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

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

Effect of starvation and diabetes on the sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate.

The sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate was decreased markedly in liver mitochondria isolated from either 48 h-starved or streptozotocin-diabetic rats. These treatments of the rat also decreased the sensitivity of fatty acid oxidation by isolated hepatocytes to inhibition by this compound. Furthermore, incubation of hepatocytes prepared from fed rats with N6O2'-dibutyryl cyclic AMP also decreased the sensitivity, whereas incubation of hepatocytes prepared from starved rats with lactate plus pyruvate had the opposite effect on 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation. The sensitivity of carnitine palmitoyltransferase I of mitochondria to 4-hydroxyphenylglyoxylate increased in a time-dependent manner, as previously reported for malonyl-CoA. Likewise, oleoyl-CoA activated carnitine palmitoyltransferase I in a time-dependent manner and prevented the sensitization by 4-hydroxyphenylglyoxylate. Increased exogenous carnitine caused a moderate increase in fatty acid oxidation by hepatocytes under some conditions and a decreased 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation at low oleate concentration, without decreasing the difference in 4-hydroxyphenylglyoxylate inhibition between fed- and starved-rat hepatocytes. Time-dependent changes in the conformation of carnitine palmitoyltransferase I or the membrane environment may be involved in differences among nutritional states in 4-hydroxyphenylglyoxylate-sensitivity of carnitine palmitoyltransferase I.     

Short-term regulation of carnitine palmitoyltransferase activity in isolated rat hepatocytes

An assay procedure for carnitine palmitoyltransferase is described which allows rapid measurement of the overt activity of this enzyme in isolated rat hepatocytes. In a one-step procedure digitonin permeabilizes the plasma membrane and at the same time carnitine palmitoyltransferase activity is measured. The use of the present procedure shows that carnitine palmitoyltransferase activity is regulated on the short term by different types of agonists. Thus, insulin, epidermal growth factor, vasopressin and the phorbol ester PMA inhibit carnitine palmitoyltransferase activity, whereas glucagon treatment renders the enzyme more active. These changes in enzyme activity coincide with corresponding changes in the rate of fatty acid oxidation.     

Simultaneous stimulation of fatty acid synthesis and oxidation in rat hepatocytes by vanadate

When added to the hepatocyte incubation medium, vanadate increased the rate of fatty acid synthesis de novo as well as the activity of acetyl-CoA carboxylase, whereas it had no effect on the activity of fatty acid synthase. On the other hand, and despite elevating the intracellular levels of malonyl-CoA, vanadate diverted exogenous fatty acids into the oxidation pathway at the expense of the esterification route. This was concomitant to an increase in carnitine palmitoyltransferase I activity. All these effects were not significantly different between periportal and perivenous hepatocytes and were also evident in cells incubated in Ca2(+)-free medium. Nevertheless, Ca2+ ions enhanced carnitine palmitoyltransferase I activity in isolated liver mitochondria. In addition, the effects of vanadate on acetyl-CoA carboxylase and carnitine palmitoyltransferase I were only evident in a permeabilized-cell assay, disappearing upon cell disruption and isolation of the corresponding cell subfraction for enzyme assay. Results show that vanadate exerts specific insulin-like and non-insulin-like effects on hepatic fatty acid metabolism, and suggest that the intracellular concentration of malonyl-CoA is not the only factor responsible for the regulation of the fatty-acid-oxidative process in the liver.     

Short-term inhibition of carnitine palmitoyltransferase I activity in rat hepatocytes incubated with ethanol

Ethanol decreased the activity of carnitine palmitoyltransferase I and the rate of fatty acid oxidation in rat hepatocytes in short-term incubations. These effects were mimicked by acetaldehyde, the product of hepatic ethanol metabolism, and were absent when ethanol oxidation was prevented by 4-methylpyrazole. Ethanol was also able to increase intracellular malonyl-CoA levels. The results suggest that inhibition of fatty acid translocation into mitochondria may play an important role in the ethanol-induced inhibition of hepatic fatty acid oxidation.     

