Evidence of a malonyl-CoA-insensitive carnitine palmitoyltransferase I activity in red skeletal muscle

Carnitine palmitoyltransferase I (CPT I), which is expressed as two distinct isoforms in liver (alpha) and muscle (beta), catalyzes the rate-limiting step in the transport of fatty acid into the mitochondria. Malonyl-CoA, a potent inhibitor of CPT I, is considered a key regulator of fatty acid oxidation in both tissues. Still unanswered is how muscle beta-oxidation proceeds despite malonyl-CoA concentrations that exceed the IC(50) for CPT Ibeta. We evaluated malonyl-CoA-suppressible [(14)C]palmitate oxidation and CPT I activity in homogenates of red (RG) and white (WG) gastrocnemius, soleus (SOL), and extensor digitorum longus (EDL) muscles. Adding 10 microM malonyl-CoA inhibited palmitate oxidation by 29, 39, 60, and 89% in RG, SOL, EDL, and WG, respectively. Thus malonyl-CoA resistance, which correlated strongly (0.678) with absolute oxidation rates (RG > SOL > EDL > WG), was greater in red than in white muscles. Similarly, malonyl-CoA-resistant palmitate oxidation and CPT I activity were greater in mitochondria from RG compared with WG. Ribonuclease protection assays were performed to evaluate whether our data might be explained by differential expression of CPT I splice variants. We detected the presence of two CPT Ibeta splice variants that were more abundant in red compared with white muscle, but the relative expression of the two mRNA species was unrelated to malonyl-CoA resistance. These results provide evidence of a malonyl-CoA-insensitive CPT I activity in red muscle, suggesting fiber type-specific expression of distinct CPT I isoforms and/or posttranslational modulations that have yet to be elucidated.     

Effects of endurance exercise on carnitine palmitoyltransferase I from rat heart, skeletal muscle and liver mitochondria

Prolonged physical exercise increased the activity of carnitine palmitoyltransferase I in rat heart and skeletal muscle mitochondria, whereas enzyme sensitivity to inhibition by malonyl-CoA remained unchanged. Nevertheless, inhibition of carnitine palmitoyltransferase I activity by small decreases in pH was attenuated in heart and skeletal muscle mitochondria from exercised animals. Liver enzyme did not suffer any alteration by endurance exercise.     

Evaluation of theophylline-stimulated changes in carnitine palmitoyltransferase activity in skeletal muscle and liver of rats

The effect of theophylline treatments on the activity of carnitine palmitoyltransferase (CPT) in skeletal muscle and the liver of rats was investigated. Theophylline was administered at 100 mg/kg bw/day and effects were monitored after a treatment period that lasted between a week and five weeks. Results showed that a significant increase in the activity of CPT was observed in skeletal muscle of theophylline-treated groups as compared to either control or placebo groups. However, there was no significant change in the activity of CPT in the hepatic tissues of theophylline-treated groups. The observed discrepancies in activity of CPT might be due to the presence of two isoenzymes, the muscle type (M-CPT) and liver type (L-CPT); it is possible that theophylline affects only M-CPT activity.     

A case of carnitine palmitoyltransferase II deficiency in human skeletal muscle

A 20-year-old man was shown to have a deficiency of carnitine palmitoyltransferase (CPT) II in skeletal muscle. The evidence was: (i) there was no significant oxidation of [9,10-3H]-palmitate or of [1-14C]palmitate in mitochondrial fractions from fresh skeletal muscle from the patient; (ii) all the CPT activity in a homogenate of fresh muscle from the patient was overt (CPT I) with no increase in activity after the inner membrane was disrupted; (iii) all the CPT activity in the patient's muscle was inhibited by malonyl-CoA; and (iv) an immunoreactive peptide of 67 kDa corresponding to CPT II, present in mitochondria from controls, was absent in those from the patient.     

