Statin Induction of Liver Fatty Acid-Binding Protein (L-FABP) Gene Expression Is Peroxisome Proliferator-activated Receptor-α-dependent

Abstract

Statins are drugs widely used in humans to treat hypercholesterolemia. Statins act by inhibiting cholesterol synthesis resulting in the activation of the transcription factor sterol-responsive element-binding protein-2 that controls the expression of genes involved in cholesterol homeostasis. Statin therapy also decreases plasma triglyceride and non-esterified fatty acid levels, but the mechanism behind this effect remains more elusive. Liver fatty acid-binding protein (L-FABP) plays a role in the influx of long-chain fatty acids into hepatocytes. Here we show that L-FABP is a target for statins. In rat hepatocytes, simvastatin treatment induced L-FABP mRNA levels in a dose-dependent manner. Moreover, L-FABP promoter activity was induced by statin treatment. Progressive 5′-deletion analysis revealed that the peroxisome proliferator-activated receptor (PPAR)-responsive element located at position –67/–55 was responsible for the statin-mediated transactivation of the rat L-FABP promoter. Moreover, treatment with simvastatin and the PPARα agonist Wy14,649 resulted in a synergistic induction of L-FABP expression (mRNA and protein) in rat Fao hepatoma cells. This effect was also observed in vivo in wild-type mice but not in PPARα-null animals demonstrating the direct implication of PPARα in L-FABP regulation by statin treatment. Statin treatment resulted in a rise in PPARα mRNA levels both in vitro and in vivo and activated the mouse PPARα promoter in a reporter assay. Altogether, these data demonstrate that L-FABP expression is up-regulated by statins through a mechanism involving PPARα. Moreover, PPARα might be a statin target gene. These effects might contribute to the triglyceride/non-esterified fatty acid-lowering properties of statins. Statins are drugs widely used in humans to treat hypercholesterolemia. Statins act by inhibiting cholesterol synthesis resulting in the activation of the transcription factor sterol-responsive element-binding protein-2 that controls the expression of genes involved in cholesterol homeostasis. Statin therapy also decreases plasma triglyceride and non-esterified fatty acid levels, but the mechanism behind this effect remains more elusive. Liver fatty acid-binding protein (L-FABP) plays a role in the influx of long-chain fatty acids into hepatocytes. Here we show that L-FABP is a target for statins. In rat hepatocytes, simvastatin treatment induced L-FABP mRNA levels in a dose-dependent manner. Moreover, L-FABP promoter activity was induced by statin treatment. Progressive 5′-deletion analysis revealed that the peroxisome proliferator-activated receptor (PPAR)-responsive element located at position –67/–55 was responsible for the statin-mediated transactivation of the rat L-FABP promoter. Moreover, treatment with simvastatin and the PPARα agonist Wy14,649 resulted in a synergistic induction of L-FABP expression (mRNA and protein) in rat Fao hepatoma cells. This effect was also observed in vivo in wild-type mice but not in PPARα-null animals demonstrating the direct implication of PPARα in L-FABP regulation by statin treatment. Statin treatment resulted in a rise in PPARα mRNA levels both in vitro and in vivo and activated the mouse PPARα promoter in a reporter assay. Altogether, these data demonstrate that L-FABP expression is up-regulated by statins through a mechanism involving PPARα. Moreover, PPARα might be a statin target gene. These effects might contribute to the triglyceride/non-esterified fatty acid-lowering properties of statins. Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. The resulting lower intracellular cholesterol concentration after statin treatment leads to a proteolytic activation of the transcription factor sterol responsive element-binding protein-2 (SREBP-2), 1The abbreviations used are: SREBP, sterol responsive element-binding protein; LDL, low density lipoproteins; LDLr, LDL receptor; TG, triglycerides; NEFA, non-esterified fatty acids; LPL, lipoprotein lipase; L-FABP, liver fatty acid-binding protein; LCFA, long-chain fatty acid; PPAR, peroxisome proliferator-activated receptor; WT, wild type; FCS, fetal calf serum; ACO, acyl-CoA oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPRE, PPAR-responsive element; CAT, chloramphenicol acetyltransferase; SRE, sterol regulatory element.1The abbreviations used are: SREBP, sterol responsive element-binding protein; LDL, low density lipoproteins; LDLr, LDL receptor; TG, triglycerides; NEFA, non-esterified