(Receivcd 11 September 1YY5) -EJB 95 1492/1In the rat, the gene for liver mitochondria1 carniline palinitoyltt-allsferase I (CP'T I), though dormant prior to birth, is rapidly activated postnatally. We sought to elucidate which hormonal and/or nutritional factors might be responsible for this induction. I n cultured hcpatocytes from 20-day-old rat fetus. the concentration of CPT I mRNA, which initially was very low, increascd dramatically in a dosc-dependent manner after exposure of the cells to dibutyryl CAMP (Bt2cAMP). Similar results were obtained when long-chain fatty acids (LCFA), but not medium-chain fatty acids, were added to the culture mediurn. l h e effects of Bt,cAMP and LCFA were antagonized by insulin, also dose dependently. In contrast, CPT 11 gene expression, which was already high in fetal hcpatocytes, was unaffected by any of the above manipulations.Bt,cAMP stimulated CPT 1 gene expression even when endogenous triacylglycerol hreakdown was suppressed by lysosomotropic agents suggesting that the iictions of CAMP and LCFA were distinct. Moreover, half-maximal concentrations of Bt,cAMP and linoleate produced an additive effect on CPT l mRNA accumulation. While linoleate and Bt,cAMP stimulated CPT 1 gene transcription by twofold and fourfold, respectively, the fatty acid also increased the half-life of CP'T I mKNA (50%). When hepatocytes were cultured in the presence of 2-bromopulmitate, (which is readily converted by cells into its non-metabolizable CoA ester) CPT I mRNA accumulation was higher than that obscrved with oleate or linoleate. Similarly, the CPT 1 inhibitor, tctradecylglycidate, which at a concentration of 20 pM did not itself influence the CPT I mRNA level, enhanced the stiinulatory effect of linolcate. 'The implication is that induction of the CPT 1 message by LCFA does not require mitochondrial nietabolisrn of these substrates; however, formation of their CoA esters i s B necessary step.Unlike linoleale, the peroxisome proliferator, clofibrate, increased both CPT I and CPT 11 IIIKNA levels and neither effect was offset by insulin. It thus appears that thc mechanism of action of LCFA differs from that utilized by clofibrate, which presumably works through the peroxisome proliferator activated receptor. Wc conclude that the rapid increase in hepatic CPT 1 mRNA level that accoinpanics the fetal to neonatal transition in the rat is triggered by the rcciprocal change in circulating insulin and LCFA ooncentrations, coupled with elevation of the livcr content of CAMP.Ke.yword.7: carnitine palmitoyllransferase I gene transcription ; CAMP; fatty acids ; peroxisonic proliferators; cultured fctal rat hepatocytes.Iintnediately after birth, the rat is fed with milk, a high-fat low-carbohydrate diet (review in [l]). To meet the energy needs of the newborn, the capacity for fatty acid oxidation develops rapidly after birth in many peripheral tissues including the liver,

The rat liver carnitine palmitoyltransferase 1 (L-CPT 1) expressed in Saccharomyces cerevisiae was correctly inserted into the outer mitochondrial membrane and shared the same folded conformation as the native enzyme found in rat liver mitochondria. Comparison of the biochemical properties of the yeast-expressed L-CPT 1 with those of the native protein revealed the same detergent lability and similar sensitivity to malonyl-CoA inhibition and affinity for carnitine. Normal Michaelis-Menten kinetics towards palmitoyl-CoA were observed when careful experimental conditions were used for the CPT assay. Thus, the expression in S. cerevisiae is a valid model to study the structure-function relationships of L-CPT 1.z 1998 Federation of European Biochemical Societies.

