Differential effects of PPARalpha activators on induction of ectopic expression of tissue-specific fatty acid binding protein genes in the mouse liver

The effect of a potent peroxisome proliferator-activated receptor (PPAR) alpha activator Wy14,643 on tissue-specific expression of fatty acid binding (FABP) genes was studied. Wy14,643 immediately induced liver-, intestine- and FABP but not PPARgamma-regulated adipose-FABP (or aP2) mRNAs in respective mouse tissues. Moreover, it gradually induced ectopic expression of heart- and adipose-FABP mRNAs to significant levels in the liver. However, ectopic expression was not induced in the liver of PPARalpha-null mouse, indicating an obligatory role of the receptor in the modulated expression. Among the four PPARalpha activators examined, only Wy14,643 induced ectopical expression of heart-FABP in the liver. Thus, tissue-specificity of the FABP gene expression is not absolute and, with a potent activator, can be distorted by PPARalpha.     

Peroxisome proliferator-activated receptor alpha-mediated pathways are altered in hepatocyte-specific retinoid X receptor alpha-deficient mice

Retinoid x receptor alpha (RXRalpha) serves as an active partner of peroxisome proliferator-activated receptor (PPARalpha). In order to dissect the functional role of RXRalpha and PPARalpha in PPARalpha-mediated pathways, the hepatocyte RXRalpha-deficient mice have been challenged with physiological and pharmacological stresses, fasting and Wy14,643, respectively. The data demonstrate that RXRalpha and PPARalpha deficiency are different in several aspects. At the basal untreated level, RXRalpha deficiency resulted in marked induction of apolipoprotein A-I and C-III (apoA-I and apoC-III) mRNA levels and serum cholesterol and triglyceride levels, which was not found in PPARalpha-null mice. Fasting-induced PPARalpha activation was drastically prevented in the absence of hepatocyte RXRalpha. Wy14,643-mediated pleiotropic effects were also altered due to the absence of hepatocyte RXRalpha. Hepatocyte RXRalpha deficiency did not change the basal acyl-CoA oxidase, medium chain acyl-CoA dehydrogenase, and malic enzyme mRNA levels. However, the inducibility of those genes by Wy14,643 was markedly reduced in the mutant mouse livers. In contrast, the basal cytochrome P450 4A1, liver fatty acid-binding protein, and apoA-I and apoC-III mRNA levels were significantly altered in the mutant mouse livers, but the regulatory effect of Wy14,643 on expression of those genes remained the same. Wy14,643-induced hepatomegaly was partially inhibited in hepatocyte RXRalpha-deficient mice. Wy14,643-induced hepatocyte peroxisome proliferation was preserved in the absence of hepatocyte RXRalpha. These data suggested that in comparison to PPARalpha, hepatocyte RXRalpha has its unique role in lipid homeostasis and that the effect of RXRalpha, -beta, and -gamma is redundant in certain aspects.     

The peroxisome proliferator-activated receptor alpha (PPARalpha) regulates bile acid biosynthesis

Fibrates are a group of hypolipidemic agents that efficiently lower serum triglyceride levels by affecting the expression of many genes involved in lipid metabolism. These effects are exerted via the peroxisome proliferator-activated receptor alpha (PPARalpha). In addition, fibrates also lower serum cholesterol levels, suggesting a possible link between the PPARalpha and cholesterol metabolism. Bile acid formation represents an important pathway for elimination of cholesterol, and the sterol 12alpha-hydroxylase is a branch-point enzyme in the bile acid biosynthetic pathway, which determines the ratio of cholic acid to chenodeoxycholic acid. Treatment of mice for 1 week with the peroxisome proliferator WY-14,643 or fasting for 24 h both induced the sterol 12alpha-hydroxylase mRNA in liver. Using the PPARalpha knockout mouse model, we show that the induction by both treatments was dependent on the PPARalpha. A reporter plasmid containing a putative peroxisome proliferator-response element (PPRE) identified in the rat sterol 12alpha-hydroxylase promoter region was activated by treatment with WY-14,643 in HepG2 cells, being dependent on co-transfection with a PPARalpha expression plasmid. The rat 12alpha-hydroxylase PPRE bound in vitro translated PPARalpha and retinoid X receptor alpha, albeit weakly, in electrophoretic mobility shift assay. Treatment of wild-type mice with WY-14,643 for 1 week resulted in an increased relative amount of cholic acid, an effect that was abolished in the PPARalpha null mice, verifying the functionality of the PPRE in vivo.     

