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Sensitivity of carnitine acyltransferase I to malonly-CoA inhibition in isolated rat liver mitochondria is quantitatively related to hepatic malonyl-CoA concentration in vivo.

ABSTRACT: The sensitivity of carnitine acyltransferase I (EC 2.3.1.21) activity to malonyl-CoA inhibition in rat liver mitochondria isolated from animals in various physiological states was quantitatively proportional to the hepatic malonyl-CoA concentration in vivo. It is suggested that this relationship between the two parameters could result in a potent amplification mechanism for the reciprocal regulation of fatty acid synthesis and oxidation.

SUBMITTER: Robinson IN

PROVIDER: S-EPMC1158567 | biostudies-other | 1982 Jul

REPOSITORIES: biostudies-other

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L-carnitine acyltransferase in intact peroxisomes is inhibited by malonyl-CoA.

Project description:Inhibition of the overt mitochondrial carnitine palmitoyltransferase by malonyl-CoA is important in the regulation of fatty acid oxidation. In the past, the contribution of peroxisomal carnitine acyltransferase activity to the generation of medium- and long-chain acylcarnitines in the cytoplasm has been ignored. On the basis of marker enzyme levels, we now estimate that peroxisomal palmitoyltransferase activity constitutes about 20% of the peroxisomal plus overt-mitochondrial pool in fed rat liver. When assayed in situ, both the palmitoyltransferase and decanoyltransferase activities of gradient-purified peroxisomes are sensitive to malonyl-CoA, with up to 90% inhibition reached at less than 10 microM-malonyl-CoA. Very similar results were obtained with intact gradient-purified mitochondria from the same livers. In addition, the acyl-CoA substrate chain-length specificity was identical in both the peroxisomes and the mitochondria, with a decanoyltransferase/palmitoyltransferase ratio of 2. Thus the overt carnitine acyltransferase activities in peroxisomes and mitochondria have the same properties. Further, the malonyl-CoA sensitivity of the peroxisomal activity is lost on solubilization, as has been observed for the overt mitochondrial enzyme. It is suggested that malonyl-CoA inhibition of the peroxisomal enzyme as well as of the mitochondrial enzyme is important for the regulation of mitochondrial fatty acid oxidation.

| S-EPMC1133344 | biostudies-other

Insulin regulates enzyme activity, malonyl-CoA sensitivity and mRNA abundance of hepatic carnitine palmitoyltransferase-I.

Project description:The regulation of hepatic mitochondrial carnitine palmitoyltransferase-I (CPT-I) was studied in rats during starvation and insulin-dependent diabetes and in rat H4IIE cells. The Vmax. for CPT-I in hepatic mitochondrial outer membranes isolated from starved and diabetic rats increased 2- and 3-fold respectively over fed control values with no change in Km values for substrates. Regulation of malonyl-CoA sensitivity of CPT-I in isolated mitochondrial outer membranes was indicated by an 8-fold increase in Ki during starvation and by a 50-fold increase in Ki in the diabetic state. Peroxisomal and microsomal CPT also had decreased sensitivity to inhibition by malonyl-CoA during starvation. CPT-I mRNA abundance was 7.5 times greater in livers of 48-h-starved rats and 14.6 times greater in livers of insulin-dependent diabetic rats compared with livers of fed rats. In H4IIE cells, insulin increased CPT-I sensitivity to inhibition by malonyl-CoA in 4 h, and sensitivity continued to increase up to 24 h after insulin addition. CPT-I mRNA levels in H4IIE cells were decreased by insulin after 4 h and continued to decrease so that at 24 h there was a 10-fold difference. The half-life of CPT-I mRNA was 4 h in the presence of actinomycin D or with actinomycin D plus insulin. These results suggest that insulin regulates CPT-I by inhibiting transcription of the CPT-I gene.

| S-EPMC1135974 | biostudies-other

Carnitine acyltransferase activities in rat brain mitochondria. Bimodal distribution, kinetic constants, regulation by malonyl-CoA and developmental pattern.