The rat liver carnitine palmitoyltransferase 1 (L-CPT1), an integral outer mitochondrial membrane (OMM) protein, is the key regulatory enzyme of fatty acid oxidation and is inhibited by malonyl-CoA. In vitro import of L-CPT1 into the OMM requires the presence of mitochondrial receptors and is stimulated by ATP but is membrane potential-independent. Its N-terminal domain (residues 1-150), which contains two transmembrane segments, possesses all of the information for mitochondrial targeting and OMM insertion. Deletion of this domain abrogates protein targeting, whereas its fusion to non-OMM-related proteins results in their mitochondrial targeting and OMM insertion in a manner similar to L-CPT1. Functional analysis of chimeric CPTs expressed in Saccharomyces cerevisiae shows that this domain also mediates in vivo protein insertion into the OMM. When the malonyl-CoA-insensitive CPT2 was anchored at the OMM either by a specific OMM signal anchor sequence (pOM29) or by the N-terminal domain of L-CPT1, its activity remains insensitive to malonylCoA inhibition. This indicates that malonyl-CoA sensitivity is an intrinsic property of L-CPT1 and that its N-terminal domain cannot confer malonyl-CoA sensitivity to CPT2. Replacement of the N-terminal domain by pOM29 results in a less folded and less active protein, which is also malonyl-CoA-insensitive. Thus, in addition to its role in mitochondrial targeting and OMM insertion, the N-terminal domain of L-CPT1 is essential to maintain an optimal conformation for both catalytic function and malonyl-CoA sensitivity.

The temporal changes in oleate oxidation, lipogenesis, malonyl-CoA concentration and sensitivity of carnitine palmitoyltransferase I (CPT 1) to malonyl-CoA inhibition were studied in isolated rabbit hepatocytes and mitochondria as a function of time after birth of the animal or time in culture after exposure to glucagon, cyclic AMP or insulin. (1) Oleate oxidation was very low during the first 6 h after birth, whereas lipogenesis rate and malonyl-CoA concentration decreased rapidly during this period to reach levels as low as those found in 24-h-old newborns that show active oleate oxidation. (2) The changes in the activity of CPT I and the IC50 (concn. causing 50% inhibition) for malonyl-CoA paralleled those of oleate oxidation. (3) In cultured fetal hepatocytes, the addition of glucagon or cyclic AMP reproduced the changes that occur spontaneously after birth. A 12 h exposure to glucagon or cyclic AMP was sufficient to inhibit lipogenesis totally and to cause a decrease in malonyl-CoA concentration, but a 24 h exposure was required to induce oleate oxidation. (4) The induction of oleate oxidation by glucagon or cyclic AMP is triggered by the fall in the malonyl-CoA sensitivity of CPT I. (5) In cultured hepatocytes from 24 h-old newborns, the addition of insulin inhibits no more than 30% of the high oleate oxidation, whereas it stimulates lipogenesis and increases malonyl-CoA concentration by 4-fold more than in fetal cells (no oleate oxidation). This poor effect of insulin on oleate oxidation seems to be due to the inability of the hormone to increase the sensitivity of CPT I sufficiently. Altogether, these results suggest that the malonyl-CoA sensitivity of CPT I is the major site of regulation during the induction of fatty acid oxidation in the fetal rabbit liver.

Liver carnitine palmitoyltransferase I (L-CPT I) catalyses the transfer of long-chain fatty acid (LCFA) for translocation across the mitochondrial membrane. Expression of the L-CPT I gene is induced by LCFAs as well as by lipid-lowering compounds such as clofibrate. Previous studies have suggested that the peroxisome-proliferator-activated receptor alpha (PPARalpha) is a common mediator of the transcriptional effects of LCFA and clofibrate. We found that free LCFAs rather than acyl-CoA esters are the signal metabolites responsible for the stimulation of L-CPT I gene expression. Using primary culture of hepatocytes we found that LCFAs failed to stimulate L-CPT I gene expression both in wild-type and PPARalpha-null mice. These results suggest that the PPARalpha-knockout mouse does not represent a suitable model for the regulation of L-CPT I gene expression by LCFAs in the liver. Finally, we determined that clofibrate stimulates L-CPT I through a classical direct repeat 1 (DR1) motif in the promoter of the L-CPT I gene while LCFAs induce L-CPT I via elements in the first intron of the gene. Our results demonstrate that LCFAs can regulate gene expression through PPARalpha-independent pathways and suggest that the regulation of gene expression by dietary lipids is more complex than previously proposed.