty acids and sterols in the same way as 3H from 3H2O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.     

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5–8-day old rats. Cultures containing 60–80% oligodendrocytes were incubated for 16h with [3-¹⁴C]acetoacetate, d-[3-¹⁴C]3-hydroxybutyrate, [U-¹⁴C]glucose, l-[U-¹⁴C]glutamine and [1-¹⁴C]pyruvate or [2-¹⁴C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO₂ was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.     

Omega oxidation of fatty acids and the pathway of 3-hydroxybutyric acid formation

Pathways followed by the carbons of long chain fatty acids in their conversion to 3-hydroxybutyric acid were traced and the contribution of ω-oxidation to fatty acid oxidation was determined in the cellular environment where ketone body formation occurs. 1-¹⁴C-, 2-¹⁴C-, and ω-¹⁴C-labeled fatty acids were injected into alloxan-induced diabetic rats in ketosis. 3-Hydroxybutyric acid was isolated from their urines and degraded. About 1.2 to 1.4 times as much ¹⁴C was found in carbon 1 as carbon 3 of 3-hydroxybutyric acid when the 1-¹⁴C-labeled fatty acids were injected and in carbon 2 as carbon 4 when the 2-¹⁴C-labeled fatty acids were injected. There was about 4 times as much incorporation into carbon 4 as carbon 2 of 3-hydroxybutyric acid formed from the ω-¹⁴C-labeled fatty acids. This means that 50% or more of the fatty acids were oxidized, so that the terminal two carbons of the fatty acids were converted to acetoacetyl-CoA without acetyl-CoA as an intermediate. Incorporation of ¹⁴C into carbons 1 and 2 of the hydroxybutyric acid reflects the distribution of ¹⁴C in acetyl-CoA. Incorporation into carbon 1 was very small when the ω-¹⁴C-labeled fatty acids were substrate. This means that ω-oxidation of fatty acids makes, at most, a small contribution to the formation of the acetyl-CoA pool from which acetoacetate is derived.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Metabolism of acetyl-CoA by isolated peroxisomal fractions: formation of acetate and acetoacetyl-CoA.

Liver peroxisomal fractions, isolated from rats treated with clofibrate, were shown to hydrolyze added [1-14C]acetyl-CoA to free [1-14C]acetate. [1-14C]Acetyl-CoA was, however, also converted to [14C]acetoacetyl-CoA. This reaction was inhibited by added ATP and by solubilization of the peroxisomes. The effect of ATP on synthesis of [14C]acetoacetyl-CoA was likely due to ATP-dependent stimulation of acetyl-CoA hydrolase (EC 3.1.2.1) activity. The inhibitory effect due to solubilizing conditions of incubation remains unexplained. During peroxisomal beta-oxidation of [1-14C]palmitoyl-CoA, [1-14C]acetyl-CoA, [1-14C]acetate, and [14C]acetoacetyl-CoA were shown to be produced. Possible metabolic implications of peroxisomal acetoacetyl-CoA synthesis are discussed.     

Peroxisomal and mitochondrial oxidation of fatty acids in the heart, assessed from the 13C labeling of malonyl-CoA and the acetyl moiety of citrate

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,12-13C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal beta-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.     

Acute Effect of Ammonia on Branched-Chain Amino Acid Oxidation and Incorporation into Proteins in Astrocytes and in Neurons in Primary Cultures

14CO2 production and incorporation of label into proteins from the labeled branched-chain amino acids, leucine, valine, and isoleucine, were determined in primary cultures of neurons and of undifferentiated and differentiated astrocytes from mouse cerebral cortex in the absence and presence of 3 mM ammonium chloride. Production of 14CO2 from [1-14C]leucine and [1-14C]valine was larger than 14CO2 production from [U-14C]leucine and [U-14C]valine in both astrocytes and neurons. In most cases more 14CO2 was produced in astrocytes than in neurons. Incorporation of labeled branched-chain amino acids into proteins varied with the cell type and with the amino acid. Addition of 3 mM ammonium chloride greatly suppressed 14CO2 production from [1-14C]-labeled branched chain amino acids but had little effect on 14CO2 production from [U-14C]-labeled branched-chain amino acids in astrocytes. Ammonium ion, at this concentration, suppressed the incorporation of label from all three branched-chain amino acids into proteins of astrocytes. In contrast, ammonium ion had very little effect on the metabolism (oxidation and incorporation into proteins) of these amino acids in neurons. The possible implications of these findings are discussed, especially regarding whether they signify variations in metabolic fluxes and/or in magnitudes of precursor pools.     