Okadaic acid stimulates carnitine palmitoyltransferase I activity and palmitate oxidation in isolated rat hepatocytes

Okadaic acid parallely increased carnitine [corrected] palmitoyltransferase I activity and the rate of palmitate oxidation in isolated rat hepatocytes. Nevertheless, okadaic acid had no significant effect on the rate of octanoate oxidation. Maximal effects of okadaic acid were similar and non-additive to those of dibutyryl-cAMP, forskolin and glucagon. Results indicate that carnitine palmitoyltransferase I activity may be controlled by a mechanism of phosphorylation/dephosphorylation.     

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.     

Overexpression of carnitine palmitoyltransferase I in skeletal muscle in vivo increases fatty acid oxidation and reduces triacylglycerol esterification

A key regulatory point in the control of fatty acid (FA) oxidation is thought to be transport of FAs across the mitochondrial membrane by carnitine palmitoyltransferase I (CPT I). To investigate the role of CPT I in FA metabolism, we used in vivo electrotransfer (IVE) to locally overexpress CPT I in muscle of rodents. A vector expressing the human muscle isoform of CPT I was electrotransferred into the right lateral muscles of the distal hindlimb [tibialis cranialis (TC) and extensor digitorum longus (EDL)] of rats, and a control vector expressing GFP was electrotransferred into the left muscles. Initial studies showed that CPT I protein expression peaked 7 days after IVE (+104%, P<0.01). This was associated with an increase in maximal CPT I activity (+30%, P < 0.001) and a similar increase in palmitoyl-CoA oxidation (+24%; P<0.001) in isolated mitochondria from the TC. Importantly, oxidation of the medium-chain FA octanoyl-CoA and CPT I sensitivity to inhibition by malonyl-CoA were not altered by CPT I overexpression. FA oxidation in isolated EDL muscle strips was increased with CPT I overexpression (+28%, P<0.01), whereas FA incorporation into the muscle triacylglycerol (TAG) pool was reduced (-17%, P<0.01). As a result, intramyocellular TAG content was decreased with CPT I overexpression in both the TC (-25%, P<0.05) and the EDL (-45%, P<0.05). These studies demonstrate that acute overexpression of CPT I in muscle leads to a repartitioning of FAs away from esterification and toward oxidation and highlight the importance of CPT I in regulating muscle FA metabolism.     

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.

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.     

Hormonal and nutritional regulation of muscle carnitine palmitoyltransferase I gene expression in vivo

Transgenic mice carrying the human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene fused to a CAT reporter gene were generated to study the regulation of M-CPTI gene expression. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. In the diabetic transgenic mice, there was a 2- to 3-fold increase in CAT activity and CAT mRNA levels in heart and skeletal muscle which upon insulin administration reverted to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet increased CAT activity and mRNA levels by 2- to 4-fold in heart and skeletal muscle of the transgenic mice compared to the control transgenic mice on regular diet. Overall, the M-CPTI promoter was found to be necessary for the tissue-specific hormonal and dietary regulation of the gene expression.     

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.     

Restoration of the properties of carnitine palmitoyltransferase I in liver mitochondria during re-feeding of starved rats.

Platelet factor 4 is a small protein (Mr 7756) from the alpha-granules of blood platelets which binds strongly to and neutralizes the anticoagulant properties of heparin. From an analysis of X-ray crystallographic data a model for the binding of platelet factor 4 to heparin is proposed.     

Novel effect of C75 on carnitine palmitoyltransferase I activity and palmitate oxidation

