Bart Staels

The organization, promoter analysis, and expression of the human PPARgamma gene

PPARγ is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPARγ cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPARγ1 and PPARγ2, were detected in man. The relative expression of human PPARγ was studied by a newly developed and sensitive reverse transcriptase-competitive polymerase chain reaction method, which allowed us to distinguish between PPARγ1 and γ2 mRNA. In all tissues analyzed, PPARγ2 was much less abundant than PPARγ1. Adipose tissue and large intestine have the highest levels of PPARγ mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPARγ is barely detectable in muscle. This high level expression of PPARγ in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPARγs, the human PPARγs are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPARγ gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPARγ1 and PPARγ2 mRNAs, which differ at their 5′-ends. PPARγ1 is encoded by eight exons, and PPARγ2 is encoded by seven exons. The 5′-untranslated sequence of PPARγ1 is comprised of exons A1 and A2, whereas that of PPARγ2 plus the additional PPARγ2-specific N-terminal amino acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPARγ1 and γ2. Knowledge of the gene structure will allow screening for PPARγ mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.

Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor alpha-deficient mice

The peroxisome proliferator-activated receptor-α (PPARα) controls gene expression in response to a diverse class of compounds collectively referred to as peroxisome proliferators. Whereas most known peroxisome proliferators are of exogenous origin and include hypolipidemic drugs and other industrial chemicals, several endogenous PPARα activators have been identified such as fatty acids and steroids. The latter finding and the fact that PPARα modulates target genes encoding enzymes involved in lipid metabolism suggest a role for PPARα in lipid metabolism. This was investigated in the PPARα-deficient mouse model. Basal levels of total serum cholesterol, high density lipoprotein cholesterol, hepatic apolipoprotein A-I mRNA, and serum apolipoprotein A-I in PPARα-deficient mice are significantly higher compared with wild-type controls. Treatment with the fibrate Wy 14,643 decreased apoA-I serum levels and hepatic mRNA levels in wild-type mice, whereas no effect was detected in the PPARα-deficient mice. Administration of the fibrate Wy 14,643 to wild-type mice results in marked depression of hepatic apolipoprotein C-III mRNA and serum triglycerides compared with untreated controls. In contrast, PPARα-deficient mice were unaffected by Wy 14,643 treatment. These studies demonstrate that PPARα modulates basal levels of serum cholesterol, in particular high density lipoprotein cholesterol, and establish that fibrate-induced modulation in hepatic apolipoprotein A-I, C-III mRNA, and serum triglycerides observed in wild-type mice is mediated by PPARα.

The Farnesoid X Receptor Modulates Hepatic Carbohydrate Metabolism during the Fasting-Refeeding Transition

The liver plays a central role in the control of blood glucose homeostasis by maintaining a balance between glucose production and utilization. The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor. Hepatic FXR expression is regulated by glucose and insulin. Here we identify a role for FXR in the control of hepatic carbohydrate metabolism. When submitted to a controlled fasting-refeeding schedule, FXR-/- mice displayed an accelerated response to high carbohydrate refeeding with an accelerated induction of glycolytic and lipogenic genes and a more pronounced repression of gluconeogenic genes. Plasma insulin and glucose levels were lower in FXR-/- mice upon refeeding the high-carbohydrate diet. These alterations were paralleled by decreased hepatic glycogen content. Hepatic insulin sensitivity was unchanged in FXR-/- mice. Treatment of isolated primary hepatocytes with a synthetic FXR agonist attenuated glucose-induced mRNA expression as well as promoter activity of L-type pyruvate kinase, acetyl-CoA carboxylase 1, and Spot14. Moreover, activated FXR interfered negatively with the carbohydrate response elements regions. These results identify a novel role for FXR as a modulator of hepatic carbohydrate metabolism.

Fibrates influence the expression of genes involved in lipoprotein metabolism in a tissue-selective manner in the rat.