fatty acids; LPL, lipoprotein lipase; L-FABP, liver fatty acid-binding protein; LCFA, long-chain fatty acid; PPAR, peroxisome proliferator-activated receptor; WT, wild type; FCS, fetal calf serum; ACO, acyl-CoA oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPRE, PPAR-responsive element; CAT, chloramphenicol acetyltransferase; SRE, sterol regulatory element. genes cholesterol the LDL receptor of in the liver of LDL resulting in plasma Statins also at in a mechanism the of density a statins the cholesterol and and in Statins also plasma and levels in and humans through not of low density by the of the lipoprotein and of the contribute to the effect of statins. is not statins the and the of Moreover, the effect of statins is not fatty acid-binding protein (L-FABP) is a protein a for long-chain fatty acids is in more of The transcription of the L-FABP is and induced by both drugs and through a peroxisome proliferator-activated receptor (PPAR)-responsive element located in the of the promoter. of show that L-FABP in the and of was that L-FABP leads to of L-FABP influx in a dose-dependent in L-FABP expression also the of of L-FABP was to be with induced activity in hepatoma In the of the L-FABP resulted in a of by the liver of mice by lower synthesis and fatty acid wild-type controls This is by the of for and fatty acid these the role by L-FABP in and In the we the effect of statins is with of L-FABP expression in was by and for triglyceride by to and for the and of animals PPARα-null mice in the of of of in a and to and a and mice for with simvastatin the by the in and at and by to the of in in and fetal calf of the was by and for with the Fao hepatoma in a in with the of for in the in the of simvastatin in the used for in a in with was with and a and to L-FABP and rat acyl-CoA with the for rat was used to that of and This was and and glyceraldehyde-3-phosphate mRNA levels by was by after The was by transcription in a of and of at the was at of PPARα and used and the PPARα of of at for by at for the for to a The was used for the mRNA of the target to and to a was with the the and the and the the and the the L-FABP in a the the of was used for L-FABP the L-FABP rat L-FABP promoter by was into the L-FABP was used to the promoter by L-FABP L-FABP L-FABP L-FABP and L-FABP of the PPAR-responsive element by the for L-FABP in are and for L-FABP by The of the mouse PPARα promoter was into in in with at of reporter and of expression by the The was and was with of with simvastatin was to the for and for and are by L-FABP mRNA in the expression of L-FABP is by rat in the of of in L-FABP mRNA levels in a dose-dependent by a and rise of L-FABP mRNA was after with and of the L-FABP by act transcription by genes sterol regulatory in the rat L-FABP promoter by in located at and and at position the effect of of the L-FABP promoter the of simvastatin to the reporter was by activation by simvastatin was observed that not This that these are not The that the L-FABP a to be for the regulation of the L-FABP by to the of this in this in the and L-FABP the activation of the L-FABP promoter by simvastatin This was not by of the promoter used the L-FABP factor was to a reporter by the not these data that the is in the of L-FABP by PPAR-responsive element statin induction of rat L-FABP promoter the rat L-FABP promoter was in The of promoter revealed the of sterol responsive and located at position and and at The is located at position with of the L-FABP promoter L-FABP L-FABP L-FABP L-FABP and L-FABP with the of the L-FABP in for for in in of activity is to are of by and the PPARα the of PPARα in the statin-mediated induction of L-FABP Fao with simvastatin the PPARα agonist This was was used to regulation of the L-FABP by observed in rat simvastatin induced L-FABP mRNA levels to with simvastatin and the PPARα agonist a synergistic rise of L-FABP mRNA levels, induced by more with controls effects also for ACO, the rate-limiting enzyme of a PPARα target These demonstrate that this regulatory is not to the L-FABP mRNA levels are induced by simvastatin and PPARα in Fao for in simvastatin both and simvastatin the with used for analysis L-FABP and for L-FABP are in the The L-FABP and data to of L-FABP in PPARα-null the of these in vitro wild-type and PPARα-null mice for with simvastatin In wild-type in with the in Fao simvastatin and up-regulated L-FABP mRNA levels, and a synergistic effect These with a rise in L-FABP protein treatment not induction These also induced mRNA levels plasma not simvastatin treatment resulted in a in plasma levels in animals in of