Kristina Schoonjans

Mechanism of Action of Fibrates on Lipid and Lipoprotein Metabolism

Treatment with fibrates, a widely used class of lipid-modifying agents, results in a substantial decrease in plasma triglycerides and is usually associated with a moderate decrease in LDL cholesterol and an increase in HDL cholesterol concentrations. Recent investigations indicate that the effects of fibrates are mediated, at least in part, through alterations in transcription of genes encoding for proteins that control lipoprotein metabolism. Fibrates activate specific transcription factors belonging to the nuclear hormone receptor superfamily, termed peroxisome proliferator-activated receptors (PPARs). The PPAR-alpha form mediates fibrate action on HDL cholesterol levels via transcriptional induction of synthesis of the major HDL apolipoproteins, apoA-I and apoA-II. Fibrates lower hepatic apoC-III production and increase lipoprotein lipase--mediated lipolysis via PPAR. Fibrates stimulate cellular fatty acid uptake, conversion to acyl-CoA derivatives, and catabolism by the beta-oxidation pathways, which, combined with a reduction in fatty acid and triglyceride synthesis, results in a decrease in VLDL production. In summary, both enhanced catabolism of triglyceride-rich particles and reduced secretion of VLDL underlie the hypotriglyceridemic effect of fibrates, whereas their effect on HDL metabolism is associated with changes in HDL apolipoprotein expression.

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.

PPARalpha and PPARgamma activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene.

Increased activity of lipoprotein lipase (LPL) may explain the hypotriglyceridemic effects of fibrates, thiazolidinediones and fatty acids, which are known activators (and/or ligands) of the various peroxisome proliferator‐activated receptors (PPARs). Treatment with compounds which activate preferentially PPARalpha, such as fenofibrate, induced LPL expression exclusively in rat liver. In contrast, the antidiabetic thiazolidinedione BRL 49653, a high affinity ligand for PPARgamma, had no effect on liver, but induced LPL expression in rat adipose tissue. In the hepatocyte cell line AML‐12, fenofibric acid, but not BRL 49653, induced LPL mRNA, whereas in 3T3‐L1 preadipocytes, the PPARgamma ligand induced LPL mRNA levels much quicker and to a higher extent than fenofibric acid. In both the in vivo and in vitro studies, inducibility by either PPARalpha or gamma activators, correlated with the tissue distribution of the respective PPARs: an adipocyte‐restricted expression of PPARgamma, whereas PPARalpha was expressed predominantly in liver. A sequence element was identified in the human LPL promoter that mediates the functional responsiveness to fibrates and thiazolidinediones. Methylation interference and gel retardation assays demonstrated that a PPARalpha or gamma and the 9‐cis retinoic acid receptor (RXR) heterodimers bind to this sequence −169 TGCCCTTTCCCCC −157. These data provide evidence that transcriptional activation of the LPL gene by fibrates and thiazolidinediones is mediated by PPAR‐RXR heterodimers and contributes significantly to their hypotriglyceridemic effects in vivo. Whereas thiazolidinediones predominantly affect adipocyte LPL production through activation of PPARgamma, fibrates exert their effects mainly in the liver via activation of PPARalpha.

The peroxisome proliferator activated receptors (PPARS) and their effects on lipid metabolism and adipocyte differentiation.

The three types of peroxisome proliferator activated receptor (PPAR), alpha, beta (or delta), and gamma, each with a specific tissue distribution, compose a subfamily of the nuclear hormone receptor gene family. Although peroxisome proliferators, including fibrates and fatty acids, activate the transcriptional activity of these receptors, only prostaglandin J2 derivatives have been identified as natural ligands of the PPAR gamma subtype, which also binds thiazolidinedione antidiabetic agents with high affinity. Activated PPARs heterodimerize with RXR and alter the transcription of target genes after binding to specific response elements or PPREs, consisting of a direct repeat of the nuclear receptor hexameric DNA core recognition motif spaced by one nucleotide. The different PPARs can be considered key messengers responsible for the translation of nutritional, pharmacological and metabolic stimuli into changes in the expression of genes, more specifically those genes involved in lipid metabolism. PPAR alpha is involved in stimulating beta-oxidation of fatty acids. In rodents, a PPAR alpha-mediated change in the expression of genes involved in fatty acid metabolism lies at the basis of the phenomenon of peroxisome proliferation, a pleiotropic cellular response, mainly limited to liver and kidney and which can lead to hepatocarcinogenesis. In addition to their role in peroxisome proliferation in rodents, PPAR is also involved in the control of HDL cholesterol levels by fibrates and fatty acids in rodents and humans. This effect is, at least partially, based on a PPAR-mediated transcriptional regulation of the major HDL apolipoproteins, apo A-I and apo A-II. The hypotriglyceridemic action of fibrates and fatty acids also involves PPARs and can be summarized as follows: (1) an increased lipolysis and clearance of remnant particles, due to changes in LPL and apo C-III levels, (2) a stimulation of cellular fatty acid uptake and their conversion to acyl-CoA derivatives by the induction of FAT, FATP and ACS activity, (3) an induction of fatty acid beta-oxidation pathways, (4) a reduction in fatty acid and triglyceride synthesis, and finally (5) a decrease in VLDL production. Hence, both enhanced catabolism of triglyceride-rich particles as well as reduced secretion of VLDL particles are mechanisms that contribute to the hypolipidemic effect of fibrates and FFAs. Whereas for PPAR beta no function so far has been identified, PPAR gamma triggers adipocyte differentiation by inducing the expression of several genes critical for adipogenesis.