Lipid metabolism. Evidence of a δ-oxidation pathway for saturated fatty acids

1. Specific radioactivities of milk triglyceride fatty acids and gamma- and delta-hydroxy fatty acids were measured after the intramammary infusion of [1-(14)C]acetate, delta-hydroxy[1-(14)C]laurate and [1-(14)C]laurate as their sodium salts into fed lactating goats. 2. Net incorporations of the radioactive tracer into the total milk lipids were comparable, being 16, 17 and 21% of the label infused respectively. 3. The specific radioactivities of the C(4)-C(8) fatty acids after [1-(14)C]acetate infusion were lower than those of the C(10)-C(14) fatty acids. 4. After delta-hydroxy[1-(14)C]laurate administration the milk triglyceride fatty acids were labelled and their specific radioactivities were characterized by decreasing values with increasing chain length of the fatty acids, implicating C(4) unit incorporation. 5. The gamma- and delta-hydroxy fatty acids isolated after [1-(14)C]laurate infusion were highly labelled and the milk triglyceride fatty acids, other than laurate, exhibited a labelling pattern similar to that of the fatty acids derived from the radioactive delta-hydroxy fatty acid. 6. Evidence is presented for the existence of saturated fatty acid delta-oxidation in the mammary gland, in which the gamma- and delta-hydroxy fatty acids are active intermediates.     

Regulation of flux through pyruvate dehydrogenase and pyruvate carboxylase in rat hepatocytes. Effects of fatty acids and glucagon

The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions. The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1-14C]pyruvate into 14CO2 plus [14C]glucose. The flux through PDH was determined from the rate of formation of 14CO2 from [1-14C]pyruvate corrected for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by subtracting the flux through PDH from the total pyruvate metabolized. With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h-starved rats. In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59-76%) and PC (80-83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats. In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC. 2[5(4-Chlorophenyl)pentyl]oxirane-2-carboxylate (100 microM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate-stimulated but not the octanoate-stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the beta-oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation. It is concluded that the effects of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.     

Regulation of gluconeogenesis and lipogenesis. The regulation of mitochondrial pyruvate metabolism in guinea-pig liver synthesizing precursors for gluconeogenesis

1. The carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase in guinea-pig liver mitochondria was determined by measuring the amount of (14)C from H(14)CO(3) (-) fixed into organic acids in the presence of pyruvate, ATP, Mg(2+) and P(i). The main products of pyruvate carboxylation were malate, fumarate and citrate. Pyruvate utilization, metabolite formation and incorporation of (14)C from H(14)CO(3) (-) into these metabolites in the presence and the absence of ATP were examined. The synthesis of phosphoenolpyruvate from pyruvate and bicarbonate is minimal during continued oxidation of pyruvate. Larger amounts of phosphoenolpyruvate are formed from alpha-oxoglutarate than from pyruvate. Addition of glutamate, alpha-oxoglutarate or fumarate did not appreciably increase formation of phosphoenolpyruvate when pyruvate was used as substrate. With alpha-oxoglutarate as substrate addition of fumarate resulted in increased formation of phosphoenolpyruvate, whereas addition of succinate inhibited phosphoenolpyruvate formation. In the presence of added oxaloacetate guinea-pig liver mitochondria synthesized phosphoenolpyruvate in amount sufficiently high to play an appreciable role in gluconeogenesis. 2. Addition of fatty acids of increasing carbon chain length caused a strong inhibition of pyruvate oxidation and phosphoenolpyruvate formation, and greatly promoted carbon dioxide fixation and malate, citrate and acetoacetate accumulation. The incorporation of (14)C from H(14)CO(3) (-), [1-(14)C]pyruvate and [2-(14)C]pyruvate into organic acids formed was examined. 3. It is concluded that guinea-pig liver pyruvate carboxylase contributes significantly to gluconeogenesis and that fatty acids and metabolites play an important role in its regulation.     