the PPARα the statin-mediated of L-FABP expression (mRNA and protein levels, and mRNA levels and the of plasma demonstrating the role of PPARα in the induction of L-FABP expression by the synergistic of L-FABP expression by PPARα PPARα mice with of simvastatin for the L-FABP mRNA levels by of The L-FABP data to L-FABP protein expression was by assay. mRNA levels by analysis of are to triglyceride are in wild-type mice after PPARα PPARα mice with of simvastatin for the triglyceride by assay. PPARα mRNA and PPARα effect of simvastatin PPARα mRNA levels was both in vitro and in in expression of this receptor was induced by simvastatin in Fao in wild-type the mechanism by this regulation a the mouse PPARα promoter of the reporter gene. of simvastatin a dose-dependent transactivation of the reporter the of a of PPARα expression by mRNA and promoter activity are up-regulated by PPARα mRNA levels in Fao hepatoma and in liver wild-type mice by to and to with the mouse PPARα promoter. in after the for in a in of activity is to are of CoA inhibitors are widely used to in Statins act through the activation of that controls genes involved in cholesterol Statin treatment also and levels, but the mechanism is not the expression of the LDLr, to be involved in the LDL is induced by statins this the of these levels are by statins in in the is not This the of statin target genes that In with this statins are to the expression of LPL, enzyme responsible for the of the and low density This effect is by a statin-mediated in expression of a of activity both and are PPARα target genes In hepatocytes, statins also the triglyceride protein that a protein in low density Moreover, statins the through that to be In the we demonstrate for the that L-FABP expression is also up-regulated by effect that might contribute to the of these drugs by influx and into hepatocytes. these data that the effect of statins of regulatory a of genes for and responsible for the of and the and of hepatocytes, the cholesterol induced by statins the proteolytic activation of the transcription after into the the transcription of sterol target genes by in analysis of the L-FABP in the of the rat promoter. statin regulation the to the L-FABP The of this was PPARα-null of the PPARα the statin-mediated of L-FABP expression (mRNA and protein demonstrating a role for activity is at levels at the by and by and The of PPARα by was to be responsible for the induction of expression This regulation was for by a of of protein to a role in the and protein is that a regulation might also for genes the induction of PPARα expression by statins might simvastatin induced a rise in PPARα mRNA levels both in vitro and in vivo These data are in with in and in with statins mechanism for this regulation is by the that simvastatin is to the PPARα promoter are to PPARα is by a direct for regulation synergistic mRNA levels was observed treatment PPARα in both hepatoma Fao and in wild-type mice by is with the of a the PPARα and statin This a and a for the of these PPARα controls fatty acid we that the rise in activity by statins is at in PPARα This is by the that the induction of mRNA levels by simvastatin is in PPARα-null mice induction of L-FABP might contribute to the of L-FABP is for and Moreover, synthesis is with concentration L-FABP by statins might also contribute to a of synthesis and to the of plasma low density the regulation of L-FABP by statins also contribute to the effects of statins be in the that are by and levels L-FABP induction by statin treatment might a mechanism to the effect of these data that PPARα is involved in this be at in by a induction of the PPARα by statins. Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. The resulting lower intracellular cholesterol concentration after statin treatment leads to a proteolytic activation of the transcription factor sterol responsive element-binding protein-2 (SREBP-2), 1The abbreviations used are: SREBP, sterol responsive element-binding protein; LDL, low density lipoproteins; LDLr, LDL receptor; TG, triglycerides; NEFA, non-esterified fatty acids; LPL, lipoprotein lipase; L-FABP, liver fatty acid-binding protein; LCFA, long-chain fatty acid; PPAR, peroxisome proliferator-activated receptor; WT, wild type; FCS, fetal calf serum; ACO, acyl-CoA oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPRE, PPAR-responsive element; CAT, chloramphenicol acetyltransferase; SRE, sterol regulatory element.1The abbreviations used are: SREBP, sterol responsive element-binding protein; LDL, low density lipoproteins; LDLr, LDL receptor; TG, triglycerides; NEFA, non-esterified