ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose.

The transcription of genes encoding proteins involved in the hepatic synthesis of lipids from glucose is strongly stimulated by carbohydrate feeding. It is now well established that in the liver, glucose is the main activator of the expression of this group of genes, with insulin having only a permissive role. While ADD1/SREBP-1 has been implicated in lipogenic gene expression through temporal association with food intake and ectopic gain-of-function experiments, no genetic evidence for a requirement for this factor in glucose-mediated gene expression has been established. We show here that the transcription of ADD1/SREBP-1c in primary cultures of hepatocytes is controlled positively by insulin and negatively by glucagon and cyclic AMP, establishing a link between this transcription factor and carbohydrate availability. Using adenovirus-mediated transfection of a powerful dominant negative form of ADD1/SREBP-1c in rat hepatocytes, we demonstrate that this factor is absolutely necessary for the stimulation by glucose of L-pyruvate kinase, fatty acid synthase, S14, and acetyl coenzyme A carboxylase gene expression. These results demonstrate that ADD1/SREBP-1c plays a crucial role in mediating the expression of lipogenic genes induced by glucose and insulin.

SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.

Biochemical properties of liver peroxisomes from rat, guinea pig and human species and the influence of hormonal status on rat liver acyl-CoA oxidase mRNA content.

Liver peroxisomes from three different species, rat, guinea pig and man, have been purified by ultracentrifugation on a discontinuous Nycodenz gradient. Several biochemical parameters were tested in order to compare the basic peroxisomal properties of liver from rat, a species strongly responsive to peroxisome proliferators, and guinea pig and man, two weakly responsive species. Polypeptide patterns were compared and the bands in guinea pig and man comigrating with the two major bands in rat, catalase at 66 kDa and urate oxidase at 35 kDa, appeared in low amounts. However, other polypeptides are similar throughout these species especially in guinea pig as revealed by cross-immunoreactivity using an anti-rat peroxisomal protein rabbit immune serum. Specific activities of peroxisome acyl-CoA oxidase and microsome omega-lauryl hydroxylase have comparable rates in rat and guinea pig liver, but in human liver the activities are much lower. There is a cross-hybridization between acyl-CoA oxidase mRNA probed by rat liver acyl-CoA oxidase cDNA among the three species at a medium stringency. But interestingly, acyl-CoA oxidase mRNA from guinea pig and man appear to be larger in size. On the other hand, the hormonal status does not seem to have a significant effect on the rat liver acyl-CoA oxidase mRNA level suggesting at most that insulin, corticosterone and estradiol have no direct effect on acyl-CoA oxidase gene expression, which contrasts with the well-known effect of peroxisome proliferators.

Transcriptional and post-transcriptional analysis of peroxisomal protein encoding genes from rat treated with an hypolipemic agent, ciprofibrate. Effect of an intermittent treatment and influence of obesity.

The treatment of rats with ciprofibrate, a potent peroxisome proliferator, led to increased levels of the peroxisomal acyl-CoA oxidase (ACO) mRNA. How ciprofibrate functions to elevate ACO mRNA is not known. To help determine the mechanism of ciprofibrate action, in vitro transcription assays were performed. It was determined that ciprofibrate was responsible for a 3.5-fold stimulation of the rate of ACO transcription within 24 hr of ingestion. It was also observed that the transcription rate stimulation following a 2-week ciprofibrate treatment of Wistar rats was maintained following 4 weeks of ciprofibrate withdrawal. Re-introduction of the drug after the 4-week pause resulted in greater stimulation than was initially observed. The results demonstrate that the effect of ciprofibrate is rapid and persists at least twice as long as the initial treatment period. In Zucker rats, both lean and obese, ACO mRNA levels were examined following 2 weeks of ciprofibrate treatment (1 or 3 mg/kg body weight/day). The presence of increased blood levels of triglycerides did not increase ciprofibrate action on transcription, although basal levels of transcription of peroxisomal enzymes were higher in obese rats. The increase in the ACO mRNA level was greater than the transcription rate stimulation suggesting a post-transcriptional regulation.

Role of thyroid state on induction by ciprofibrate of laurate hydroxylase and peroxisomal enzymes in rat liver microsomes.