The acyl-CoA thioesterase I is regulated by PPARalpha and HNF4alpha via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.     

PPARalpha activation increases triglyceride mass and adipose differentiation-related protein in hepatocytes

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed in various tissues. In mice treated with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist Wy14,643 (Wy), hepatic mRNA and protein levels of ADRP as well as hepatic triglyceride content increased. Also in primary mouse hepatocytes, Wy increased ADRP expression and intracellular triglyceride mass. The triglyceride mass increased in spite of unchanged triglyceride biosynthesis and increased palmitic acid oxidation. However, Wy incubation decreased the secretion of newly synthesized triglycerides, whereas apolipoprotein B secretion increased. Thus, decreased availability of triglycerides for VLDL assembly could help to explain the cellular accumulation of triglycerides after Wy treatment. We hypothesized that this effect could be mediated by increased ADRP expression. Similar to PPARalpha activation, adenovirus-mediated ADRP overexpression in mouse hepatocytes enhanced cellular triglyceride mass and decreased the secretion of newly synthesized triglycerides. In ADRP-overexpressing cells, Wy incubation resulted in a further decrease in triglyceride secretion. This effect of Wy was not attributable to decreased cellular triglycerides after increased fatty acid oxidation because the triglyceride mass in Wy-treated ADRP-overexpressing cells was unchanged. In summary, PPARalpha activation prevents the availability of triglycerides for VLDL assembly and increases hepatic triglyceride content in part by increasing the expression of ADRP.     

WY14,643, a peroxisome proliferator-activated receptor alpha (PPARalpha ) agonist, improves hepatic and muscle steatosis and reverses insulin resistance in lipoatrophic A-ZIP/F-1 mice

WY14,643 is a specific peroxisome proliferator-activated receptor alpha (PPARalpha) agonist with strong hypolipidemic effects. Here we have examined the effect of WY14,643 in the A-ZIP/F-1 mouse, a model of severe lipoatrophic diabetes. With 1 week of treatment, all doses of WY14,643 that were tested normalized serum triglyceride and fatty acid levels. Glucose and insulin levels also improved but only with high doses and longer treatment duration. WY14,643 reduced liver and muscle triglyceride content and increased levels of mRNA encoding fatty acid oxidation enzymes. In liver, the elevated lipogenic mRNA profile (including PPARgamma) in A-ZIP/F-1 mice remained unchanged. These results suggest that WY14,643 acts by increasing beta-oxidation rather by than decreasing lipogenesis or lipid uptake. Hyperinsulinemic euglycemic clamp studies indicated that WY14,643 treatment improved liver more than muscle insulin sensitivity and that hepatic mRNA levels of gluconeogenic enzymes were reduced. Combination treatment with both WY14,643 and a PPARgamma ligand, rosiglitazone, did not lower glucose levels more effectively than did treatment with WY14,643 alone. These data support the hypothesis that reducing intracellular triglycerides in non-adipose tissues improves insulin sensitivity and suggest that further investigation of the role of PPARalpha agonists in the treatment of lipoatrophic diabetes is warranted.     

Absence of spontaneous peroxisome proliferation in enoyl-CoA Hydratase/L-3-hydroxyacyl-CoA dehydrogenase-deficient mouse liver. Further support for the role of fatty acyl CoA oxidase in PPARalpha ligand metabolism.

Peroxisomes contain a classical L-hydroxy-specific peroxisome proliferator-inducible beta-oxidation system and also a second noninducible D-hydroxy-specific beta-oxidation system. We previously generated mice lacking fatty acyl-CoA oxidase (AOX), the first enzyme of the L-hydroxy-specific classical beta-oxidation system; these AOX-/- mice exhibited sustained activation of peroxisome proliferator-activated receptor alpha (PPARalpha), resulting in profound spontaneous peroxisome proliferation in liver cells. These observations implied that AOX is responsible for the metabolic degradation of PPARalpha ligands. In this study, the function of enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-PBE), the second enzyme of this peroxisomal beta-oxidation system, was investigated by disrupting its gene. Mutant mice (L-PBE-/-) were viable and fertile and exhibited no detectable gross phenotypic defects. L-PBE-/- mice showed no hepatic steatosis and manifested no spontaneous peroxisome proliferation, unlike that encountered in livers of mice deficient in AOX. These results indicate that disruption of classical peroxisomal fatty acid beta-oxidation system distal to AOX step does not interfere with the inactivation of endogenous ligands of PPARalpha, further confirming that the AOX gene is indispensable for the physiological regulation of this receptor. The absence of appreciable changes in lipid metabolism also indicates that enoyl-CoAs, generated in the classical system in L-PBE-/- mice are diverted to D-hydroxy-specific system for metabolism by D-PBE. When challenged with a peroxisome proliferator, L-PBE-/- mice showed increases in the levels of hepatic mRNAs and proteins that are regulated by PPARalpha except for appreciable blunting of peroxisome proliferative response as compared with that observed in hepatocytes of wild type mice similarly treated. This blunting of peroxisome proliferative response is attributed to the absence of L-PBE protein in L-PBE-/- mouse liver, because all other proteins are induced essentially to the same extent in both wild type and L-PBE-/- mice.     

