Long-chain acyl-CoA Esters Research Articles

Effect of dietary fat on individual long-chain fatty acyl-CoA esters in rat liver and skeletal muscle.

The effect of dietary fat on the long-chain acyl-CoA ester profile of liver and skeletal muscle was investigated by feeding weanling rats 12%-fat diets composed of high-linoleic safflower oil (73% 18:2n-6), high-oleic safflower oil (70% 18:1n-9) or olive oil (70% 18:1n-9) for six and ten weeks. Approximately 50% of both hepatic and skeletal muscle acyl-CoA esters comprised linoleoyl-CoA or oleoyl-CoA with high-linoleic or oleic feeding, respectively. Total hepatic acyl-CoA ester concentration was 40% higher (p less than 0.05) in rats fed 12% fat compared with controls fed a 4%-fat diet. These data demonstrate that the long-chain acyl-CoA ester profile of liver and skeletal muscle reflects the dietary fatty acid profile.

Determination of individual long-chain fatty acyl-CoA esters in heart and skeletal muscle.

A method has been developed for determination of individual long-chain fatty acyl-CoA esters from heart and skeletal muscle using high performance liquid chromatography (HPLC). The esters were extracted from freeze-clamped tissue of pig and rat hearts and rat skeletal muscle for analysis on a radially compressed C18 5mu reverse-phase column. Nine peaks in the extract with carbon chain lengths from C12 to C20 that subsequently disappeared on alkaline hydrolysis were identified. The major acyl-CoA peaks were 14:1, 18:2, 16:0 and 18:1 and additionally in rat heart 18:0. Total long-chain acyl-CoA esters obtained by summation of the individual molecular species was 11.34 +/- 1.48 nmol/g wet wt. pig heart; 14.51 +/- 2.11 nmol/g wet wt. in rat heart, and 4.35 +/- 0.71 nmol/g wet wt. in rat skeletal muscle. These values were approximately 132% of those obtained using a separate procedure that measured total CoA by HPLC after alkaline hydrolysis of the esters. The described method demonstrates the quantitation of individual acyl-CoA species in muscle tissue. Therefore, it has a number of advantages in that it permits information to be obtained on the individual molecular species under various nutritional and metabolic conditions.

Adaptive changes in individual acyl-CoA esters from hamster BAT during cold acclimation

Long-chain fatty acyl-CoA esters (LCFACoAE) were extracted from freeze-clamped powdered brown adipose tissue (BAT) obtained from thermoneutral control and cold-acclimated hamsters and the CoA esters individually separated by high-performance liquid chromatography. LCFACoAE of carbon chain length C12 to C20 were identified by increasing column retention time in the following order: C12:0, C14:1, C14:0, C16:1, C18:2, C16:0, C18:1, C18:0, and C20:4. The mean total LCFACoAE concentrations were 235 +/- 40 nmol/g protein for the control hamsters and 648 +/- 105 nmol/g protein for the 22-day cold-acclimated hamsters. A rapid fourfold increase in the levels of C16:0, C18:0, and C18:1 occurred within hours after initiation of the cold temperature, whereas the concentrations of the other six LCFACoAE either increased only slightly or remained unchanged. Almost 50% of the total LCFACoAE in the BAT of cold-acclimated hamsters was made up of C16:0, C18:0, and C18:1. These results, which demonstrate some dynamic changes in adipose tissue LCFACoAE, are consistent with their proposed role in the initiation and maintenance of BAT thermogenesis.

Peroxisomal beta-oxidation of long-chain fatty acids possessing different extents of unsaturation.