Intramitochondrial factors controlling hepatic fatty acid oxidation at weaning in the rat

Fatty acid oxidation was studied in isolated liver mitochondria of rats during the suckling-weaning transition. The oxidation rate of oleyl-CoA and palmitoylcarnitine was reduced 2.5-fold in rats weaned on a high-carbohydrate diet compared to suckling rats, when acetyl-CoA produced by beta-oxidation was directed towards ketone-body synthesis. Weaning on a high-fat diet minimized this change. Channeling of acetyl-CoA towards citrate synthesis doubled the oxidation rate of both substrates in HC-weaned rats. Thus, in addition to changes in carnitine palmitoyltransferase I activity, the beta-hydroxymethylglutaryl-CoA synthase pathway is also involved in the decreased fatty acid oxidation at weaning. This was confirmed by measurement of beta-hydroxymethylglutaryl-CoA synthase pathway activity.     

Study of some factors controlling fatty acid oxidation in liver mitochondria of obese Zucker rats.

Livers of genetically obese Zucker rats showed, compared with lean controls, hypertrophy and enrichment in triacylglycerols, indicating that fatty acid metabolism was directed towards lipogenesis and esterification rather than towards fatty acid oxidation. Mitochondrial activities of cytochrome c oxidase and monoamine oxidase were significantly lower when expressed per g wet wt. of liver, whereas peroxisomal activities of urate oxidase and palmitoyl-CoA-dependent NAD+ reduction were unchanged. Liver mitochondria were able to oxidize oleic acid at the same rate in both obese and lean rats. For reactions occurring inside the mitochondria, e.g. octanoate oxidation and palmitoyl-CoA dehydrogenase, no difference was found between both phenotypes. Total carnitine palmitoyl-, octanoyl- and acetyl-transferase activities were slightly higher in mitochondria from obese rats, whereas the carnitine content of both liver tissue and mitochondria was significantly lower in obese rats compared with their lean littermates. The carnitine palmitoyltransferase I activity was slightly higher in liver mitochondria from obese rats, but this enzyme was more sensitive to malonyl-CoA inhibition in obese than in lean rats. The above results strongly suggest that the impaired fatty acid oxidation observed in the whole liver of obese rats is due to the diminished transport of fatty acids across the mitochondrial inner membrane via the carnitine palmitoyltransferase I. This effect could be reinforced by the decreased mitochondrial content per g wet wt. of liver. The depressed fatty acid oxidation may explain in part the lipid infiltration of liver observed in obese Zucker rats.     

Two mechanisms produce tissue-specific inhibition of fatty acid oxidation by oxfenicine.

Oxfenicine [S-2-(4-hydroxyphenyl)glycine] is transaminated in heart and liver to 4-hydroxyphenylglyoxylate, an inhibitor of fatty acid oxidation shown in this study to act at the level of carnitine palmitoyltransferase I (EC 2.3.1.21). Oxfenicine was an effective inhibitor of fatty acid oxidation in heart, but not in liver. Tissue specificity of oxfenicine inhibition of fatty acid oxidation was due to greater oxfenicine transaminase activity in heart and to greater sensitivity of heart carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate [I50 (concentration giving 50% inhibition) of 11 and 510 microM for the enzymes of heart and liver mitochondria, respectively]. Branched-chain-amino-acid aminotransferase (isoenzyme I, EC 2.6.1.42) was responsible for the transamination of oxfenicine in heart. A positive correlation was found between the capacity of various tissues to transaminate oxfenicine and the known content of branched-chain-amino-acid aminotransferase in these tissues. Out of three observed liver oxfenicine aminotransferase activities, one may correspond to asparagine aminotransferase, but the major activity could not be identified by partial purification and characterization. As reported previously for malonyl-CoA inhibition of carnitine palmitoyltransferase I, 4-hydroxyphenylglyoxylate inhibition of this enzyme was found to be very pH-dependent. In striking contrast with the kinetics of malonyl-CoA inhibition, 4-hydroxyphenylglyoxylate inhibition was not affected by oleoyl-CoA concentration, but was partially reversed by increasing carnitine concentrations.     

Effect of pH on malonyl-CoA inhibition of carnitine palmitoyltransferase I.

Malonyl-CoA inhibition of carnitine palmitoyltransferase I was found to be very pH-dependent. Malonyl-CoA concentrations causing 50% inhibition (I50) at pH 6.0, 6.5, 7.0, 7.5 and 8.0 were 0.04, 1, 9, 40 and 200 microM respectively. It is suggested that a lowering of intracellular pH, such as might occur in ketoacidosis, may attenuate hepatic fatty acid oxidation by increasing malonyl-CoA sensitivity of carnitine palmitoyltransferase I.     