Modulation of the hepatic fatty acid pool in peroxisomal 3-ketoacyl-CoA thiolase B-null mice exposed to the selective PPARalpha agonist Wy14,643

The peroxisomal 3-ketoacyl-CoA thiolase B (Thb) gene was previously identified as a direct target gene of PPARalpha, a nuclear hormone receptor activated by hypolipidemic fibrate drugs. To better understand the role of ThB in hepatic lipid metabolism in mice, Sv129 wild-type and Thb null mice were fed or not the selective PPARalpha agonist Wy14,643 (Wy).Here, it is shown that in contrast to some other mouse models deficient for peroxisomal enzymes, the hepatic PPARalpha signaling cascade in Thb null mice was normal under regular conditions. It is of interest that the hypotriglyceridemic action of Wy was reduced in Thb null mice underlining the conclusion that neither thiolase A nor SCPx/SCP2 thiolase can fully substitute for ThB in vivo. Moreover, a significant increased in the expression of lipogenic genes such as Stearoyl CoA Desaturase-1 (SCD1) was observed in Thb null mice fed Wy. Elevation of Scd1 mRNA and protein levels led to higher SCD1 activity, through a molecular mechanism that is probably SREBP1 independent. In agreement with higher SCD1, enrichment of liver mono-unsaturated fatty acids of the n-7 and n-9 series was found in Thb null mice fed Wy.Overall, we show that the reduced peroxisomal β-oxidation of fat observed in Thb null mice fed Wy is associated with enhanced hepatic lipogenesis, through the combined elevation of microsomal SCD1 protein and activity. Ultimately, not only the amount but also the quality of the hepatic fatty acid pool is modulated upon the deletion of Thb.     

Glucose directly links to lipid metabolism through high affinity interaction with peroxisome proliferator-activated receptor alpha

The pathophysiology of diabetes is characterized not only by elevated glucose but also elevated long chain fatty acid levels. We show for the first time that the peroxisome proliferator-activated receptor-alpha (PPARalpha) binds glucose and glucose metabolites with high affinity, resulting in significantly altered PPARalpha secondary structure. Glucose decreased PPARalpha interaction with fatty acid metabolites and steroid receptor coactivator-1 while increasing PPARalpha interaction with DNA. Concomitantly, glucose increased PPARalpha interaction with steroid receptor coactivator-1, DNA binding, and transactivation of beta-oxidation pathways in the presence of activating ligands. Heterodimerization of PPARalpha to the retinoid X receptor-alpha resulted in even larger increases in transactivation with the addition of glucose. These data suggest that PPARalpha is responsible for maintaining energy homeostasis through a concentration-dependent regulation of both lipids and sugars and that hyperglycemic injury mediated by PPARalpha occurs not only indirectly through elevated long chain fatty acid levels but also through direct action of glucose on PPARalpha.     

Metabolism of phytol to phytanic acid in the mouse, and the role of PPARalpha in its regulation

Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha). To investigate the metabolism of phytol and the role of PPARalpha in its regulation, wild-type and PPARalpha knockout (PPARalpha-/-) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARalpha agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARalpha-/- mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARalpha agonist resulted in a PPARalpha-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARalpha and is specific for the breakdown of (E)-phytol.     