Rates of peroxisomal beta-oxidation were measured as fatty acyl-CoA-dependent NAD+ reduction, by using solubilized peroxisomal fractions isolated from livers of rats treated with clofibrate. Medium- to long-chain saturated fatty acyl-CoA esters as well as long-chain polyunsaturated fatty acyl-CoA esters were used. Peroxisomal beta-oxidation shows optimal specificity towards long-chain polyunsaturated acyl-CoA esters. Eicosa-8,11,14-trienoyl-CoA, eicosa-11,14,17-trienoyl-CoA and docosa-7,10,13,16-tetraenoyl-CoA all gave Vmax. values of about 150% of that obtained with palmitoyl-CoA. The Km values obtained with these fatty acyl-CoA esters were 17 +/- 6, 13 +/- 4 and 22 +/- 3 microM respectively, which are in the same range as the value for palmitoyl-CoA (13.8 +/- 1 microM). Myristoyl-CoA gave the higher Vmax. (110% of the palmitoyl-CoA value) of the saturated fatty acyl-CoAs tested. Substrate inhibition was mostly observed with acyl-CoA esters giving Vmax. values higher than 50% of that given by palmitoyl-CoA.

Quantitative determination of acyl chain composition of subnanomole amounts of cellular long-chain acyl-coenzyme A esters

A procedure for the quantitative determination of the acyl chain composition of cellular long-chain acyl-CoA esters in subnanomole amounts is described. The abundant cellular lipids of samples are removed by extraction with organic solvents, and the proteins are precipitated from the aqueous phase by the addition of acetonitrile. The CoA thiolesters are adsorbed on neutral alumimum oxide and reduced with sodium borohydride to the corresponding alcohols that are then converted to t-butyldimethylsilyl ethers and analyzed quantitatively by gas chromatography. Saturated and unsaturated acyl chains behaved similarly throughout the procedure, and the common lipid esters do not interfere with the analysis of the CoA esters in the final assay procedure described. This simple and relatively rapid method is suitable for analyzing a large number of samples at a time.

Extraction of tissue long-chain acyl-CoA esters and measurement by reverse-phase high-performance liquid chromatography

Long-chain acyl-CoA esters were extracted from freeze-clamped livers of fed and fasted rats according to the method of Mancha et al. [ M. Mancha, G. B. Stokes, and P. K. Stumpf (1975) Anal. Biochem. 68, 600–608] and analyzed on a radially compressed C 18, 5 μm, reverse-phase column using a gradient system consisting of acetonitrile and 25 m m KH 2PO 4, pH 5.3, at 254 nm. Total analysis time was 25 min. Eight peaks in the extract with carbon chain lengths of 12 to 18, which subsequently disappeared on alkaline hydrolysis, were identified. The major acyl-CoA peaks in the extract in order of increasing retention times were 14:0, 16:1, 18:2, 16:0, 18:1, and 18:0. Total liver long-chain acyl-CoA esters were 108 ± 11 and 248 ± 19 nmol/g protein for fed and fasted rats, respectively. On fasting (48 h) the levels of 18:2, 16:0, and 18:1 increased two-to threefold and that of 18:0 sixfold. The advantages of this method are that it not only provides a more direct determination of total tissue long-chain acyl-CoA esters, in that no decomposition of the CoA ester is involved, but it also detects the constituent molecular species.

Recovery of ventricular function in reperfused ischemic rat hearts exposed to fatty acids.

The relationship between tissue levels of fatty acid metabolites in ischemic and reperfused hearts and recovery of mechanical function of these hearts on reperfusion was studied. Isolated rat hearts were exposed to global ischemia for periods up to 60 min under various conditions of coronary flow, O2 supply, and fatty acid concentrations and were then reperfused for either 15 or 30 min under aerobic conditions both with and without fatty acids present. Tissue levels of ATP, creatine phosphate, long-chain acyl CoA, and long-chain acyl carnitine were determined at the end of the ischemic and reperfusion periods. In some experiments K+ arrest during ischemia was used to prevent adenine nucleotide depletion both in the absence and presence of high fatty acids. Although the ability of these hearts to recover their preischemic mechanical function varied from 8 to 90% and tissue levels of acyl CoA and acyl carnitine during ischemia varied from 3- to 10-fold depending on the condition, no correlation was found between the recovery of function during reperfusion and either the presence of fatty acid or high levels of tissue long-chain acyl CoA and carnitine esters during ischemia.