The influence of different fibrates on apolipoprotein metabolism was investigated. Administration of fenofibrate provoked a dose-dependent decrease in plasma cholesterol concentration that was already evident after 1 day. Intestinal apolipoprotein (apo) A-I and apo A-IV mRNA levels remained fairly constant. In contrast, liver apo A-I, apo A-II, and apo A-IV mRNA levels decreased in a dose-dependent fashion, which was associated with a lower transcription rate of the apo A-I but not the apo A-II gene. The decline in hepatic apo A-I, apo A-II, and apo A-IV mRNA had already started after 1 day and was associated with a drop in plasma apo A-I and apo A-IV concentrations. Plasma apo E had already decreased after 1 day of fenofibrate, whereas apo B initially remained constant and increased only after 14 days of fenofibrate at the highest dose. Hepatic and intestinal apo B mRNA contents and liver, heart, kidney, and testis apo E mRNA contents were only marginally affected after treatment with fenofibrate. Liver low density lipoprotein receptor mRNA levels rose slightly after a 3-day administration of the highest dose of fenofibrate. Both clofibrate and gemfibrozil had effects comparable to those of fenofibrate on liver and intestinal apolipoprotein mRNA levels except for liver apo A-II mRNA, which decreased only marginally. Compared with fenofibrate, clofibrate caused similar changes in plasma cholesterol, apo A-I, apo A-IV, and apo E concentrations, whereas gemfibrozil increased plasma cholesterol and apo E without changing apo A-I and apo A-IV concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxisome proliferator-activated receptor-alpha regulates lipid homeostasis, but is not associated with obesity: studies with congenic mouse lines

Considerable controversy exists in determining the role of peroxisome proliferator-activated receptor-α (PPARα) in obesity. Two purebred congenic strains of PPARα-null mice were developed to study the role of this receptor in modulating lipid transport and storage. Weight gain and average body weight in wild-type and PPARα-null mice on either an Sv/129 or a C57BL/6N background were not markedly different between genotypes from 3 to 9 months of age. However, gonadal adipose stores were significantly greater in both strains of male and female PPARα-null mice. Hepatic accumulation of lipids was greater in both strains and sexes of PPARα-null mice compared with wild-type controls. Administration of the peroxisome proliferator WY-14643 caused hepatomegaly, alterations in mRNAs encoding proteins that regulate lipid metabolism, and reduced serum triglycerides in a PPARα-dependent mechanism. Constitutive differences in serum cholesterol and triglycerides in PPARα-null mice were found between genetic backgrounds. Results from this work establish that PPARα is a critical modulator of lipid homeostasis in two congenic mouse lines. This study demonstrates that disruption of the murine gene encoding PPARα results in significant alterations in constitutive serum, hepatic, and adipose tissue lipid metabolism. However, an overt, obese phenotype in either of the two congenic strains was not observed. In contrast to earlier published work, this study establishes that PPARα is not associated with obesity in mice.

Changes in IgG Fc receptor expression induced by phorbol 12-myristate 13-acetate treatment of THP-1 monocytic leukemia cells

We studied changes in the three types of Fc gamma receptor (FcR) on the THP-1 human monocytic leukemia cells, after incubation with the phorbol ester, PMA, which has been shown to alter the expression of several genes in these cells. THP-1 cells constitutively express FcRI and FcRII, and PMA down-regulated the expression of both FcRI and FcRII. The FcRIII expression was not detected on either untreated or PMA-treated cells. Addition of PMA to THP-1 cells also resulted in a dose-dependent decrease of CD4 expression, as well as in an increased expression of activation-associated antigens. PMA treatment was followed by a progressive decrease in the steady state level of FcRI mRNA, while FcRII mRNA levels did not change, pointing to different regulatory mechanisms at the pre- and post-transcriptional level respectively. The FcRIII mRNA was undetectable. In order to further delineate the mechanism by which PMA induces alterations in FcR expression, we treated cells with stimulators of protein kinase C, of Ca2+ calmodulin-dependent kinase, and of protein kinase A. Since stimulation of none of these second messenger systems induced similar alterations in FcR expression as PMA we next tested the effects of PMA on differentiation and arrest of proliferation. The changes in FcR only occurred at PMA concentrations capable of inducing cell adherence and an arrest of proliferation, and showed a relatively slow time pattern. This suggested that the alterations in FcR expression may be linked to partial differentiation into a more macrophage-like cell. The changes in FcR expression could furthermore be reproduced by 1,25(OH)2 vitamin D3, another agent capable of differenting monocytes. In conclusion, PMA treatment of THP-1 cells decreases FcRI gene transcription and membrane expression and reduces membrane expression of FcRII. Both changes might be linked with an arrest of cell growth and induction of differentiation.

Down-regulation of hepatic lipase gene expression and activity by fenofibrate

The influence of the hypolipidemic drug, fenofibrate, on hepatic lipase (HL) gene expression and activity was investigated in the rat. Fenofibrate treatment provoked a dose-dependent decrease in HL mRNA levels. At a dose of 0.5% (w/w), HL mRNA levels were reduced to nearly 50% the levels in untreated controls. This decrease was parallelled by a comparable reduction in liver HL activity. The decrease in HL mRNA levels was already observed after 1 day of fenofibrate treatment. Whole liver perfusion experiments showed that the heparin-releasable HL activity in fenofibrate-treated livers dropped to 10% the activity in control livers. In conclusion, treatment with fenofibrate decreases HL gene expression, leading to a lowered activity of endothelium bound HL in fenofibrate-treated livers.