fatty acids; LPL, lipoprotein lipase; L-FABP, liver fatty acid-binding protein; LCFA, long-chain fatty acid; PPAR, peroxisome proliferator-activated receptor; WT, wild type; FCS, fetal calf serum; ACO, acyl-CoA oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPRE, PPAR-responsive element; CAT, chloramphenicol acetyltransferase; SRE, sterol regulatory element. genes cholesterol the LDL receptor of in the liver of LDL resulting in plasma Statins also at in a mechanism the of density a statins the cholesterol and and in Statins also plasma and levels in and humans through not of low density by the of the lipoprotein and of the contribute to the effect of statins. is not statins the and the of Moreover, the effect of statins is not Liver fatty acid-binding protein (L-FABP) is a protein a for long-chain fatty acids is in more of The transcription of the L-FABP is and induced by both drugs and through a peroxisome proliferator-activated receptor (PPAR)-responsive element located in the of the promoter. of show that L-FABP in the and of was that L-FABP leads to of L-FABP influx in a dose-dependent in L-FABP expression also the of of L-FABP was to be with induced activity in hepatoma In the of the L-FABP resulted in a of by the liver of mice by lower synthesis and fatty acid wild-type controls This is by the of for and fatty acid Altogether, these the role by L-FABP in and In the we the effect of statins is with of L-FABP expression in was by and for triglyceride by to and for the and of animals PPARα-null mice in the of of of in a and to and a and mice for with simvastatin the by the in and at and by to the of in in and fetal calf of the was by and for with the Fao hepatoma in a in with the of for in the in the of simvastatin in the used for in a in with was with and a and to L-FABP and rat acyl-CoA with the for rat was used to that of and This was and and glyceraldehyde-3-phosphate mRNA levels by was by after The was by transcription in a of and of at the was at of PPARα and used and the PPARα of of at for by at for the for to a The was used for the mRNA of the target to and to a was with the the and the and the the and the the L-FABP in a the the of was used for L-FABP the L-FABP rat L-FABP promoter by was into the L-FABP was used to the promoter by L-FABP L-FABP L-FABP L-FABP and L-FABP of the PPAR-responsive element by the for L-FABP in are and for L-FABP by The of the mouse PPARα promoter was into in in with at of reporter and of expression by the The was and was with of with simvastatin was to the for and for and are by was by and for triglyceride by to and for the and of animals PPARα-null mice in the of of of in a and to and a and mice for with simvastatin the by the in and at and by to the of in in and fetal calf of the was by and for with the Fao hepatoma in a in with the of for in the in the of simvastatin in the used for in a in with was with and a and to L-FABP and rat acyl-CoA with the for rat was used to that of and This was and and glyceraldehyde-3-phosphate mRNA levels by was by after The was by transcription in a of and of at the was at of PPARα and used and the PPARα of of at for by at for the for to a The was used for the mRNA of the target to and to a was with the the and the and the the and the the L-FABP in a the the of was used for L-FABP the L-FABP rat L-FABP promoter by was into the L-FABP was used to the promoter by L-FABP L-FABP L-FABP L-FABP and L-FABP of the PPAR-responsive element by the for L-FABP in are and for L-FABP by The of the mouse PPARα promoter was into in in with at of reporter and of expression by the The was and was with of with simvastatin was to the for and for and are by L-FABP mRNA in the expression of L-FABP is by rat in the of of in L-FABP mRNA levels in a dose-dependent by a and rise of L-FABP mRNA was after with and of the L-FABP by act transcription by genes sterol regulatory in the rat L-FABP promoter by in located at and and at position the effect of of the L-FABP promoter the of simvastatin to the reporter was by activation by simvastatin was observed that not This that these are not The that the L-FABP a to be for the regulation of the L-FABP by to the of this in this in the and L-FABP the activation of the L-FABP promoter by simvastatin This was not by of the promoter used the L-FABP factor was to a reporter by the not these data that the is in the of L-FABP by by and the PPARα the of PPARα in the statin-mediated induction of L-FABP Fao with simvastatin the PPARα agonist This was was used to regulation of the L-FABP by observed in rat simvastatin induced L-FABP mRNA levels