Fatty Acids and Fibrates are Potent Inducers of Transcription of the Phosphenol pyruvate Carboxykinase Gene in Adipocytes

Abstract

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.

Acute and selective regulation of glyceroneogenesis and cytosolic phosphoenolpyruvate carboxykinase in adipose tissue by thiazolidinediones in type 2 diabetes

“…As expected, a strong correlation was also shown between the glyceroneogenic flux of the tissue and the release of NEFA in the incubation medium. As a consequence, we observed a large decrease in plasma NEFA concentration in agreement with the role that glyceroneogenesis is playing in G3P synthesis required for fatty acid esterification after a high-fat or high-protein carbohydrate-free diet and for reesterification during fasting [36,37]. According to the minor role of glycerol phosphorylation in this process, we could not detect any variation in glycerol release whether rats were treated with rosiglitazone or not, in agreement with our previous studies, in which either a very mild decrease or no variation in glycerol release was observed [18].…”

Selectivity of fatty acids on lipid metabolism and gene expression

“…This finding indicates that PUFA-mediated regulation of adipose tissue gene expression would not necessarily need extra-adipose factors. For instance, phosphoenolpyruvate carboxykinase (EC 4.1.1.32) mRNA was strongly induced (about tenfold) by fibrates and DHA compared with oleic acid in adipocyte cell line 3T3-F442A, and the role of a PPAR in fatty acid signalling has been postulated (Antras-Ferry et al 1995). It is interesting to note that DHA is a strong activator of PPARγ2, which is a transcription factor specifically expressed in adipose tissue.…”

Modulation of Glutathione S-Transferase Subunits A2, M1, and P1 Expression by Interleukin-1β in Rat Hepatocytes in Primary Culture

“…Nuclei were prepared essentially as described by Shapiro (26) except that they were purified on a 2 M sucrose cushion. Briefly, the nuclear pellet resuspended in 2 M sucrose was layered on a 2 M sucrose cushion and then centrifuged at 40,000 rpm in an SW41 rotor at 4°C for 1 h. Nuclear run-on transcription was assayed as described by AntrasFerry et al (27). About 50 ϫ 10 6 cpm were hybridized per membrane.…”