(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 mRNA and the activity of glucose-6-phosphatase (Glc-6-Pase) were present in the liver, kidney, and small intestine of 15-day-old suckling rats, but were absent from the stomach, colon, lung, white and brown adipose tissues, muscle, heart, brain, and spleen. The mRNA encoding Glc-6-Pase was present in the liver of 21-day-old fetal rats and increased markedly immediately after birth. From 5 days after birth to the end of the suckling period, it returned to 50% of the level found in the liver of 48-h starved adult rats. When rats were weaned at 21 days onto a high-carbohydrate, low-fat (HCLF) diet, the concentration of liver Glc-6-Pase mRNA was markedly increased. In the fetal rat jejunum, the activity and mRNA of Glc-6-Pase were very low. It increased during the 5 days after birth and then declined to reach very low levels. Neither mRNA nor activity of Glc-6-Pase was present in the fetal kidney. They appeared and increased slowly during the suckling period to reach maximal levels 15 days after birth and then remained constant. Weaning onto the HCLF diet did not change the Glc-6-Pase gene expression, neither in the jejunum nor in the kidney. The regulation of Glc-6-Pase gene expression by hormones and nutrients was studied in cultured hepatocytes from 20-day-old rat fetuses. Bt2cAMP stimulated the Glc-6-Pase gene expression in a dose-dependent manner. This probably resulted from an increased gene transcription since the half-life of the transcript was not affected by dibutyryl cAMP (Bt2cAMP). The Bt2cAMP-induced Glc-6-Pase mRNA accumulation was antagonized by insulin in a dose-dependent manner. Long-chain fatty acids (LCFAs), but not medium-chain fatty acids, induced the accumulation of Glc-6-Pase mRNA and the stabilization of the transcript. The peroxisome proliferator, clofibrate, induced a threefold increase in Glc-6-Pase mRNA concentration. Both stimulation of Glc-6-Pase mRNA by LCFAs and clofibrate were inhibited by insulin. Increasing concentrations of glucose (from 0 to 20 mmol/l) did not affect the Bt2cAMP-induced Glc-6-Pase gene expression. By contrast, high glucose concentration (25 mmol/l) markedly induced the Glc-6-Pase gene expression in fed adult rat hepatocytes. The difference in the response to glucose between fetal and adult rat hepatocytes is discussed. We conclude that the rapid increase in hepatic Glc-6-Pase mRNA levels that accompanies the fetal-to-neonatal transition in the rat is triggered by the reciprocal change in circulating insulin and LCFA concentrations, coupled to the rise in liver cAMP concentration.

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.

resents the main site of control for the entry of LCFA into the mitochondria [Z] and mitochondrial Volume 23