Carnitine Palmitoyltransferase System Research Articles

Regulation of fatty acid oxidation in skeletal muscle.

Researchers using animals are beginning to elucidate the control of fatty acid metabolism in muscle at the molecular and enzymatic level. This review examines the physiological data that has been collected from human subjects in the context of the proposed control mechanisms. A number of factors, including the availability of free fatty acids and the abundance of fatty acid transporters, may influence the rate of muscle fatty acid oxidation. However, the predominant point of control appears to be the rate at which fatty acyl-coenzyme A is transported into the mitochondria by the carnitine palmitoyl transferase system. In turn, evidence suggests that the intracellular concentration of malonyl-coenzyme A in muscle is an important regulator of carnitine palmitoyl transferase-I activity. Malonyl-coenzyme A is increased by glucose, which is likely the mechanism whereby glucose intake suppresses the transfer of fatty acids into the mitochondria for subsequent oxidation. In contrast, malonyl-coenzyme A levels decrease during exercise, which enables increased fatty acid oxidation. However, for any given carnitine palmitoyl transferase-I activity, there may be an effect of free fatty acid availability on fatty acid oxidation, particularly at low levels of free fatty acids. Nonetheless, the rate of glucose or glycogen metabolism is probably the primary regulator of the balance between glucose and fatty acid oxidation in muscle.

The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis.

First conceptualized as a mechanism for the mitochondrial transport of long-chain fatty acids in the early 1960s, the carnitine palmitoyltransferase (CPT) system has since come to be recognized as a pivotal component of fuel homeostasis. This is by virtue of the unique sensitivity of the outer membrane CPT I to the simple molecule, malonyl-CoA. In addition, both CPT I and the inner membrane enzyme, CPT II, have proved to be loci of inherited defects, some with disastrous consequences. Early efforts using classical approaches to characterize the CPT proteins in terms of structure/function/regulatory relationships gave rise to confusion and protracted debate. By contrast, recent application of molecular biological tools has brought major enlightenment at an exponential pace. Here we review some key developments of the last 20 years that have led to our current understanding of the physiology of the CPT system, the structure of the CPT isoforms, the chromosomal localization of their respective genes, and the identification of mutations in the human population.

Mammalian mitochondrial beta-oxidation.

The enzymic stages of mammalian mitochondrial beta-oxidation were elucidated some 30-40 years ago. However, the discovery of a membrane-associated multifunctional enzyme of beta-oxidation, a membrane-associated acyl-CoA dehydrogenase and characterization of the carnitine palmitoyl transferase system at the protein and at the genetic level has demonstrated that the enzymes of the system itself are incompletely understood. Deficiencies of many of the enzymes have been recognized as important causes of disease. In addition, the study of these disorders has led to a greater understanding of the molecular mechanism of beta-oxidation and the import, processing and assembly of the beta-oxidation enzymes within the mitochondrion. The tissue-specific regulation, intramitochondrial control and supramolecular organization of the pathway is becoming better understood as sensitive analytical and molecular techniques are applied. This review aims to cover enzymological and organizational aspects of mitochondrial beta-oxidation together with the biochemical aspects of inherited disorders of beta-oxidation and the intrinsic control of beta-oxidation.