Carnitine palmitoyltransferase in the heart is controlled by a different mechanism than the hepatic enzyme

Diminished sensitivity of hepatic carnitine palmitoyltransferase to inhibition by malonyl-CoA in the fasting and diabetic states is a well-recognized aspect of the regulatory mechanism forhepatic fatty acid oxidation. Inhibition of myocardial carnitine palmitoyltransferase by malonyl-CoA may play an important role in regulation of fatty acid oxidation in the heart, but there has been a discrepancy in data relating to changes in malonyl-CoA sensitivity of the myocardial carnitine palmitoyltransferase during fasting. Analysis of malonyl-CoA inhibition of myocardial carnitine palmitoyltransferase in fasting and fed states under a variety of conditions has indicated that under no condition could any difference be found in malonyl-CoA sensitivity that was attributable to fasting. Proteolysis of the outer carnitine palmitoyltransferase led to artifactual changes in sensitivity due to the appearance of partial inhibition. We have concluded that the sensitivity of myocardial carnitine palmitoyltransferase to malonyl-CoA does not change during fasting. Changes in fatty acid oxidation in the heart are probably due to changes in malonyl-CoA concentrations or to other inhibitors. (Mol Cell Biochem 116: 39–45, 1992)     

Some aspects of fatty acid oxidation in isolated fat-cell mitochondria from rat.

Mitochondrial were prepared from fat-cells isolated from rat epididymal adipose tissues of fed and 48 h-starved rats to study some aspects of fatty acid oxidation in this tissue. The data were compared with values obtained in parallel experiments with liver mitochondria that were prepared and incubated under identical conditions. 2. In the presence of malonate, fluorocitrate and arsenite, malate, but not pyruvate-bicarbonate, facilitated palmitoyl-group oxidation in both types of mitochondria. In the presence of malate, fat-cell mitochondria exhibited slightly higher rates of palmitoylcarnitine oxidation than liver. Rates of octanoylcarnitine oxidation were similar in liver and fat-cell mitochondria. Uncoupling stimulated acylcarnitine oxidation in liver, but not in fat-cell mitochondria. Oxidation of palmitoyl- and octanoyl-carnitine was partially additive in fat-cell but not in liver mitochondria. Starvation for 48 h significantly decreased both palmitoylcarnitine oxidation and latent carnitine palmitoyltransferase activity in fat-cell mitochondria. Starvation increased latent carnitine palmitoyltransferase activity in liver mitochondria but did not alter palmitoylcarnitine oxidation. These results suggested that palmitoylcarnitine oxidation in fat-cell but not in liver mitochondria may be limited by carnitine palmitoyltransferase 2 activity. 3. Fat-cell mitochondria also differed from liver mitochondria in exhibiting considerably lower rates of carnitine-dependent oxidation of palmitoyl-CoA or palmitate, suggesting that carnitine palmitoyltransferase 1 activity may severely rate-limit palmitoyl-CoA oxidation in adipose tissue.     

A novel brain-expressed protein related to carnitine palmitoyltransferase I

Malonyl-CoenzymeA acts as a fuel sensor, being both an intermediate of fatty acid synthesis and an inhibitor of the two known isoforms of carnitine palmitoyltransferase I (CPT I), which control mitochondrial fatty acid oxidation. We describe here a novel CPT1 family member whose mRNA is present predominantly in brain and testis. Chromosomal locations and genome organization are reported for the mouse and human genes. The protein sequence contains all the residues known to be important for both carnitine acyltransferase activity and malonyl-CoA binding in other family members. Yeast expressed protein has no detectable catalytic activity with several different acyl-CoA esters that are good substrates for other carnitine acyltransferases, including the liver isoform of CPT I, which is also expressed in brain; however, it displays high-affinity malonyl-CoA binding. Thus this new CPT I related protein may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.     

The localization of palmitoyl-CoA: Carnitine palmitoyltransferase in rat liver

1. The distribution of palmitoyl-CoA:carnitine palmitoyltransferase has been studied in subcellular fractions of rat liver. By using two different estimations for the enzyme activity and by differential centrifugation and linear sucrose density gradient centrifugation, the enzyme is shown to be localized both in mitochondria and microsomes.

2. The mitochondrial palmitoyl-CoA: carnitine palmitoyltransferase is localized in the inner membrane plus matrix fraction.