Selective inhibition of cholesterol synthesis by cell-free preparations of rat liver by using inhibitors of cytoplasmic acetoacetyl-coenzyme A thiolase.

Cytoplasmic acetoacetyl-CoA thiolase (acetyl-CoA C-acetyltransferase, EC 2.3.1.9) was partially purified from rat liver. The enzyme was irreversibly inactivated by 4-bromocrotonyl-CoA, but-3-ynoyl-CoA, pent-3-ynoyl-CoA and dec-3-ynoyl-CoA. In the case of the alk-3-ynoyl-CoA esters the potency as alkylating agents of acetoacetyl-CoA thiolase decreased with increased chain length of the alk-3-ynoyl moiety. Advantage was taken of the specific action of alk-3-ynoyl-CoA esters on acetoacetyl-CoA thiolase to show that in a postmitochondrial fraction from rat liver they are effective inhibitors of cholesterol synthesis from sodium [2-14C]acetate under conditions when mevalonate conversion into cholesterol and fatty acid synthesis are unafffected. Short-chain alk-3-ynoic acids have little effect on sterol synthesis, although dec-3-ynoic acid is an effective inhibitor owing to its conversion into the CoA ester by the microsomal fatty acyl-CoA synthetase.     

Role of acetoacetyl-CoA synthetase in acetoacetate utilization by tumor cells

Tumors of peripheral tissues contain low levels of succinyl CoA-acetoacetate CoA transferase activity which is not induced in vitro by prolonged cultivation in 2.5 mM DL-3-hydroxybutyrate. Although this enzyme is considered to be the main agent controlling the extent to which ketone bodies serve as metabolic substrates such tumors metabolize D(-)-3-hydroxy[3(14)C]butyrate to 14CO2. Also addition of 3-hydroxybutyrate and/or acetoacetate reduces the amount of 14CO2 produced from D-[U-14C] glucose suggesting a common metabolic intermediate. These observations can be accounted for by the presence of acetoacetyl-CoA synthetase, an enzyme which is able to synthesize acetoacetyl-CoA directly from acetoacetate, ATP and coenzyme A. This is the first demonstration of this enzyme in tumor tissue. The rate of metabolism of acetoacetate by this enzyme is sufficient to account for the production of CO2 from 3-hydroxybutyrate.     

omega-Oxidation of fatty acids and the acetylation p-aminobenzoic acid.

p-Aminobenzoic acid was fed to normal and alloxan-induced diabetic rats injected with [omega-14C]labeled and [2-14C]labeled fatty acids. The p-acetamidobenzoic acid that was excreted was hydrolyzed to yield acetate which was degraded. The distribution of 14C in the acetates formed when an [omega-14C]labeled fatty acid was injected was similar to that when a [2-14C]labeled fatty acid was injected. This contrasts with the finding that in acetates from 2-acetamido-4-phenylbutyric acid excreted when 2-amino-4-phenylbutyric acid was fed, there was a difference in the distributions of 14C, a difference attributable to omega-oxidation of the fatty acid. Acetylation of p-aminobenzoic acid is then concluded to occur in a different cellular environment than that of 2-amino-4-phenylbutyric acid, one in which omega-oxidation is not functional. When 2-amino-4-phenylbutyric acid was fed and [6-14C]palmitic acid injected, rather than [16-14C]palmitic acid, the distribution of 14C in acetate was the same as when [2-14C]palmitic acid was injected. This indicates that the dicarboxylic acid formed on omega-oxidation of palmitic acid does not undergo beta-oxidation to form succinyl-CoA. Thus, glucose is not formed via omega-oxidation of long-chain fatty acid.     