Phosphofructokinase and the Regulation of the Flux of Carbon from Glucose to Lipid in the Oleaginous Yeast Rhodosporidium toruloides

Summary: Changes in cell composition of Rhodosporidium toruloides CBS 14 were monitored during growth of batch cultures with NH4Cl and glutamate as nitrogen sources. Carbohydrate was synthesized at the expense of lipid in NH4 +-grown cells, whereas in glutamate-grown cells lipid accumulation was predominant. Total biomass and protein concentration were similar in both cultures. Uptake of [U-14C], [1-14C] and [6-14C]glucose, and evolution of 14CO2 from these sources, by washed suspensions of cells grown on glutamate revealed the flux of carbon during glucose dissimilation was principally via the Embden-Meyerhof pathway (72%), with 28% being metabolized by the pentose phosphate pathway. Both urea and glutamate, when added to the cell suspensions, significantly stimulated glucose catabolism, with the flux of carbon via the former pathway increasing to about 89% of the total. Phosphofructokinase (PFK) was implicated as the likely controlling enzyme to explain these events. PFK was only detected in extracts prepared from the yeast grown in a carbon-limited (nitrogen-excess) medium; no activity was detected in extracts of cells grown in nitrogen-limited medium. The presence of a protease in these latter extracts was revealed. PFK was purified 92-fold to a final specific activity (in the presence of 10 mM-NH4 +) of 4.2 units (mg protein)−1 and exhibited one broad band on polyacrylamide gel electrophoresis. The apparent mol. wt (Mt ) of the enzyme was approx. 700000. The major properties of the enzyme were examined to determine its regulatory role during lipid biosynthesis. Unlike the enzyme from Saccharomyces cerevisiae, no inhibition was found with 10 mM-ATP. ADP was not inhibitory either. NH4 + ions increased activity 11-fold by increasing the affinity of the enzyme for both fructose 6-phosphate and ATP. K+ ions also stimulated activity but to a lesser extent. Activity was severely inhibited by citrate, isocitrate and cis-aconitate but this inhibition was dramatically alleviated by NH4 +. Inhibition by citrate was competitive with fructose 6-phosphate in the absence of NH4 + ions. The K i values for citrate were 1.0 mM (with no NH4 +) and 7.2 mM (with 10 mM-NH4 +). Long-chain fatty acyl-CoA esters had no significant inhibitory effect. It is concluded that the interplay between the prevailing intracellular concentrations of NH4 + and citrate is the major determinant of the activity of PFK in vivo and thus governs the extent to which glucose is converted either to lipid or carbohydrate.

Inhibition of the adenine nucleotide translocator by matrix-localized palmityl-CoA in rat heart mitochondria

The activity of the adenine nucleotide translocator in rat heart mitochondria was quantitatively determined by the rate of [ 14C]ATP transport at 2°C using the carboxyatractyloside inhibitor-stop technique. Linear uptake was obtained for 15 s, and with differing protein concentrations. The effect of matrix long-chain acyl-CoA esters upon the adenine nucleotide translocator activity was determined in these mitochondria. Incubation with palmitylcarnitine produced an increase in matrix long-chain acyl-CoA esters and decreased the velocity of [ 14C]ATP transport. Mitochondria isolated in the presence of KCN showed elevated levels of long-chain acyl-CoA esters, decreased transportable nucleotides, and very low adenine nucleotide translocator activity. Addition of potassium ferricyanide to these mitochondria caused a reduction in matrix acyl-CoA esters and partially restored adenine nucleotide translocator activity. Potassium ferricyanide also lowered matrix acyl-CoA in freshly isolated mitochondria and increased [ 14C]ATP transport. These findings show that the level of long-chain acyl-CoA esters within the mitochondrial matrix affects adenine nucleotide translocator activity and regulates mitochondrial activity.