Peroxisome proliferator-activated receptor alpha activates transcription of the brown fat uncoupling protein-1 gene. A link between regulation of the thermogenic and lipid oxidation pathways in the brown fat cell

High expression of the peroxisome proliferator-activated receptor alpha (PPARalpha) differentiates brown fat from white, and is related to its high capacity of lipid oxidation. We analyzed the effects of PPARalpha activation on expression of the brown fat-specific uncoupling protein-1 (ucp-1) gene. Activators of PPARalpha increased UCP-1 mRNA levels severalfold both in primary brown adipocytes and in brown fat in vivo. Transient transfection assays indicated that the (-4551)UCP1-CAT construct, containing the 5'-regulatory region of the rat ucp-1 gene, was activated by PPARalpha co-transfection in a dose-dependent manner and this activation was potentiated by Wy 14,643 and retinoid X receptor alpha. The coactivators CBP and PPARgamma-coactivator-1 (PGC-1), which is highly expressed in brown fat, also enhanced the PPARalpha-dependent regulation of the ucp-1 gene. Deletion and point-mutation mapping analysis indicated that the PPARalpha-responsive element was located in the upstream enhancer region of the ucp-1 gene. This -2485/-2458 element bound PPARalpha and PPARgamma from brown fat nuclei. Moreover, this element behaved as a promiscuous responsive site to either PPARalpha or PPARgamma activation, and we propose that it mediates ucp-1 gene up-regulation associated with adipogenic differentiation (via PPARgamma) or in coordination with gene expression for the fatty acid oxidation machinery required for active thermogenesis (via PPARalpha).     

Peroxisome proliferator-activated receptor alpha-retinoid X receptor agonists induce beta-cell protection against palmitate toxicity

Fatty acids can stimulate the secretory activity of insulin-producing beta-cells. At elevated concentrations, they can also be toxic to isolated beta-cells. This toxicity varies inversely with the cellular ability to accumulate neutral lipids in the cytoplasm. To further examine whether cytoprotection can be achieved by decreasing cytoplasmic levels of free acyl moieties, we investigated whether palmitate toxicity is also lowered by stimulating its beta-oxidation. Lower rates of palmitate-induced beta-cell death were measured in the presence of L-carnitine as well as after addition of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, conditions leading to increased palmitate oxidation. In contrast, inhibition of mitochondrial beta-oxidation by etomoxir increased palmitate toxicity. A combination of PPARalpha and retinoid X receptor (RXR) agonists acted synergistically and led to complete protection; this was associated with enhanced expression levels of genes involved in mitochondrial and peroxisomal beta-oxidation, lipid metabolism, and peroxisome proliferation. PPARalpha-RXR protection was abolished by the carnitine palmitoyl transferase 1 inhibitor etomoxir. These observations indicate that PPARalpha and RXR regulate beta-cell susceptibility to long-chain fatty acid toxicity by increasing the rates of beta-oxidation and by involving peroxisomes in fatty acid metabolism.     

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARalpha-dependent and -independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.     

Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor alpha (PPARalpha)-regulated peroxisomal acyl-CoA thioesterases

Peroxisomes are organelles that function in the beta-oxidation of long- and very long-chain acyl-CoAs, bile acid-CoA intermediates, prostaglandins, leukotrienes, thromboxanes, dicarboxylic fatty acids, pristanic acid, and xenobiotic carboxylic acids. The very long- and long-chain acyl-CoAs are mainly chain-shortened and then transported to mitochondria for further metabolism. We have now identified and characterized two peroxisomal acyl-CoA thioesterases, named PTE-Ia and PTE-Ic, that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A. PTE-Ia and PTE-Ic show 82% sequence identity at the amino acid level, and a putative peroxisomal type 1 targeting signal of -AKL was identified at the carboxyl-terminal end of both proteins. Localization experiments using green fluorescent fusion protein showed PTE-Ia and PTE-Ic to be localized in peroxisomes. Despite their high level of sequence identity, we show that PTE-Ia is mainly active on long-chain acyl-CoAs, whereas PTE-Ic is mainly active on medium-chain acyl-CoAs. Lack of regulation of enzyme activity by free CoASH suggests that PTE-Ia and PTE-Ic regulate intraperoxisomal levels of acyl-CoA, and they may have a function in termination of beta-oxidation of fatty acids of different chain lengths. Tissue expression studies revealed that PTE-Ia is highly expressed in kidney, whereas PTE-Ic is most highly expressed in spleen, brain, testis, and proximal and distal intestine. Both PTE-Ia and PTE-Ic were highly up-regulated in mouse liver by treatment with the peroxisome proliferator WY-14,643 and by fasting in a peroxisome proliferator-activated receptor alpha-dependent manner. These data show that PTE-Ia and PTE-Ic have different functions based on different substrate specificities and tissue expression.