Opposite regulation of hepatic lipase and lecithin: cholesterol acyltransferase by glucocorticoids in rats.

Rats were treated with hydrocortisone, dexamethasone or triamcinolone for 4 days. The effect of treatment on hepatic lipase and lecithin:cholesterol acyltransferase (LCAT) mRNA levels and catalytic activities was determined. Hepatic lipase mRNA was not affected by hydrocortisone, but was decreased after dexamethasone (-28%) and triamcinolone (-54%). Hepatic lipase activity followed the same pattern, it was not affected by hydrocortisone and lowered by dexamethasone (-38%) and triamcinolone (-70%). The LCAT mRNA level in the liver was also not affected by hydrocortisone, but increased upon treatment with dexamethasone (+22%) and triamcinolone (+72%). Plasma LCAT, determined with an excess exogenous substrate (designated LCAT-II), tended to decrease after hydrocortisone treatment (-11%) and was higher after dexamethasone (+21%) and triamcinolone (+22%). The plasma cholesterol esterification rate (designated LCAT-I), determined by incubation of the plasma at 37 degrees C, followed the same pattern. The activity ratio of hepatic lipase/LCAT-II decreased from 1 in the controls to 0.51 after dexamethasone and 0.25 in the triamcinolone-treated animals. The plasma HDL cholesterol concentration in the different groups changed oppositely to the hepatic lipase/LCAT activity ratio. It is concluded that HDL cholesterol is raised by synthetic glucocorticoids due, among other factors, to a lowered hepatic lipase and an increased plasma LCAT activity. The influence of glucocorticoids on these enzymes is, at least partly, explained by the effects on the hepatic mRNA contents.

Effects of sex steroids on hepatic and lipoprotein lipase activity and mRNA in the rat

In humans, sex steroids have been implicated in the regulation of hepatic and lipoprotein lipase activity. Therefore, the effects of orchidectomy and subsequent androgen or estrogen administration on hepatic lipase (HL) and adipose tissue and heart lipoprotein lipase (LPL) were examined. Relative to intact controls, orchidectomy of male rats resulted in no significant change in HL activity and mRNA, or in heart and adipose tissue LPL activity and mRNA levels. Subsequently, a subcutaneous silastic tubing, delivering either testosterone, dihydrotestosterone, nandrolone, or 17 beta-estradiol, was implanted for 5 weeks. All substitution treatments had a tendency to reduce HL activity and to induce HL mRNA levels. This effect was, however, only significant for testosterone which resulted in a decrease in HL activity (238 +/- 15 vs. 328 +/- 31 mU/g tissue; p vs. control < 0.05) and an increase in HL mRNA (166 +/- 11 vs. 100 RAU; p vs. control < 0.01). No significant effects of androgens on LPL expression either in heart or adipose tissue were observed. Adipose tissue LPL activity (20 +/- vs. 35 +/- 4 mU/g; p vs. control < 0.05) and mRNA (28 +/- 4 vs. 100 RAU; p vs. control < 0.001) levels, but not heart LPL, however, were diminished substantially after 17 alpha-estradiol treatment. In conclusion, rat HL is influenced by testosterone, while adipose tissue, but not heart LPL, is reduced after estrogen administration.

Neonatal extinction of liver lipoprotein lipase expression

In contrast to the complete absence of lipoprotein lipase (LPL) mRNA in adult rat liver, fetal and neonatal rat liver contain substantial amounts of LPL mRNA, which is translated in active LPL protein as can be deduced from the presence of LPL activity in this organ. At this neonatal stage, both the relative abundance of LPL mRNA and LPL activity increased with starvation. During the suckling period, LPL mRNA and LPL activity gradually decreased until both parameters were undetectable. While the administration of L-thyroxine or hydrocortisone enhanced the disappearance of LPL mRNA, induced hypothyroidism delayed its disappearance. In adult animals induced hypothyroidism could not reactivate LPL mRNA production in the liver. The data presented suggest that liver LPL production responds to changes in the nutritional state and becomes extinguished during development, in a fashion reminiscent to the extinction of alpha-fetoprotein. This extinction of LPL gene expression is influenced by hormonal factors.