Increase in mitochondrial content of long-chain acyl-CoA in brown adipose tissue during cold-acclimation

1. The mitochondrial content of long-chain acyl-CoA esters in the brown adipose tissue of guinea pigs increased 3.5-fold from a level of 92 ± 17 pmol per mg protein (± S.E.; n = 7) in the control animals adapted at 22°C to a new steady-state level of 328 ± 20 pmol per mg protein (± S.E.; n = 46) after 10 days of cold-acclimation (5°C). These low values of long-chain acyl-CoA species and the slow adaptive response for their increase do not support the proposal (Cannon, B., Sindin, U. and Romert, L. (1977) FEBS Lett. 4, 43–46) that the fatty acid CoA-esters have a physiological function in the regulation of the H + (or OH −) permeability of the mitochondrial inner membrane. 2. Experimental evidence is presented supporting the proposal that the long-chain acyl-CoA species are largely confined to the cytosolic side of the inner membrane. 3. The activity of the adenine nucleotide translocase, as estimated at 25°C in the reverse direction, was found to increase 5-fold upon depletion of the mitochondria of fatty acids (free and esterified) by preincubation with bovine serum albumin. The presence of potent inhibitors i.e., long-chain acyl-CoA species, of adenine nucleotide translocation in brown adipose tissue of thermogenically active animals further supports the conclusion that ATP hydrolyzing mechanisms contribute insignificantly to long-term thermogenesis. 4. The low values of long-chain acyl-CoA hydrolase (EC 3.1.2.1) activity, as measured in intact mitochondria and on a mitochondrial matrix fraction (i.e., 1.6 nmol · min −1 per mg protein), do not support the proposal that the hydrolase activity plays a significant role in the loose-coupling of brown adipose tissue mitochondria, either by a futile cycle mechanism or promoted by free fatty acid-induced uncoupling.

Partial Purification and Some Properties of ATP: Citrate Lyase from the Oleaginous Yeast Lipomyces starkeyi

ATP:citrate lyase has been purified some 20-fold from the oleaginous yeast, Lipomyces starkeyi CBS 1809. The enzyme, which is labile, was stabilized by the addition of ATP and citrate to extraction buffers. Michaelis-Menten kinetics were exhibited towards ATP and citrate. The molecular weight of the enzyme was determined to be approximately 510000. There was a specific requirement for ATP for activity. No activity was observed in the absence of Mg2+, although Co2+ and Mn2+ could partially substitute for Mg2+. ADP inhibited activity (50% inhibition at 1 mM), as did long-chain fatty acyl-CoA esters (50% inhibition with 4m oleoyl-CoA). The possibility of control of ATP:citrate lyase activity by fluctuations in the prevailing energy charge or by changes in the intracellular concentrations of long-chain fatty acyl-CoA esters is discussed.

Regulation of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts by long-chain fatty acyl-CoA esters.

1. Citrate efflux from a wide range of yeast mitochondria was inhibited by long-chain fatty-acyl-CoA esters. 2. Fatty-acyl-CoA esters with chain lengths of between 14 and 18 carbons were the most potent inhibitors of citrate efflux which was unaffected by the fatty acids themselves. 3. 50% inhibition of citrate transport was observed using palmitoyl-CoA and oleoyl-CoA at approx. 4-5 microM when the tricarboxylate carrier was saturated with L-malate as counter-anion. 4. The inhibition with palmitoyl-CoA and oleoyl-CoA was competitive with L-malate. 5. The possibility that the fatty-acyl-CoA esters were exerting their effect by acting as detergent was eliminated because of the low concentrations used and appropriate comparisons being made with non-specific detergents. Although detergents inhibited citrate efflux they also released citrate by causing membrane damage. 6. The effect of fatty-acyl-CoA esters on citrate efflux could be decreased by using higher mitochondrial protein levels and by adding bovine serum albumin. 7. The possibility is discussed that this inhibition represents a genuine feedback inhibition which could regulate the amount of lipid being synthesized by an oleaginous yeast.