Project description:Carnitine palmitoyltransferase and carnitine octanoyltransferase activities in brain mitochondrial fractions were approx. 3-4-fold lower than activities in liver. Estimated Km values of CPT1 and CPT2 (the overt and latent forms respectively of carnitine palmitoyltransferase) for L-carnitine were 80 microM and 326 microM, respectively, and K0.5 values for palmitoyl-CoA were 18.5 microM and 12 microM respectively. CPT1 activity was strongly inhibited by malonyl-CoA, with I50 values (concn. giving 50% of maximum inhibition) of approx. 1.5 microM. In the absence of other ligands, [2-14C]malonyl-CoA bound to intact brain mitochondria in a manner consistent with the presence of two independent classes of binding sites. Estimated values for KD(1), KD(2), N1 and N2 were 18 nM, 27 microM, 1.3 pmol/mg of protein and 168 pmol/mg of protein respectively. Neither CPT1 activity, nor its sensitivity towards malonyl-CoA, was affected by 72 h starvation. Rates of oxidation of palmitoyl-CoA (in the presence of L-carnitine) or of palmitoylcarnitine by non-synaptic mitochondria were extremely low, indicating that neither CPT1 nor CPT2 was likely to be rate-limiting for beta-oxidation in brain. CPT1 activity relative to mitochondrial protein increased slightly from birth to weaning (20 days) and thereafter decreased by approx. 50%.

| S-EPMC1144709 | biostudies-other

The effect of malonyl-CoA on overt and latent carnitine acyltransferase activities in rat liver and adipocyte mitochondria.

Project description:1. Carnitine palmitoyltransferase and carnitine octanoyltransferase activities were measured in mitochondria at various acyl-CoA concentrations before and after sonication, thus permitting assessment of both overt and latent activities. 2. Overt carnitine palmitoyltransferase in liver and adipocyte mitochondria and overt carnitine octanoyltransferase in liver mitochondria were inhibited by malonyl-CoA. None of the latent activities were affected by this metabolite. 3. 5,5'-Dithiobis-(2-nitrobenzoic acid) stimulated latent hepatic carnitine palmitoyltransferase at low [palmitoyl-CoA]. 4. Starvation (24 h) decreased overt carnitine palmitoyltransferase activity in adipocyte mitochondria, but did not alter the sensitivity of this activity to malonyl-CoA.

| S-EPMC1154261 | biostudies-other

Importance of experimental conditions in evaluating the malonyl-CoA sensitivity of liver carnitine acyltransferase. Studies with fed and starved rats.

Project description:The experiments reconfirm the powerful inhibitory effect of malonyl-CoA on carnitine acyltransferase I and fatty acid oxidation in rat liver mitochondria (Ki 1.5 microM). Sensitivity decreased with starvation (Ki after 18 h starvation 3.0 microM, and after 42 h 5.0 microM). Observations by Cook, Otto & Cornell [Biochem. J. (1980) 192, 955--958] and Ontko & Johns [Biochem. J. (1980) 192, 959--962] have cast doubt on the physiological role of malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. The high Ki values obtained in the cited studies are shown to be due to incubation conditions that cause substrate depletion, destruction of malonyl-CoA or generation of excessively high concentrations of unbound acyl-CoA (which offsets the competitive inhibition of malonyl-CoA towards carnitine acyltransferase I). The present results are entirely consistent with the postulated role of malonyl-CoA as the primary regulatory of fatty acid synthesis and oxidation in rat liver.

| S-EPMC1163527 | biostudies-other

Carnitine palmitoyltransferase in human erythrocyte membrane. Properties and malonyl-CoA sensitivity.

Project description:Carnitine palmitoyltransferase located in the erythrocyte plasma membrane is sensitive to inhibition by malonyl-CoA and 2-bromopalmitoyl-CoA plus carnitine. Although this inhibition and other properties suggest similarities to the intracellular enzymes in other tissues, no cross-reaction was observed with antisera to the peroxisomal or to the mitochondrial inner-membrane enzyme. The activity was solubilized by and was stable in Triton X-100, which destroys the enzymes found in microsomes and in the mitochondrial outer membrane. The substrate specificity is broader than for the intracellular enzymes, the activities with stearoyl-CoA (114%) and arachidonoyl-CoA (97%) being equal to that with palmitoyl-CoA, and the activities with linoleoyl-CoA (44%) and erucoyl-CoA (46%) about half that with palmitoyl-CoA. The function of this carnitine palmitoyltransferase is probably to buffer the acyl-CoA present in the erythrocyte for turnover of the fatty acyl groups of the membrane lipids.

| S-EPMC1150109 | biostudies-other

Increased sensitivity of carnitine palmitoyltransferase I activity to malonyl-CoA inhibition after preincubation of intact rat liver mitochondria with micromolar concentrations of malonyl-CoA in vitro.