Coordinate regulation of the expression of the fatty acid transport protein and acyl-CoA synthetase genes by PPARalpha and PPARgamma activators

Intracellular fatty acid (FA) concentrations are in part determined by a regulated import/export system that is controlled by two key proteins, i.e. fatty acid transport protein (FATP) and acyl-CoA synthetase (ACS), which respectively facilitate the transport of FAs across the cell membrane and their esterification to prevent their efflux. The aim of this investigation was to analyze the expression pattern of FATP and ACS and to determine whether their expression was altered by agents that affect FA metabolism through the activation of peroxisome proliferator-activated receptors (PPAR) such as the fibrates and thiazolidinediones. FATP mRNA was ubiquitously expressed, with highest levels being detected in adipose tissue, heart, brain, and testis. Fibrate treatment, which is known to preferentially activate PPARα, induced FATP mRNA levels in rat liver and intestine and induced ACS mRNA levels in liver and kidney. The antidiabetic thiazolidinedione BRL 49653, which is a high-affinity ligand for the adipocyte-specific PPARγ form, caused a small induction of muscle but a robust induction of adipose tissue FATP mRNA levels. BRL 49653 did not affect liver FATP and had a tendency to decrease heart FATP mRNA levels. ACS mRNA levels in general showed a similar pattern after BRL 49653 as FATP except for the muscle where ACS mRNA was induced. This regulation of FATP and ACS expression by PPAR activators was shown to be at the transcriptional level and could also be reproduced in vitro in cell culture systems. In the hepatocyte cell lines AML-12 or Fa 32, fenofibric acid, but not BRL 49653, induced FATP and ACS mRNA levels, whereas in the 3T3-L1 preadipocyte cell line, the PPARγ ligand induced FATP and ACS mRNA levels quicker than fenofibric acid. Inducibility of ACS and FATP mRNA by PPARα or γ activators correlated with the tissue-specific distribution of the respective PPARs and was furthermore associated with a concomitant increase in FA uptake. Most interestingly, thiazolidinedione antidiabetic agents seem to favor adipocyte-specific FA uptake relative to muscle, perhaps underlying in part the beneficial effects of these agents on insulin-mediated glucose disposal.

Regulation of Peroxisome Proliferator-Activated Receptor γ Expression by Adipocyte Differentiation and Determination Factor 1/Sterol Regulatory Element Binding Protein 1: Implications for Adipocyte Differentiation and Metabolism

ABSTRACT Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor implicated in adipocyte differentiation and insulin sensitivity. We investigated whether PPARγ expression is dependent on the activity of adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1 (ADD-1/SREBP-1), another transcription factor associated with both adipocyte differentiation and cholesterol homeostasis. Ectopic expression of ADD-1/SREBP-1 in 3T3-L1 and HepG2 cells induced endogenous PPARγ mRNA levels. The related transcription factor SREBP-2 likewise induced PPARγ expression. In addition, cholesterol depletion, a condition known to result in proteolytic activation of transcription factors of the SREBP family, induced PPARγ expression and improved PPRE-driven transcription. The effect of the SREBPs on PPARγ expression was mediated through the PPARγ1 and -3 promoters. Both promoters contain a consensus E-box motif that mediates the regulation of the PPARγ gene by ADD-1/SREBP-1 and SREBP-2. These results suggest that PPARγ expression can be controlled by the SREBP family of transcription factors and demonstrate new interactions between transcription factors that can regulate different pathways of lipid metabolism.