Adenine nucleotide transport in hepatoma mitochondria. Characterization of factors influencing the kinetics of ADP and ATP uptake

Initial velocity measurements of [ 3H]ADP and [ 3H]ATP uptake have been made with mitochondria isolated from Morris hepatomas of differing growth rates, and factors known to influence the rates of nucleotide exchange have been examined in an effort to determine whether the elevated rates of aerobic glycolysis in these tumors can be attributed to altered carrier activity. These studies included the determination of the apparent kinetic constants for nucleotide uptake as a function of the mitochondrial energy state and the dependence of transport rates on temperature. Also included in these studies were measurements of the mitochondrial levels of endogenous inhibitors, divalent cations and internal adenine nucleotides. Results obtained showed that with mitochondria isolated from the various tumor lines, the apparent kinetic constants for nucleotide uptake are different from those of control rat or regenerating liver mitochondria; the apparent V max values for both ADP and ATP uptake are significantly lower. Furthermore, under conditions of a high-energy state, the K m and V max values for ATP uptake are greater than the K m and V max value for ADP uptake but that under uncoupled conditions, the opposite is observed. Comparison of the levels of mitochondrial Ca 2+, Mg 2+, long-chain acyl-CoA ester and adenine nucleotide from the various mitochondria showed that important differences exist between liver and hepatoma mitochondria in the levels of Ca 2+, long-chain acyl-CoA ester and AMP. Mitochondrial Ca 2+ levels are elevated 3–5-fold in all tumor lines, and for Morris 7777 hepatoma (a rapidly growing tumor) by a remarkable 70-fold; whereas the levels of acyl-CoA ester and AMP are significantly lower in the more rapidly growing tumors. Arrhenius plots for nucleotide uptake in mitochondria from liver and hepatoma are characterized as being biphasic, having similar activation energies above and below the break point temperature (28–38 and 6–16 kcal/mol, respectively). However, the transition temperature for mitochondria from the various hepatomas is uniformly 4–5°C lower than mitochondria from control liver. The latter difference may reflect a variation in membrane composition, most probably lipid components. It is concluded that the presence of elevated levels of Ca 2+ and lower levels of AMP in hepatoma mitochondria and difference of membrane compositions may play an important role in limiting adenine nucleotide transport activity in vivo and that the impaired carrier activity may contribute to higher rates of aerobic glycolysis observed in these tumors.

Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.

Inhibition of hepatic gluconeogenesis and lipogenesis by benzoic acid, p-tert.-butylbenzoic acid, and a structurally related hypolipidemic agent SC-33459

Benzoic acid, p-tert.-butylbenzoic acid, and a structurally related hypolipidemic agent SC-33459 were found to inhibit glucose synthesis by hepatocytes isolated from 48-h fasted rats as well as fatty acid synthesis by hepatocytes isolated from meal-fed rats. Glucose synthesis was less sensitive than fatty acid synthesis. Benzoic acid was the least effective inhibitor of both processes; SC-33459 and p-tert.-butylbenzoic acid were very potent inhibitors with similar efficacy. Glycine prevented the inhibition of fatty acid synthesis caused by benzoic acid, but had no effect on that caused by p-tert.-butylbenzoic acid. Octanoate opposed the inhibitory effects of both benzoic acid and p-tert.-butylbenzoic acid. Oxidation of [1- 14C]oleate to ketone bodies and acid-soluble radioactive products was inhibited by both p-tert.-butylbenzoic acid and SC-33459. Preincubation of hepatocytes with SC-33459 was required for the latter effect, suggesting catabolism of this compound may be involved. SC-33459 is a p-tert.-butylphenyl derivative which should be readily converted to p-tert.-butylbenzoic acid by β oxidation. Both p-tert.-butylbenzoic acid and SC-33459 decreased citrate levels dramatically. All three compounds reduced CoA and acetyl-CoA levels and increased medium-chain acyl-CoA ester levels. p-tert.-Butylbenzoic acid and SC-33459 also increased long-chain acyl-CoA ester levels. The increase in medium-chain acyl-CoA levels presumably reflects benzoyl-CoA formation from benzoic acid and p-tert.-butylbenzoyl-CoA formation from p-tert.-butylbenzoic acid and SC-33459. Inhibition of glucose and fatty acid synthesis by these compounds may be due to effects on specific enzymes or to CoA sequestration.