The effects of hypothyroidism and hyperthyroidism upon liver microsomal omega-laurate hydroxylase activity (cytochrome P450 IV A1-dependent), peroxisome proliferation marker enzyme activities and acyl CoA oxidase (AOX) expression induced by ciprofibrate (2 mg/kg/day during 8 days) were studied in the male Wistar rat so as to clarify firstly the possible involvement of thyroid hormones in the modification of peroxisomal ciprofibrate-induced enzyme activities in relation to hepatic microsomal cytochrome P450 IV A1 induction, and secondly the possible direct effect of thyroid hormones on the gene expression of specific peroxisomal enzymes. No significant change was found in the ciprofibrate-induced omega-laurate hydroxylase activity in hypothyroid rats or in rats that had received a large dose of triiodothyronine (LT3), suggesting that the thyroid hormone does not interfere with the peroxisome proliferation process through such an indirect mechanism. The induction by ciprofibrate [2-(4-(2-2dichlorocyclopropyl)phenoxyl-2methyl-propion ic acid)] of mitochondrial alpha-glycerolphosphate dehydrogenase and microsomal bilirubin UDPGT was decreased about 3-fold and 1.5-fold, respectively, while the induction of peroxisomal AOX, carnitine acetyl transferase and enoyl CoA hydratase enzyme activities was decreased by 36%, 34% and 22% in thyroidectomized animals, as compared to euthyroid animals. However, no significant changes in the quantity of peroxisomal proteins and in the AOX mRNA level were noted. The administration of large doses of LT3 to normal rats decreased the peroxisomal ciprofibrate AOX enzyme induction with a marked concomitant decrease in the AOX mRNA level. This suggests that high doses of LT3 enhance the turnover of some specific mRNAs or down regulate the peroxisome proliferator receptor. Our results also do not exclude inhibition of catabolic activity towards AOX which depends on thyroid hormone.

Effect of different hypolipemic agents on rat liver peroxisomal and mitochondrial functions and biogenesis.

The effect of four fibrate analogues (i.e. clofibrate, ciprofibrate, clobuzarit and 2,4-dichlorophenoxyacetic acid (2,4-D), an active herbicide molecule) were tested on the biogenesis of liver mitochondrial and peroxisomal proteins by rat in vivo treatment at 100 ppm for 26 weeks. The evaluations were done at different levels: somatic index, histochemistry electron microscopy, enzymatic activities on purified peroxisomes and mitochondria, polypeptides electrophoresis and immunolabeling, and finally mRNA hybridization with specific DNA probes. This work shows that the tested hypolipemic agents are strong peroxisomal proliferators especially ciprofibrate, while mitochondria are weakly affected. However, the four fibrates gave different effects, especially 2,4-D which modifies mitochondrial polypeptide pattern. Post-transcriptional study of mRNAs level shows a slight increase in catalase mRNA despite the potential of hypolipemic agents. The peroxisomal acyl-CoA oxidase mRNA content is enhanced with ciprofibrate treatment as well as mitochondrial R-3-hydroxybutyrate dehydrogenase (BDH) mRNA level. Finally, the dual action of ciprofibrate on content and on enzymatic activity of BDH (a lipid metabolism related enzyme) reveals that such a molecule may have differential regulatory effects (positive on gene transcription or mRNA stability and negative on catalytic enzyme activity).

Response of genetically obese Zucker rats to ciprofibrate, a hypolipidemic agent, with peroxisome proliferation activity as compared to Zucker lean and Sprague-Dawley rats.

Genetically obese Zucker (fa/fa) rats were used as an experimental model to study the effects of hypolipidemic agents on peroxisome proliferation; comparison was made with Zucker lean phenotype (Fa/-) and Sprague-Dawley strain/phenotype. The pharmacokinetics of a single administration of ciprofibrate (1 or 3 mg/kg), appeared to be similar in all strains/phenotypes. After a 2-week oral administration at the same dosages, there were dosage-related increases in hepatocellular peroxisomal yield and in the hepatic enzymes' cyanide-insensitive acyl-CoA oxidase and catalase. The peroxisomal yield was less increased in Zucker than in Sprague-Dawley rats, while the enzyme activities were similarly increased. Although the absolute specific activity of microsomal omega-lauryl hydroxylase (cytochrome P4504A1) was lower in Zucker rats, it was increased more in this strain than in Sprague-Dawley rats in response to drug exposure. The hypolipidemic effect (cholesterol and triglyceride reduction) was more pronounced in Zucker obese rats. Based on biochemical and morphological results, no major differences between strains/phenotypes in terms of peroxisome proliferation were observed following a 2-week administration of ciprofibrate.

Difference between guinea pig and rat in the liver peroxisomal response to equivalent plasmatic level of ciprofibrate.