3. During palmitate oxidation by isolated mitochondria, in the presence of a physiological concentration of carnitine, palmitoylcarnitine accumulates. From this and experiments with sonicated mitochondria, it is concluded that the capacities of long-chain fatty acid activation and of palmitoyl-CoA:carnitine palmitoyltransferase in vitro by far exceed the capacity of fatty acid oxidation.     

The effect of di(ethylhexyl)phthalate on fatty acid oxidation and carnitine palmitoyltransferase in various rat tissues

Male rats were fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. Total and peroxisomal oxidation rates of palmitic and arachidonic acid were increased in homogenates of liver and kidney after DEHP administration. The relative peroxisomal contribution to the total oxidation was only higher in liver. The activities of acyl-CoA oxidase and carnitine palmitoyltransferase were also higher in both tissues. Immunoblots showed that the increase of fatty acid oxidation was associated with a higher concentration of enzymes of peroxisomal and mitochondrial beta-oxidation. DEHP did not change total and peroxisomal fatty acid oxidation and activity of carnitine palmitoyltransferase of homogenates of heart and skeletal muscle. The cause for the tissue-specific response is discussed.     

Ethanol feeding to rats reversibly decreases hepatic carnitine palmitoyltransferase activity and increases enzyme sensitivity to malonyl-CoA

The effects of prolonged ethanol feeding on both carnitine palmitoyltransferase I activity and enzyme sensitivity to inhibition by malonyl-CoA were studied in rat liver, heart, skeletal muscle and kidney cortex mitochondria. Heart and skeletal muscle enzymes showed the highest specific activity and sensitivity to malonyl-CoA. Carnitine palmitoyltransferase I in extrahepatic tissues showed no changes on ethanol feeding. Only the liver enzyme activity was altered after long term ethanol administration, by suffering a progressive decrease in activity and a parallel increase in sensitivity to malonyl-CoA. These alterations reversed after 10 days of ethanol withdrawal. These results are discussed in relation to the control of carnitine palmitoyltransferase I and the effects of ethanol on fatty acid metabolism.     

Zonation of fatty acid metabolism in rat liver.

Fatty acid metabolism was studied in periportal and perivenous hepatocytes isolated by the method of Chen & Katz [Biochem. J. (1988) 255, 99-104]. The rate of fatty acid synthesis and the activity of acetyl-CoA carboxylase were markedly enhanced in perivenous hepatocytes as compared with periportal cells. However, the response of these two parameters to short-term modulation by cellular effectors such as the hormones insulin and glucagon, the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate and the xenobiotics ethanol and acetaldehyde was similar in the two zones of the liver. In addition, perivenous hepatocytes showed a higher capacity of esterification of exogenous fatty acids into both cellular and very-low-density-lipoprotein lipids. Nevertheless, no difference between the two cell sub-populations seemed to exist in relation to the secretion of very-low-density lipoproteins. On the other hand, the rate of fatty acid oxidation was increased in periportal cells. This could be accounted for by a higher activity of carnitine palmitoyltransferase I and a lower sensitivity of this enzyme to inhibition by malonyl-CoA in the periportal zone. No differences were observed between periportal and perivenous hepatocytes in relation to the short-term response of fatty acid oxidation and carnitine palmitoyltransferase I activity to the cellular modulators mentioned above. In conclusion, our results show that: (i) lipogenesis is achieved at higher rates in the perivenous zone of the liver, whereas the fatty-acid-oxidative process occurs with a certain preference in the periportal area of this organ; (ii) the short-term response of the different fatty-acid-metabolizing pathways to cellular effectors is quantitatively similar in the two zones of the liver.     

Metoprolol improves cardiac function and modulates cardiac metabolism in the streptozotocin-diabetic rat