Role of the Blood-Brain Barrier in the Formation of Long-Chain ω-3 and ω-6 Fatty Acids from Essential Fatty Acid Precursors

Elongated, more highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis and mechanism of entry are not well characterized. To investigate the role of the blood-brain barrier in this process, cultured murine cerebromicrovascular endothelia were incubated with [1-14C]18:2 omega-6 or [1-14C]18:3 omega-3 and their elongation/desaturation products determined. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary product from 18:3 omega-3 was 20:5 omega-3. Although these products were found primarily in cell lipids, they were also released from the cells and gradually accumulated in the extracellular fluid. Eicosanoid production was observed from the 20:4 omega-6 and 20:5 omega-3 that were formed. No 22:5 omega-6 or 22:6 omega-3 fatty acids were detected, suggesting that these endothelial cells are not the site of the final desaturation step. Although the uptake of 18:3 omega-3 and 18:2 omega-6 was nearly identical, 18:3 omega-3 was more extensively elongated and desaturated. Competition experiments demonstrated a preference for 18:3 omega-3 by the elongation/desaturation pathway. These findings suggest that the blood-brain barrier can play an important role in the elongation and desaturation of omega-3 and omega-6 essential fatty acids during their transfer from the circulation into the brain.     

3-Hydroxy-3-methylgutaryl-CoA synthase. Participation of acetyl-S-enzyme and enzyme-S-hydroxymethylgutaryl-SCoA intermediates in the reaction.

Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.     

Activities of enzymes of acetoacetate metabolism in rat brown adipose tissue during development.

The activities of two mitochondrial enzymes concerned in the utilization of acetoacetate, namely 3-oxoacid CoA-transferase and acetoacetyl-CoA thiolase, were high throughout the suckling and weanling period in brown adipose tissue of the rat. In contrast, 3-hydroxybutyrate dehydrogenase activity was comparatively low during this period. The activity of cytosolic acetoacetyl-CoA synthetase (involved in lipogenesis) declined after birth and remained low until the pups were weaned. Experiments with brown-adipose-tissue slices from weanling rats indicated that 70% of the [3-14C]acetoacetate utilized was oxidized to 14CO2, and this value was not altered appreciably by the addition of glucose and insulin.     

Pyruvate metabolism by lymphocytes: evidence for an additional ketogenic tissue

The rate of utilization of pyruvate (at various concentrations) was measured in lymphocytes prepared from rat mesenteric lymph nodes. The quantitative contribution of pyruvate to CO2, lactate, aspartate, alanine, citrate, acetate, acetyl-CoA and ketone bodies accounted for the pyruvate metabolized. Pyruvate utilization was depressed by increasing concentrations of pyruvate. The maximum catalytic activities and selected intracellular distributions of the following enzymes of pyruvate, citrate and acetyl-CoA metabolism were measured: citrate synthase, ATP-citrate lyase, lactate dehydrogenase, acetyl-CoA hydrolase, acetylcarnitine transferase, NAD+- and NADP+- isocitrate dehydrogenases, HMG-CoA lyase, HMG-CoA synthase, Pyruvate dehydrogenase, acetoacetyl-CoA thiolase, 3-oxoacid-CoA transferase, 3-hydroxybutyrate dehydrogenase and pyruvate carboxylase. Acetyl-CoA formed from pyruvate did not contribute to the respiratory energy metabolism of resting lymphocytes. Instead acetyl-CoA was converted to acetoacetate by reactions which may favour the pathway catalyzed by acetoacetyl-CoA thiolase and 3-oxoacid-CoA transferase. Acetate, acetyl- and palmitoyl-carnitine inhibited the decarboxylation of [1-14C] pyruvate. These observations may be connected with the suppression of pyruvate utilization by increased pyruvate substrate concentration. Only very small amounts of either pyruvate or acetate were incorporated into lipids in resting lymphocytes. The amounts incorporated were partitioned in approximately the same pattern into FFA, T.G., cholesterol and cholesterol esters. Taken together the data show that pyruvate metabolism is directed inter alia at the formation of acetoacetate which may serve as a lipid synthesis precursor. When pyruvate utilization and metabolism was enhanced by concanavalin A, then acetoacetate formation was not favoured and from this it is proposed that the acetyl units may then be directed into lipid synthesis and may also make a contribution to the energy metabolism of the activated lymphocyte.