Retinol esterification by mammary gland microsomes from the lactating rat.

Because vitamin A in milk is largely present as esterified retinol while blood plasma predominantly contains unesterified retinol, experiments were conducted to determine whether membranes from the lactating mammary gland are able to synthesize retinyl esters in vitro. When microsomes from rats lactating for 7 to 14 days were incubated with [(3)H]retinol dispersed in dimethylsulfoxide, some [(3)H]retinol esterification was observed (147 pmol/5 min per 0.5 mg protein). However, 3- to 7-fold increases in retinyl ester synthesis could be achieved by supplying either a fatty acyl CoA-generating system or preformed fatty acyl CoA thioesters; thus, the major activity in vitro has the characteristics of a fatty acyl CoA: retinol acyltransferase. Both long-chain and medium-chain fatty acyl CoA esters stimulated [(3)H]-labeled retinyl ester synthesis in vitro. Concordantly, analysis of the retinyl ester pattern of rat milk demonstrated the presence of eight different esters of retinol ranging in fatty acyl chain length from 8 to 18 carbons. Retinol esterification by microsomes was maximal at neutral pH (7.1) in the presence of approximately 50 micro M palmitoyl CoA, and was linear with time of incubation for at least 5 min. Retinyl ester synthesis increased with the apparent concentration of [(3)H]retinol to approximately 200 nmol/ml, but was also dependent on the ratio of retinol relative to total microsomal protein in the incubation mixture. These experiments demonstrate for the first time retinol esterification by mammary gland membranes and point to the hypothesis that free retinol from plasma is esterified in this organ before secretion of retinyl esters in milk.-Ross, A. C., Retinol esterification by mammary gland microsomes from the lactating rat.

Carnitine biosynthesis: nutritional implications.

Much interest in carnitine metabolism and function has been shown in recent years with the recognition of its catalytic role in the intramitochondrial transport of fatty acids. Figure 1 shows a series of events requisite for the activation and transport of extramitochondrial long-chain fatty acids such as palmitic acid to the site of β-oxidation in the mitochondrial matrix [cf. Bremer (1977) for discussion and relevant references]. Palmitic acid released from adipose tissue or derived from the diet is activated by outer membrane ATP-dependent palmitoyl-CoA synthetase to form palmitoyl-CoA. Such long-chain fatty acyl-CoA esters have only a limited ability to cross the mitochondrial membrane, but their entry is facilitated by “outer” carnitine palmitoyl transferase which catalyzes a transes-terification reaction in which the palmitoyl moiety from CoA is transferred to carnitine, forming palmitoylcarnitine. The latter ester is then thought to cross the inner mitochondrial membrane through the action of a translocase. A second transesterification reaction now takes place wherein “inner” palmitoyltransferase, located in the inner mitochondrial membrane, regenerates palmitoyl-CoA for subsequent β-oxidation and releases carnitine for a repetition of its catalytic role in overall fatty acid transport.

ATP: Citrate Lyase - The Regulatory Enzyme for Lipid Biosynthesis in Lipomyces starkeyi?

ATP: citrate lyase from Lipomyces starkeyi was inhibited by oleoyl-CoA (50% inhibition at approx. 3 μm) and other long-chain fatty acyl-CoA esters but not by other fatty acyl compounds. The inhibition was readily reversed by adding bovine serum albumin. The molecular size of the protein was not altered even in the presence of 200 μm-oleoyl-CoA. It is concluded that this is a genuine feedback inhibition effect which may be significant in regulating the amount of triacylglycerol being produced by an oleaginous yeast.