The effects of diabetes on heart function may be initiated or compounded by the exaggerated reliance of the diabetic heart on fatty acids and ketones as metabolic fuels. beta-Blocking agents such as metoprolol have been proposed to inhibit fatty acid oxidation. We hypothesized that metoprolol would improve cardiac function by inhibiting fatty acid oxidation and promoting a compensatory increase in glucose utilization. We measured ex vivo cardiac function and substrate utilization after chronic metoprolol treatment and acute metoprolol perfusion. Chronic metoprolol treatment attenuated the development of cardiac dysfunction in streptozotocin (STZ)-diabetic rats. After chronic treatment with metoprolol, palmitate oxidation was increased in control hearts but decreased in diabetic hearts without affecting myocardial energetics. Acute treatment with metoprolol during heart perfusions led to reduced rates of palmitate oxidation, stimulation of glucose oxidation, and increased tissue ATP levels. Metoprolol lowered malonyl-CoA levels in control hearts only, but no changes in acetyl-CoA carboxylase phosphorylation or AMP-activated protein kinase activity were observed. Both acute metoprolol perfusion and chronic in vivo metoprolol treatment led to decreased maximum activity and decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA. Metoprolol also increased sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and prevented the reexpression of atrial natriuretic peptide in diabetic hearts. These data demonstrate that metoprolol ameliorates diabetic cardiomyopathy and inhibits fatty acid oxidation in streptozotocin-induced diabetes. Since malonyl-CoA levels are not increased, the reduction in total carnitine palmitoyltransferase I activity is the most likely factor to explain the decrease in fatty acid oxidation. The metabolism changes occur in parallel with changes in gene expression.     

Effect of dietary alpha-linolenic acid on the activity and gene expression of hepatic fatty acid oxidation enzymes

The activities of hepatic fatty acid oxidation enzymes in rats fed linseed and perilla oils rich in alpha-linolenic acid (alpha-18:3) were compared with those in the animals fed safflower oil rich in linoleic acid (18:2) and saturated fats (coconut or palm oil). Mitochondrial and peroxisomal palmitoyl-CoA (16:0-CoA) oxidation rates in the liver homogenates were significantly higher in rats fed linseed and perilla oils than in those fed saturated fats and safflower oil. The fatty oxidation rates increased as dietary levels of alpha-18:3 increased. Dietary alpha-18:3 also increased the activity of fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. Unexpectedly, dietary alpha-18:3 caused great reduction in the activity of 3-hydroxyacyl-CoA dehydrogenase measured with short- and medium-chain substrates but not with long-chain substrate. Dietary alpha-18:3 significantly increased the mRNA levels of hepatic fatty acid oxidation enzymes including carnitine palmitoyltransferase I and II, mitochondrial trifunctional protein, acyl-CoA oxidase, peroxisomal bifunctional protein, mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, 2, 4-dienoyl-CoA reductase and delta3, delta2-enoyl-CoA isomerase. Fish oil rich in very long-chain n-3 fatty acids caused similar changes in hepatic fatty acid oxidation. Regarding the substrate specificity of beta-oxidation pathway, mitochondrial and peroxisomal beta-oxidation rate of alpha-18:3-CoA, relative to 16:0- and 18:2-CoAs, was higher irrespective of the substrate/albumin ratios in the assay mixture or dietary fat sources. The substrate specificity of carnitine palmitoyltransferase I appeared to be responsible for the differential mitochondrial oxidation rates of these acyl-CoA substrates. Dietary fats rich in alpha-18:3-CoA relative to safflower oil did not affect the hepatic activity of fatty acid synthase and glucose 6-phosphate dehydrogenase. It was suggested that both substrate specificities and alterations in the activities of the enzymes in beta-oxidation pathway play a significant role in the regulation of the serum lipid concentrations in rats fed alpha-18:3.     

Fatty acid oxidation and related gene expression in heart depleted of carnitine by mildronate treatment in the rat

The metabolic and genic effects induced by a 20-fold lowering of carnitine content in the heart were studied in mildronate-treated rats. In the perfused heart, the proportion of palmitate taken up then oxidized was 5-10% lower, while the triacylglycerol (TAG) formation was 100% greater than in controls. The treatment was shown to increase the maximal capacity of heart homogenates to oxidize palmitate, the mRNA level of carnitine palmitoyltransferase I (CPT-I) isoforms, the specific activity of CPT-I in subsarcolemmal mitochondria and the total carnitine content of isolated mitochondria. Concomitantly, the increased mRNA expression of lipoprotein lipase, fatty acid translocase and enzymes of TAG synthesis was associated with a 5- and 2-times increase in serum TAG and free fatty acid contents, respectively. The compartmentation of carnitine at its main functional location was expected to allow the increased CPT-I activity to ensure in vivo correct fatty acid oxidation rates. All the inductions related to fatty acid transport, oxidation and esterification most likely stem from the abundance of blood lipids providing cardiomyocytes with more fatty acids.