Guinea pig was previously classified as a species nonresponsive to peroxisome proliferators. However, none of the previous reports was based on pharmacokinetic data. Here, after a comparative pharmacokinetic study between the guinea pig and rat, we evaluate the guinea pig liver peroxisomal response to ciprofibrate, a hypolipemic agent and a potent peroxisome proliferator in rat. (1) Pharmacokinetic results show equivalent in guinea pig and rat when guinea pigs are treated with ciprofibrate at 30 mg/kg twice a day and rats are treated at 3 mg/kg once a day. (2) The treatment of guinea pigs at 30 mg/kg twice a day for 2 weeks leads to a significant increase in the liver peroxisomal palmitoyl-CoA oxidase activity (x 1.6) and also in the microsomal omega-laurate hydroxylase activity (x 1.8). These increases are in accordance with the changes in polypeptide patterns of isolated liver peroxisomes as well as in the immunoblotting of acyl-CoA oxidase. It is deduced that a weak, but significant, peroxisome proliferation can occur in guinea pig liver after a ciprofibrate treatment at dosages corresponding to equivalent plasmic concentrations of the drug between guinea pig and rat. (3) The hybridization of guinea pig liver RNA with the rat liver-inducible acyl-CoA oxidase cDNA probe shows a decrease in the corresponding heterologous mRNA content after treatment with ciprofibrate at 30 mg/kg twice a day. This result contrasts with the slight increase observed in immunodetection and in enzymatic assays, suggesting the existence of at least two different acyl-CoA oxidases in guinea pig liver peroxisomes.

Differential regulation by a peroxisome proliferator of the different multifunctional proteins in guinea pig: cDNA cloning of the guinea pig D-specific multifunctional protein 2.

After our previous report on the cloning of two cDNA species in guinea pig, both encoding the same hepatic 79 kDa multifunctional protein 1 (MFP-1) [Caira, Cherkaoui-Malki, Hoefler and Latruffe (1996) FEBS Lett. 378, 57-60], here we report the cloning of a cDNA encoding a second multifunctional peroxisomal protein (MFP-2) in guinea-pig liver. This 2356 nt cDNA encodes a protein of 735 residues (79.7 kDa) whose sequence shows 83% identity with rat MFP-2 [Dieuaide-Noubhani, Novikov, Baumgart, Vanhooren, Fransen, Goethals, Vandekerckhove, Van Veldhoven and Mannaerts (1996) Eur. J. Biochem. 240, 660-666]. In parallel, we studied the effect of ciprofibrate, a hypolipaemic agent also known as peroxisome proliferator in rodent, on the expression of MFP-1 and MFP-2 (2.6 kb) in rats and guinea pigs. By Northern blotting analysis we demonstrated that three MFP-1-related mRNA species are expressed in the guinea-pig liver. The expression of two of them (3.5 and 2.6 kb) is slightly increased by ciprofibrate, whereas the 3.0 kb MFP-1 mRNA is, unlike the rat one, strongly down-regulated in guinea pigs treated with ciprofibrate. In a similar way, the hepatic expression of the guinea-pig 2.6 kb MFP-2 mRNA is also down-regulated in guinea pigs treated with ciprofibrate. These results demonstrate (1) that in contrast with the unique 3.0 kb MFP-1 rat mRNA, at least three hepatic MFP-1-related mRNA species are co-expressed in guinea pig; and (2) that, opposed to the accepted idea of non-responsiveness of the guinea pig to ciprofibrate, this drug affects MFP-1 and MFP-2 gene expression in this species. Also, the mRNA species for acyl-CoA oxidase and thiolase, two other enzymes of the peroxisomal beta-oxidation pathway that are induced severalfold in responsive species are down-regulated in guinea pig. This paper is the first, to our knowledge, reporting the down-regulation of the expression of genes encoding enzymes involved in the peroxisomal beta-oxidation of fatty acids (MFP-1) and bile acid synthesis (MFP-2) in mammals.

Critical roles of PPAR beta/delta in keratinocyte response to inflammation.

The immediate response to skin injury is the release of inflammatory signals. It is shown here, by use of cultures of primary keratinocytes from wild-type and PPAR beta/delta(-/-) mice, that such signals including TNF-alpha and IFN-gamma, induce keratinocyte differentiation. This cytokine-dependent cell differentiation pathway requires up-regulation of the PPAR beta/delta gene via the stress-associated kinase cascade, which targets an AP-1 site in the PPAR beta/delta promoter. In addition, the pro-inflammatory cytokines also initiate the production of endogenous PPAR beta/delta ligands, which are essential for PPAR beta/delta activation and action. Activated PPAR beta/delta regulates the expression of genes associated with apoptosis resulting in an increased resistance of cultured keratinocytes to cell death. This effect is also observed in vivo during wound healing after an injury, as shown in dorsal skin of PPAR beta/delta(+/+) and PPAR beta/delta(+/-) mice.