Mitochondrial Acyl-CoA, adenine nucleotide translocase activity and oxidative phosphorylation in myocardial ischaemia

The hypothesis that inhibition of mitochondria) adenine nucleotide translocase activity by accumulation of long-chain acyl-CoA esters might be responsible for the depression in mitochondria) energy metabolism in myocardial ischaemia was reinvestigated. Hypoxic, low-flow perfusion (substrate-free) of the isolated rat heart resulted in increases in tissue and mitochondria) acyl-CoA contents together with a reduction in adenine nucleotide translocase activity and oxidative phosphorylation function of mitochondria isolated from the tissue. Addition of exogenous substrates together with insulin or propranolol to the perfusate (glucose and insulin glucose, glycerol and insulin; glucose, propranolol and insulin) caused significant further increases in tissue and mitochondria) acyl-CoA contents. In contrast, the mitochondria) adenine nucleotide translocase activity and oxidative phosphorylation function improved and were similar to those of control perfused hearts. The difference between the in vivo and in vitro effects of acyl-CoA on mitochondria) adenine nucleotide translocase and oxidative phosphorylation could be due to in vivo binding to intracellular fatty acid binding proteins.

Regulation of hepatic fatty acid metabolism. The activities of mitochondrial and microsomal acyl-CoA:sn-glycerol 3-phosphate O-acyltransferase and the concentrations of malonyl-CoA, non-esterified and esterified carnitine, glycerol 3-phosphate, ketone bodies and long-chain acyl-CoA esters in livers of fed or starved pregnant, lactating and weaned rats.

1. The concentrations of malonyl-CoA, glycerol 3-phosphate, non-esterified carnitine, acid-soluble and acid-insoluble acylcarnitines, acetoacetate, 3-hydroxybutyrate and acid-insoluble acyl-CoA were measured in rapidly-frozen liver samples from fed or starved (24h) virgin, pregnant (19-20 days), lactating (2, 10-12 and 18-20 days) and weaned (for 24h, on 10th day of lactation) rats. The activities of total and N-ethylmaleimide-sensitive and -insensitive glycerophosphate acyltransferase (acyl-CoA:sn-glycerol 3-phosphate O-acyltransferase; EC 2.3.1.15) were also measured. 2. The concentration of malonyl-CoA was significantly higher in liver of fed pregnant, mid- and late-lactating rats than in liver of fed virgin rats. After starvation for 24h hepatic malonyl-CoA concentrations were higher in mid-lactating rats and lower in pregnant and weaned rats than in virgin animals. 3. After starvation for 24h the hepatic concentrations of glycerol 3-phosphate, ketone bodies, acid-soluble acylcarnitines and the value for the [3-hydroxybutyrate]/[acetoacetate] ratio were all highest in pregnant rats, intermediate in virgin, 2-day lactating and weaned animals and lowest in mid- and late-lactating rats. The concentrations of acid-insoluble acylcarnitines also increased most in pregnant rats, after starvation. The concentration of acid-insoluble acyl-CoA increased equally after starvation in virgin and pregnant animals but did not increase significantly in all other animals studied. 4. The total concentration of carnitine was similar in livers of fed virgin, pregnant and 2-day lactating animals but fell markedly by the 10th day of lactation and remained low in late-lactating animals. The concentration of non-esterified carnitine followed the same pattern. After starvation for 24h the hepatic concentration of non-esterified carnitine decreased significantly in virgin, pregnant and 2-day lactating animals, but remained unchanged in mid- and late-lactating or weaned animals. 5. The activities of N-ethylmaleimide-sensitive and -insensitive glycerophosphate acyltransferase both increased significantly in livers of mid-lactating animals. After starvation for 24h the activity of the N-ethylmaleimide-insensitive O-acyltransferase decreased in livers of virgin, pregnant and mid-lactating animals, whereas the activity of the N-ethylmaleimide-sensitive O-acyltransferase was unchanged in virgin animals but decreased markedly in livers of pregnant and lactating rats. 6. The results are discussed in relation to the importance of different metabolic parameters in the regulation of long-chain acyl-CoA metabolism in the liver.