The enzyme phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in gluconeogenesis in liver and in glyceroneogenesis in adipose tissue. These processes, and PEPCK, are regulated by a number of hormones, some of which have different effects on the enzyme in liver and adipose tissue. To explore this phenomenon, PEPCK gene expression was studied in 3T3-F442A adipocytes maintained in a serum-free medium. The beta-adrenergic agonist isoprenaline (isoproterenol) and a cyclic AMP analogue (8-CPT-cAMP) increased PEPCK mRNA. A maximal 3-fold induction occurred in 2 h. Dexamethasone decreased PEPCK mRNA by 80% in 4 h. Dexamethasone also counteracted the inductive effects of isoprenaline and 8-CPT-cAMP. Run-on transcription experiments showed that the isoprenaline and dexamethasone actions were, at least in part, exerted at the level of PEPCK gene transcription. These effects were further analysed by using transient and stable transfection of adipocytes with a plasmid containing bp -2100 to 69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene. In such cells isoprenaline stimulated CAT expression, an effect that was prevented if the cells were also exposed to dexamethasone.

Cytosolic phosphoPnolpyruvate carboxykinase (PEPCK) plays a critical role in adipose tissue glyccroneogenesis. We havc previously shown that transcription of the PEPCK gene was stimulated by isoprend i n e and relinoic acid in 3T3-E'442A adipocytes. We. also showed that oleate increased PEPCK mRNA. Here, we analysed the effect that fatty acids of various chain lengths and unsaturation degrces exerted on PEPCK gene expression in 3T3-F442A adipocytes. Whcn maintained in scrum-free, glucose-free medium, differentiated cells responded to unsaturated long-chain fatty acids by a large increase in PEPCK mRNA whereas saturated fatty acids were inefficient. A maximum fivefold stimulation by oleate was attained at 4 h of treatment with 1 mM fatty acid bound to albumin i n a 6: 1 ratio. The poly-unsaturated very longchain fatty acid all-cis-4,7,10,'13,16,19-docosahcxaenoic acid (C,,,,) was even more potent and produced a tenfold increase. The expression of the genes encoding glycerol-3-phosphate dehydrogenase, hormonesensitive lipase or actin remained unaffected by oleate exposure. A 4-h treatment by the hypolipidemic drug clofibrate, 0.5-2 mM, also produccd a large (3-9-fold) increase in PEPCK mRNA. When used at non-saturating concentrations, oleate and clofibrate acted in an additive manner. At maximally effective concentrations, additivity was lost, suggesting that fatty acids and fibrates might act through similar mechanisms. Nuclear transcription experiments showed that olcate and clofibrate stimulated the transcription rate of the gene. 3T3-F442A cells were stably transfected with it plasmid containing the base pairs -2100 to +69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase gene. These differentiated stable transfectants responded to oleate and clofibrrite by a specific increase in chloramphenicol acetyltransferasc activity. Adipocytes express various isoforms of peroxisomc-proliferator-activated receptors that can be activated by fibrates and fatty acids. Potcntial recognition sequences for peroxisomeproliferator-activated receptors are present in the -2100 to +69 fragment of thc PEPCK gene promoter. Thus, this gene represents an ideal molecular target for undersianding the complex transcriptional control excrted by fatty acids and peroxisomc proliferators.

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