to with simvastatin and the PPARα agonist a synergistic rise of L-FABP mRNA levels, induced by more with controls effects also for ACO, the rate-limiting enzyme of a PPARα target These demonstrate that this regulatory is not to the L-FABP mRNA levels are induced by simvastatin and PPARα in Fao for in simvastatin both and simvastatin the with used for analysis L-FABP and for L-FABP are in the The L-FABP and data to of L-FABP in PPARα-null the of these in vitro wild-type and PPARα-null mice for with simvastatin In wild-type in with the in Fao simvastatin and up-regulated L-FABP mRNA levels, and a synergistic effect These with a rise in L-FABP protein treatment not induction These also induced mRNA levels plasma not simvastatin treatment resulted in a in plasma levels in animals in of the PPARα the statin-mediated of L-FABP expression (mRNA and protein levels, and mRNA levels and the of plasma demonstrating the role of PPARα in the induction of L-FABP expression by the synergistic of L-FABP expression by PPARα PPARα mice with of simvastatin for the L-FABP mRNA levels by of The L-FABP data to L-FABP protein expression was by assay. mRNA levels by analysis of are to triglyceride are in wild-type mice after PPARα PPARα mice with of simvastatin for the triglyceride by assay. PPARα mRNA and PPARα effect of simvastatin PPARα mRNA levels was both in vitro and in in expression of this receptor was induced by simvastatin in Fao in wild-type the mechanism by this regulation a the mouse PPARα promoter of the reporter gene. of simvastatin a dose-dependent transactivation of the reporter the of a of PPARα expression by mRNA and promoter activity are up-regulated by PPARα mRNA levels in Fao hepatoma and in liver wild-type mice by to and to with the mouse PPARα promoter. in after the for in a in of activity is to are of L-FABP mRNA in the expression of L-FABP is by rat in the of of in L-FABP mRNA levels in a dose-dependent by a and rise of L-FABP mRNA was after with and of the L-FABP by act transcription by genes sterol regulatory in the rat L-FABP promoter by in located at and and at position the effect of of the L-FABP promoter the of simvastatin to the reporter was by activation by simvastatin was observed that not This that these are not The that the L-FABP a to be for the regulation of the L-FABP by to the of this in this in the and L-FABP the activation of the L-FABP promoter by simvastatin This was not by of the promoter used the L-FABP factor was to a reporter by the not these data that the is in the of L-FABP by L-FABP by and the PPARα the of PPARα in the statin-mediated induction of L-FABP Fao with simvastatin the PPARα agonist This was was used to regulation of the L-FABP by observed in rat simvastatin induced L-FABP mRNA levels to with simvastatin and the PPARα agonist a synergistic rise of L-FABP mRNA levels, induced by more with controls effects also for ACO, the rate-limiting enzyme of a PPARα target These demonstrate that this regulatory is not to the L-FABP of L-FABP in PPARα-null the of these in vitro wild-type and PPARα-null mice for with simvastatin In wild-type in with the in Fao simvastatin and up-regulated L-FABP mRNA levels, and a synergistic effect These with a rise in L-FABP protein treatment not induction These also induced mRNA levels plasma not simvastatin treatment resulted in a in plasma levels in animals in of the PPARα the statin-mediated of L-FABP expression (mRNA and protein levels, and mRNA levels and the of plasma demonstrating the role of PPARα in the induction of L-FABP expression by statins. PPARα mRNA and PPARα effect of simvastatin PPARα mRNA levels was both in vitro and in in expression of this receptor was induced by simvastatin in Fao in wild-type the mechanism by this regulation a the mouse PPARα promoter of the reporter gene. of simvastatin a dose-dependent transactivation of the reporter the of a of PPARα expression by statins. CoA inhibitors are widely used to in Statins act through the activation of that controls genes involved in cholesterol Statin treatment also and levels, but the mechanism is not the expression of the LDLr, to be involved in the LDL is induced by statins this the of these levels are by statins in in the is not This the of statin target genes that In with this statins are to the expression of LPL, enzyme responsible for the of the and low density This effect is by a statin-mediated in expression of a of activity both and are PPARα target genes In hepatocytes, statins also the triglyceride protein that a protein in low density Moreover, statins the through that to be In the we demonstrate for