Pierre Robin

Peroxisome proliferator activated receptor-γ, leptin and tumor necrosis factor-α mRNA expression during very low calorie diet in subcutaneous adipose tissue in obese women

PPARγ, leptin and TNFα are three major factors that play a key role in influencing adipocyte differentiation and both adipose tissue function and metabolism. However, the regulation of these three genes during a dynamic period of weight loss is unknown. We therefore investigated the concomitant regulation of the mRNA expression of PPARγ, leptin and TNFα in adipose tissue during a 21‐day very low calorie diet (VLCD) in 12 non‐diabetic obese women.

Glucocorticoids repress induction by thiazolidinediones, fibrates, and fatty acids of phosphoenolpyruvate carboxykinase gene expression in adipocytes

Phosphoenolpyruvate carboxykinase (PEPCK) exerts a glyceroneogenic function in adipocytes in which transcription of its gene is increased by unsaturated fatty acids and fibrates. We used cultured rat adipose tissue fragments and 3T3‐F442A adipocytes to show that the antidiabetic thiazolidinedione BRL 49653, a ligand and an activator of the γ isoform of peroxisome proliferator activated receptors (PPARγ), is a potent inducer of PEPCK mRNA. In 3T3‐F442A adipocytes, the effect of BRL 49653 is rapid and concentration dependent, with a maximum reached at 1 μM and a half‐maximum at 10–100 nM. PEPCK mRNA is similarly induced by the natural ligand of PPARγ, the 15‐deoxy‐Δ12–14 prostaglandin J2. These observations strongly suggest that PPARγ is a primary regulator of PEPCK gene expression in adipocytes. Dexamethasone at 10 nM repress induction of PEPCK mRNA by 1 μM BRL 49653, 0.32 mM oleate, or 1 mM clofibrate, in a cycloheximide‐independent manner. The antiglucocorticoid RU 38486 prevents dexamethasone action, demonstrating involvement of the glucocorticoid receptor. Stable transfectants of 3T3‐F442A adipocytes bearing −2100 to +69 base pairs of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene respond to 1 μM BRL 49653 or 1 mM clofibrate by a large increase in CAT activity, which is prevented by the simultaneous addition of 10 nM dexamethasone. Hence, in adipocytes, glucocorticoids act directly through the 5′‐flanking region of the PEPCK gene to repress, in a dominant fashion, the stimulation of PEPCK gene transcription by thiazolidinediones and fibrates. J. Cell. Biochem. 68:298–308, 1998.

Isolation and characterization of the mouse cytosolic phosphoenolpyruvate carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites

A 72 kilobase pair DNA fragment that contains the mouse phosphoenolpyruvate carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from approximately -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.

Transcriptional and posttranscriptional mechanisms of glucocorticoid-mediated repression of phosphoenolpyruvate carboxykinase gene expression in adipocytes

Glucocorticoids exert pleiotropic effects, among which negative regulation of transcription has been recognized as of crucial importance. While glucocorticoids induce phosphoenolpyruvate carboxykinase (PEPCK) gene expression in liver cells, it represses gene activity in adipose cells. We used the 3T3‐F442A adipocytes to analyze the underlying mechanisms. In these cells, the synthetic glucocorticoid dexamethasone exerts a dominant repression either on basal or on β‐agonist stimulation of PEPCK gene expression. To determine whether glucocorticoid action required protein synthesis, we employed cycloheximide, anisomycin, and puromycin, three different translation inhibitors. None of these affected induction by isoprenaline or repression by dexamethasone of isoprenaline stimulation. In contrast, dexamethasone inhibitory action on basal PEPCK mRNA was totally prevented by the three translation inhibitors. Time courses of glucocorticoid action on basal and on induction by β‐agonist were similar. Half‐maximal effect of dexamethasone on isoprenaline‐induced PEPCK mRNA was obtained at about 10 nM, a tenfold higher concentration than that observed for the reduction of basal mRNA. Using the transcription inhibitor DRB, we showed that dexamethasone did not alter mRNA half‐life, while isoprenaline strongly stabilized mRNA. In a 3T3‐F442A stable transfectant bearing −2,100 base pairs of the PEPCK promoter fused to the chloramphenicol acetyltransferase (CAT) gene, isoprenaline stimulated CAT activity, whereas dexamethasone reduced basal and isoprenaline‐induced CAT expression. Hence, β‐agonists exert both transcriptional and posttranscriptional regulation, while glucocorticoid action is purely transcriptional. However, mechanisms of glucocorticoid repression of basal and of β‐agonist stimulation appear different. J. Cell. Biochem. 66:386–393, 1997.

Intramitochondrial factors controlling hepatic fatty acid oxidation at weaning in the rat

Fatty acid oxidation was studied in isolated liver mitochondria of rats during the suckling‐weaning transition. The oxidation rate of oleyl‐CoA and palmitoylcarnitine was reduced 2.5‐fold in rats weaned on a high‐carbohydrate diet compared to suckling rats, when acetyl‐CoA produced by β‐oxidation was directed towards ketone‐body synthesis. Weaning on a high‐fat diet minimized this change. Channeling of acetyl‐CoA towards citrate synthesis doubled the oxidation rate of both substrates in HC‐weaned rats. Thus, in addition to changes in carnitine palmitoyltransferase I activity, the β‐hydroxymethylglutaryl‐CoA synthase pathway is also involved in the decreased fatty acid oxidation at weaning. This was confirmed by measurement of β‐hydroxymethylglutaryl‐CoA synthase pathway activity.