Project description:Carnitine palmitoyltransferase I in rat liver mitochondria preincubated with malonyl-CoA was more sensitive to inhibition by malonyl-CoA than was the enzyme in mitochondria preincubated in the absence of malonyl-CoA. For carnitine palmitoyltransferase I in mitochondria from starved animals this increase also resulted in the enzyme becoming significantly more sensitive than that in mitochondria assayed immediately after their isolation. Concentrations of malonyl-CoA that induced half the maximal degree of sensitization observed were 1-3 microM.

| S-EPMC1154313 | biostudies-other

Effect of membrane environment on the activity and inhibitability by malonyl-CoA of the carnitine acyltransferase of hepatic microsomal membranes.

Project description:We have investigated the extent to which membrane environment affects the catalytic properties of the malonyl-CoA-sensitive carnitine acyltransferase of liver microsomal membranes. Arrhenius-type plots of activity were linear in the absence and presence of malonyl-CoA (2.5 microM). Sensitivity to malonyl-CoA increased with decreasing assay temperature. Partly purified enzyme displayed an increased K0.5 (substrate concentration supporting half the maximal reaction rate) for myristoyl-CoA and a reduced sensitivity to malonyl-CoA compared with the enzyme in situ in membranes. Reconstitution with liposomes of a range of compositions restored the K0.5 for myristoyl-CoA to values similar to that seen in native membranes. The lipid requirements for restoration of sensitivity to malonyl-CoA were more stringent. When animals were starved for 24 h the specific activity of carnitine acyltransferase in microsomal membrane residues was increased 3.3-fold, whereas sensitivity to malonyl-CoA was decreased to 1/2.8. When enzymes partly purified from fed and starved animals were reconstituted into crude soybean phosphatidylcholine liposomes there was no difference in sensitivity to malonyl-CoA. When partly purified enzyme from fed rats was reconstituted into liposomes prepared from microsomal membrane lipids from fed animals it was 2.2-fold more sensitive to malonyl-CoA than when reconstituted with liposomes prepared from microsomal membrane lipids from starved animals. This suggests that the physiological changes in sensitivity to malonyl-CoA are mediated via changes in membrane lipid composition rather than via modification of the enzyme protein itself. The increased specific actvity of acyltransferase observed on starvation could not be attributed to changes in membrane lipid composition.

| S-EPMC1218209 | biostudies-other

Sensitivity of brown-adipose-tissue carnitine palmitoyltransferase to inhibition by malonyl-CoA.

Project description:Overt carnitine palmitoyltransferase in mitochondria isolated from interscapular brown adipose tissue of cold-adapted rats or rats maintained at normal temperature is extremely sensitive to inhibition by malonyl-CoA.

| S-EPMC1158357 | biostudies-other

Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non-hepatic tissues of the rat.

Project description:The requirement for carnitine and the malonyl-CoA sensitivity of carnitine palmitoyl-transferase I (EC 2.3.1.21) were measured in isolated mitochondria from eight tissues of animal or human origin using fixed concentrations of palmitoyl-CoA (50 microM) and albumin (147 microM). The Km for carnitine spanned a 20-fold range, rising from about 35 microM in adult rat and human foetal liver to 700 microM in dog heart. Intermediate values of increasing magnitude were found for rat heart, guinea pig liver and skeletal muscle of rat, dog and man. Conversely, the concentration of malonyl-CoA required for 50% suppression of enzyme activity fell from the region of 2-3 microM in human and rat liver to only 20 nM in tissues displaying the highest Km for carnitine. Thus, the requirement for carnitine and sensitivity to malonyl-CoA appeared to be inversely related. The Km of carnitine palmitoyltransferase I for palmitoyl-CoA was similar in tissues showing large differences in requirement for carnitine. Other experiments established that, in addition to liver, heart and skeletal muscle of fed rats contain significant quantities of malonyl-CoA and that in all three tissues the level falls with starvation. Although its intracellular location in heart and skeletal muscle is not known, the possibility is raised that malonyl-CoA (or a related compound) could, under certain circumstances, interact with carnitine palmitoyltransferase I in non-hepatic tissues and thereby exert control over long chain fatty acid oxidation.

| S-EPMC1152205 | biostudies-other

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