C75 is a potential drug for the treatment of obesity. It was first identified as a competitive, irreversible inhibitor of fatty acid synthase (FAS). It has also been described as a malonyl-CoA analogue that antagonizes the allosteric inhibitory effect of malonyl-CoA on carnitine palmitoyltransferase I (CPT I), the main regulatory enzyme involved in fatty acid oxidation. On the basis of MALDI-TOF analysis, we now provide evidence that C75 can be transformed to its C75-CoA derivative. Unlike the activation produced by C75, the CoA derivative is a potent competitive inhibitor that binds tightly but reversibly to CPT I. IC50 values for yeast-overexpressed L- or M-CPT I isoforms, as well as for purified mitochondria from rat liver and muscle, were within the same range as those observed for etomoxiryl-CoA, a potent inhibitor of CPT I. When a pancreatic INS(823/13), muscle L6E9, or kidney HEK293 cell line was incubated directly with C75, fatty acid oxidation was inhibited. This suggests that C75 could be transformed in the cell to its C75-CoA derivative, inhibiting CPT I activity and consequently fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 in mice produced short-term inhibition of CPT I activity in mitochondria from the liver, soleus, and pancreas, indicating that C75 could be transformed to its C75-CoA derivative in these tissues. Finally, in silico molecular docking studies showed that C75-CoA occupies the same pocket in CPT I as palmitoyl-CoA, suggesting an inhibiting mechanism based on mutual exclusion. Overall, our results describe a novel role for C75 in CPT I activity, highlighting the inhibitory effect of its C75-CoA derivative.     

Tandem mass spectrometric assay for the determination of carnitine palmitoyltransferase II activity in muscle tissue

Carnitine palmitoyltransferase II (CPT-II) mediates the import of long-chain fatty acids into the mitochondrial matrix for subsequent beta-oxidation. Defects of CPT-II manifest as a severe neonatal hepatocardiomuscular form or as a mild muscular phenotype in early infancy or adolescence. CPT-II deficiency is diagnosed by the determination of enzyme activity in tissues involving the time-dependent conversion of radiolabeled CPT-II substrates (isotope-exchange assays) or the formation of chromogenic reaction products. We have established a mass spectrometric assay (MS/MS) for the determination of CPT-II activity based on the stoichiometric formation of acetylcarnitine in a coupled reaction system. In this single-tube reaction system palmitoylcarnitine is converted by CPT-II to free carnitine, which is subsequently esterified to acetylcarnitine by carnitine acetyltransferase. The formation of acetylcarnitine directly correlates with the CPT-II activity. Comparison of the MS/MS method (y) with our routine spectrophotometric assay (x) revealed a linear regression of y = 0.58x + 0.12 (r = 0.8369). Both assays allow one to unambiguously detect patients with the muscular form of CPT-II deficiency. However, the higher specificity and sensitivity as well as the avoidance of the drawbacks inherent in the use of radiolabeled substrates make this mass spectrometric method most suitable for the determination of CPT-II activity.     

Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids

Morphological and biochemical effects were induced at the subcellular level in the skeletal muscle, heart and liver of male rats as a result of feeding with EPA, DHA, and 3-thia fatty acids. The 3-thia fatty acid, tetradecylthioacetic acid (TTA) and EPA induced mitochondrial growth in type I muscle fibers in both the diaphragm and soleus muscle, and the size distribution of mitochondrial areas followed a similar pattern. Only the 3-thia fatty acid induced mitochondrial growth in type II muscle fibers. The mean area occupied by the mitochondria and the size distribution of mitochondrial areas in both fiber types were highly similar in DHA-treated and control animals. Only the 3-thia fatty acid increased the gene-expression of carnitine palmitoyltransferase (CPT)-II in the diaphragm. In the heart, however, the gene expression decreased. In hepatocytes an increase in the mean size of mitochondria was observed after EPA treatment, concomitant with an increase in mitochondrial CPT-II gene expression. Administration of 2-methyl-substituted EPA (methyl-EPA) induced a higher rate of growth of mitochondria than EPA. At the peroxisomal level in the hepatocytes a 3-thia fatty acid, EPA, and DHA increased the areal fraction concomitant with the induction of gene expression of peroxisomal fatty acyl-CoA oxidase (FAO). In the diaphragm, mRNA levels of FAO were not affected by EPA or DHA treatment, whereas gene expression was significantly increased after 3-thia fatty acid treatment. In the heart, both 3-thia fatty acid, EPA and DHA tended to decrease the levels of FAO mRNA. The areal fraction of fat droplets in all three tissue types was significantly lower in the groups treated with 3-thia fatty acid. In the group treated with EPA a lower areal fraction of fat droplets was observed, while the DHA group was similar to the control. This indicates that EPA and DHA have different effects on mitochondrial biogenesis.