the that L-FABP expression is also up-regulated by effect that might contribute to the of these drugs by influx and into hepatocytes. these data that the effect of statins of regulatory a of genes for and responsible for the of and the and of hepatocytes, the cholesterol induced by statins the proteolytic activation of the transcription after into the the transcription of sterol target genes by in analysis of the L-FABP in the of the rat promoter. statin regulation the to the L-FABP The of this was PPARα-null of the PPARα the statin-mediated of L-FABP expression (mRNA and protein demonstrating a role for activity is at levels at the by and by and The of PPARα by was to be responsible for the induction of expression This regulation was for by a of of protein to a role in the and protein is that a regulation might also for genes the induction of PPARα expression by statins might simvastatin induced a rise in PPARα mRNA levels both in vitro and in vivo These data are in with in and in with statins mechanism for this regulation is by the that simvastatin is to the PPARα promoter are to PPARα is by a direct for regulation synergistic mRNA levels was observed treatment PPARα in both hepatoma Fao and in wild-type mice by is with the of a the PPARα and statin This a and a for the of these PPARα controls fatty acid we that the rise in activity by statins is at in PPARα This is by the that the induction of mRNA levels by simvastatin is in PPARα-null mice induction of L-FABP might contribute to the of L-FABP is for and Moreover, synthesis is with concentration L-FABP by statins might also contribute to a of synthesis and to the of plasma low density the regulation of L-FABP by statins also contribute to the effects of statins be in the that are by and levels L-FABP induction by statin treatment might a mechanism to the effect of these data that PPARα is involved in this be at in by a induction of the PPARα by statins. CoA inhibitors are widely used to in Statins act through the activation of that controls genes involved in cholesterol Statin treatment also and levels, but the mechanism is not the expression of the LDLr, to be involved in the LDL is induced by statins this the of these levels are by statins in in the is not This the of statin target genes that In with this statins are to the expression of LPL, enzyme responsible for the of the and low density This effect is by a statin-mediated in expression of a of activity both and are PPARα target genes In hepatocytes, statins also the triglyceride protein that a protein in low density Moreover, statins the through that to be In the we demonstrate for the that L-FABP expression is also up-regulated by effect that might contribute to the of these drugs by influx and into hepatocytes. these data that the effect of statins of regulatory a of genes for and responsible for the of and the and of In hepatocytes, the cholesterol induced by statins the proteolytic activation of the transcription after into the the transcription of sterol target genes by in analysis of the L-FABP in the of the rat promoter. statin regulation the to the L-FABP The of this was PPARα-null of the PPARα the statin-mediated of L-FABP expression (mRNA and protein demonstrating a role for PPARα. PPARα activity is at levels at the by and by and The of PPARα by was to be responsible for the induction of expression This regulation was for by a of of protein to a role in the and protein is that a regulation might also for genes the induction of PPARα expression by statins might simvastatin induced a rise in PPARα mRNA levels both in vitro and in vivo These data are in with in and in with statins mechanism for this regulation is by the that simvastatin is to the PPARα promoter are to PPARα is by a direct for regulation synergistic mRNA levels was observed treatment PPARα in both hepatoma Fao and in wild-type mice by is with the of a the PPARα and statin This a and a for the of these PPARα controls fatty acid we that the rise in activity by statins is at in PPARα This is by the that the induction of mRNA levels by simvastatin is in PPARα-null mice induction of L-FABP might contribute to the of L-FABP is for and Moreover, synthesis is with concentration L-FABP by statins might also contribute to a of synthesis and to the of plasma low density the regulation of L-FABP by statins also contribute to the effects of statins be in the that are by and levels In L-FABP induction by statin treatment might a mechanism to the effect of these data that PPARα is involved in this be at in by a induction of the PPARα by statins. for the of rat hepatocytes.