Transcriptional control of triglyceride metabolism: fibrates and fatty acids change the expression of the LPL and apo C-III genes by activating the nuclear receptor PPAR

The development of atherosclerosis is often associated with altered concentrations of systemic lipoproteins, which are determined by the concentration and/or activity of three groups of different proteins, i.e. apolipoproteins (apo), enzymes, and receptors. The effects of diet or therapeutic interventions on lipid metabolism are mediated by changes in activity or concentrations of these three components. Fibrates have been shown to activate nuclear receptors belonging to the steroid hormone receptor super-family, termed peroxisome proliferator activated receptor (PPAR). These activated PPARs are potent transcription factors which influence the expression of several target genes implicated in lipoprotein homeostasis, e.g. LPL, apo C-III and apo A-1. Fibrates decrease apo C-III transcription and increase LPL production via these PPARs resulting in a profound hypotriglyceridaemic effect. Apolipoproteins and enzymes are important in governing lipid metabolism, thus therapeutically altering the expression of these genes constitutes an efficient therapeutic option.

PPARγ activators improve glucose homeostasis by stimulating fatty acid uptake in the adipocytes

It is currently thought that the effects of PPARgamma activation on glucose homeostasis may be due to the effect of this nuclear receptor on the production of adipocyte-derived signalling molecules, which affect muscle glucose metabolism. Potential signalling molecules derived from adipocytes and modified by PPARgamma activation include TNFalpha and leptin, which both interfere with glucose homeostasis. In addition to its effects on these proteins, PPARgamma also profoundly affects fatty acid metabolism. Activation of PPARgamma will selectively induce the expression of several genes involved in fatty acid uptake, such as lipoprotein lipase, fatty acid transport protein and acyl-CoA synthetase, in adipose tissue without changing their expression in muscle tissue. This co-ordinate regulation of fatty acid partitioning by PPARgamma results in an adipocyte FFA steal causing a relative depletion of fatty acids in the muscle. Based on the well established interference of muscle fatty acid and glucose metabolism it is hypothesized that reversal of muscle fatty acid accumulation will contribute to the improvement in whole body glucose homeostasis.

3-Hydroxy-3-methylglutaryl CoA reductase inhibitors reduce serum triglyceride levels through modulation of apolipoprotein C-III and lipoprotein lipase

Statins are hypolipidemic drugs which not only improve cholesterol but also triglyceride levels. Whereas their cholesterol‐reducing effect involves inhibition of de novo biosynthesis of cellular cholesterol through competitive inhibition of its rate‐limiting enzyme 3‐hydroxy‐3‐methylglutaryl CoA reductase, the mechanism by which they lower triglycerides remains unknown and forms the subject of the current study. Treatment of normal rats for 4 days with simvastatin decreased serum triglycerides significantly, whereas it increased high density lipoprotein cholesterol moderately. The decrease in triglyceride concentrations after simvastatin was caused by a reduction in the amount of very low density lipoprotein particles which were of an unchanged lipid composition. Simvastatin administration increased the lipoprotein lipase mRNA and activity in adipose tissue and heart. This effect on lipoprotein lipase was accompanied by decreased mRNA as well as plasma levels of the lipoprotein lipase inhibitor apolipoprotein C‐III. These results suggest that the triglyceride‐lowering effect of statins involves a stimulation of lipoprotein lipase‐mediated clearance of triglyceride‐rich lipoproteins.

The effects of fibrates and thiazolidinediones on plasma triglyceride metabolism are mediated by distinct peroxisome proliferator activated receptors (PPARs)

The hypolipidemic fibrates and antidiabetic thiazolidinediones display potent triglyceride-lowering activities. Studies on the molecular action mechanisms of these compounds indicate that thiazolidinediones and fibrates exert their action by activating distinct transcription factors of the peroxisome proliferator activated receptor (PPAR) family, resulting in increased expression of lipoprotein lipase (LPL) and decreased expression of apolipoprotein (apo) C-III, both key-players in plasma triglyceride metabolism. Fibrates, on the one hand, are PPAR alpha activators, which selectively induce LPL mRNA levels and activity in the liver. Furthermore, hepatic apo C-III mRNA levels and protein production strongly decrease after fibrate treatment. On the other hand, thiazolidinediones, which are high affinity ligands for PPAR gamma, have no effect in the liver, but act primarily on adipose tissue, where they induce LPL mRNA levels and activity. The modulation of the expression of the LPL and apo C-III genes in liver and adipose tissue is correlated with the tissue-specific distribution of the respective PPARs (PPAR gamma expression being restricted to adipose tissue, whereas PPAR alpha is expressed predominantly in liver) confirming that fibrates and thiazolidinediones exert their effects primarily through PPAR alpha and PPAR gamma respectively. This distinct tissue-specific transcriptional regulation of genes involved in lipid metabolism by fibrates and thiazolidinediones indicates that research of compounds displaying combined PPAR alpha and PPAR gamma activation potential should lead to the discovery of more potent triglyceride-lowering drugs, which may be of use in the treatment of hypertriglyceridemia.