RAT SOLEUS MUSCLE MITOCHONDRIAL CARNITINE PALMITOYLTRANSFERASE ACTIVITY AFTER SWIMMING 363

Endogenous triglyceride avaiable to active muscle cells both from plasma as well as from intramuscular depots consist of a major fuel contributing to maintainance of low to moderate-intensity exercise. After training, an enhanced density of mitochondria in muscle fiber is observed, accompanied by an increase of the oxidative enzyme content, reflecting an augmented fatty acid oxidative capacity. Fatty acid oxidation however, is limited during exercise, and beyond a certain threshold, glucose will be also an important fuel. Oxidation of long chain fatty acids (LCFA) is restricted by the activity of fatty acid binding proteins and the enzymatic carnitine palmitoyltransferase system (CPT). Controversial results of CPT activity have been obtained with different exercise protocols. This study investigates the activity of CPT I and II in the soleus muscle of control rats (C), rats trained (T) under a swimming protocol (5 days/week, 5 weeks, at 70% VO2max, killed after exhaustion, swimming with an extra load corresponding to 8% body weight tied to the tail), and sedentary rats submitted to a single exercise bout until exhaustion (TE). A radioassay was used for enzyme activity measurement after CPT I and II separation. The results showed an increase in CPT II activity (2.1-fold) in the T group as compared with C. In the TE group CPT II activity was not different from that of C, suggesting that the modification of CPT II activity results from long term training, and a session of exercise will not per se, influence the enzyme. CPT I activity was not modified in the T group compared with C. However, although not statistically significant, the result for CPT I activity in the TE group was lower than that for the C group (13.75±4.43 and 6.70±8.90 nmol/min.mg protein, respectively). In conclusion, training as a result of the swimming protocol presently adopted seems hence, to affect solely CPT II activity. A single, acute bout of exercise may apparently decrease CPT I activity in sedentary animals, what could imply in a diminished capacity for LCFA utilization under these conditions. Financial Support: CNPq and Laboratorios Baldacci S/A.

Interactive regulation of the pyruvate dehydrogenase complex and the carnitine palmitoyltransferase system.

The review examines the mechanisms regulating the activities of the two key enzymes determining rates of glucose and fatty acid oxidation, i.e., the pyruvate dehydrogenase (PDH) complex and the carnitine palmitoyltransferase (CPT) system. The review also evaluates the regulatory importance of gene expression in the control of tissue fuel selection within the context of substrate competition between glucose and fatty acids. It identifies a strong indirect input of nutrient-gene interactions in the control of pyruvate oxidation through the regulated provision of pyruvate as a substrate for PDH and as an inhibitor of PDH kinase. Nutrient-gene interactions are also identified in relation to the regulation of CPT I activity by malonyl-CoA (inhibitor) and by the provision of long-chain acyl-CoA (substrate/activator), the latter via the hydrolysis of plasma or tissue triacylglycerol (by lipoprotein lipase and hormone-sensitive lipase, respectively). We discuss how such regulation is reinforced by long-term modulation of PDH kinase-specific activity and CPT I maximal activity. We also explore the role of mechanisms operating at the levels of the PDH complex and the CPT system that act to promote and accelerate a switch in fuel utilization once a committed change in nutrient supply has been established. In particular, we discuss the regulatory influences exerted by altered sensitivities of PDH kinase to inhibition by pyruvate and CPT I to inhibition by malonyl-CoA, respectively.

Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes

1. The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. 2. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48 h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. 6. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.

Studies on carnitine palmitoyl transferase: The similar nature of CPT I (inner form) and CPT O (outer form)

Rat liver mitochondrial carnitine palmitoyl transferase (CPT) was found to reside in two mitochondrial locations. Twenty to twenty-five percent of the total CPT activity was easily released and solubilized by digitonin. This activity appeared to be the outer form of CPT (CPT O). The remainder of the activity or the inner CPT (CPT I) was tightly membrane bound. Trypsin digestion of the digitonin prepared mitoplast did not affect residual CPT activity indicating that this activity probably resided on the inner side of the membrane. Following their separation by digitonin treatment, CPT O and CPT I were partially purified 14.7-and 16.7-fold, respectively. The purification of each enzyme involved extraction from the membrane with Tween 80, ammonium sulfate fractionation, gel filtration, and another ammonium sulfate fractionation. The partially purified CPT O and CPT I were found to have identical elution volumes from a G-200 column corresponding to a molecular weight of 430,000. Also they both were found to have nearly identical K m values for palmitoyl-CoA, palmitoyl-carnitine, CoA, and carnitine suggesting they were identical enzymes. The V values could not be compared due to differences in purity, but the ratio of V in the forward direction to V in the reverse direction was identical for CPT I and CPT O again suggesting enzyme identity. Assay of the CPT system “ in situ” by following the reduction of the acyl-CoA dehydrogenase, a flavoprotein, suggested that the activity of CPT I was 450-fold greater than the activity of CPT O when both were present in the intact membrane. These data suggest that “ in situ” factors exist which greatly change the catalytic properties of CPT I compared to CPT O.