High power custom toric intraocular lens for correcting high corneal astigmatism in post-keratoplasty and keratoconus patients with cataract.

Purpose: To analyze the outcomes of phacoemulsification with high power customized toric intraocular lens implantation (IOL) in patients with high corneal astigmatism (6-10 D Cyl) post keratoplasty and keratoconus eyes with cataract. Methods: Five eyes post keratoplasty with clear graft, four eyes with stable keratoconus, one eye with pterygium excision scar with visually significant cataract were included in this retrospective study. Phacoemulsification was done followed by implantation of custom made high power toric IOL in all patients. Outcomes included uncorrected and best-corrected distance visual acuity (UDVA, BCVA), pre-operative astigmatism at the corneal plane and IOL plane, post-operative residual astigmatism, mean torus of all IOLs used were calculated. Results: The minimum follow-up time was 12 months. At the last follow-up visit, there was a significant improvement (pre-operative vs post-operative) of UDVA (1.5 ± 0.47 vs 0.28 ± 0.14 logMAR; P < 0.05), cylindrical refraction (-9.0 ± 1.80 D vs -1.1 ± 0.45 vs ; P < 0.05). Range of IOL powers used was 1.0-26.50 DSph and 9.0-15.5 DCyl. Post-operative mean residual spherical equivalent was 0.75 ± 0.5. Conclusion: This novel study describes the effectiveness of custom toric IOLs in high astigmatism in the range of 9.0-15.5 DCyl. Phacoemulsification with implantation of a customized high power toric IOL was effective in correcting high astigmatism in complex cases in our study.

Severe focal stromal degeneration up to Descemet membrane after corneal collagen cross-linking.

Corneal collagen cross-linking (CXL) is an effective treatment for arresting progression in keratoconus cases. It is considered safe despite a few complications that have been recorded earlier. In this case series, we report a rare and late complication caused due to severe stromal thinning up to Descemet's membrane in three patients who underwent CXL 3 to 6 years back for keratoconus. Deep anterior lamellar keratoplasty (DALK) was then done for the affected eye with good outcomes. This case series highlights the possible late effects of UVA irradiation post CXL.

The inflammatory response in acyl-CoA oxidase 1 deficiency (pseudoneonatal adrenoleukodystrophy).

Among several peroxisomal neurodegenerative disorders, the pseudoneonatal adrenoleukodystrophy (P-NALD) is characterized by the acyl-coenzyme A oxidase 1 (ACOX1) deficiency, which leads to the accumulation of very-long-chain fatty acids (VLCFA) and inflammatory demyelination. However, the components of this inflammatory process in P-NALD remain elusive. In this study, we used transcriptomic profiling and PCR array analyses to explore inflammatory gene expression in patient fibroblasts. Our results show the activation of IL-1 inflammatory pathway accompanied by the increased secretion of two IL-1 target genes, IL-6 and IL-8 cytokines. Human fibroblasts exposed to very-long-chain fatty acids exhibited increased mRNA expression of IL-1α and IL-1ß cytokines. Furthermore, expression of IL-6 and IL-8 cytokines in patient fibroblasts was down-regulated by MAPK, p38MAPK, and Jun N-terminal kinase inhibitors. Thus, the absence of acyl-coenzyme A oxidase 1 activity in P-NALD fibroblasts triggers an inflammatory process, in which the IL-1 pathway seems to be central. The use of specific kinase inhibitors may permit the modulation of the enhanced inflammatory status.

The neuronal migration defect in mice with Zellweger syndrome (Pex5 knockout) is not caused by the inactivity of peroxisomal beta-oxidation.

The purpose of this study was to investigate whether deficient peroxisomal beta-oxidation is causally involved in the neuronal migration defect observed in Pex5 knockout mice. These mice are models for Zellweger syndrome, a peroxisome biogenesis disorder. Neocortical development was evaluated in mice carrying a partial or complete defect of peroxisomal beta-oxidation at the level of the second enzyme of the pathway, namely, the hydratase-dehydrogenase multifunctional/bifunctional enzymes MFP1/L-PBE and MFP2/D-PBE. In contrast to patients with multifunctional protein 2 deficiency who present with neocortical dysgenesis, impairment of neuronal migration was not observed in the single MFP2 or in the double MFP1/MFP2 knockout mice. At birth, the double knockout pups displayed variable growth retardation and about one half of them were severely hypotonic, whereas the single MFP2 knockout animals were all normal in the perinatal period. These results indicate that in the mouse, defective peroxisomal beta-oxidation does not cause neuronal migration defects by itself. This does not exclude that the inactivity of this metabolic pathway contributes to the brain pathology in mice and patients with complete absence of functional peroxisomes.

Identification of the peroxisomal beta-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid.

DHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by beta-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the beta-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in beta-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal beta-oxidation.

Nonalcoholic steatosis and steatohepatitis. III. Peroxisomal beta-oxidation, PPAR alpha, and steatohepatitis.

Peroxisomes are involved in the beta-oxidation chain shortening of long-chain and very-long-chain fatty acyl-CoAs, long-chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs, and the CoA esters of the bile acid intermediates, and in the process, they generate H(2)O(2). There are two complete sets of beta-oxidation enzymes present in peroxisomes, with each set consisting of three distinct enzymes. The classic PPAR alpha-regulated and inducible set participates in the beta-oxidation of straight-chain fatty acids, whereas the second noninducible set acts on branched-chain fatty acids. Long-chain and very-long-chain fatty acids are also metabolized by the cytochrome P-450 CYP4A omega-oxidation system to dicarboxylic acids that serve as substrates for peroxisomal beta-oxidation. Evidence derived from mouse models of PPAR alpha and peroxisomal beta-oxidation deficiency highlights the critical importance of the defects in PPAR alpha-inducible beta-oxidation in energy metabolism and in the development of steatohepatitis.

Identification of toxicologically predictive gene sets using cDNA microarrays.

We have developed an approach to classify toxicants based upon their influence on profiles of mRNA transcripts. Changes in liver gene expression were examined after exposure of mice to 24 model treatments that fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Analysis of 1200 transcripts using both a correlation-based approach and a probabilistic approach resulted in a classification accuracy of between 50 and 70%. However, with the use of a forward parameter selection scheme, a diagnostic set of 12 transcripts was identified that provided an estimated 100% predictive accuracy based on leave-one-out cross-validation. Expansion of this approach to additional chemicals of regulatory concern could serve as an important screening step in a new era of toxicological testing.

Human peroxisome proliferator-activated receptor alpha (PPARalpha) supports the induction of peroxisome proliferation in PPARalpha-deficient mouse liver.

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, induce peroxisomal, microsomal, and mitochondrial fatty acid oxidation enzymes, in conjunction with peroxisome proliferation, in liver cells. Sustained activation of PPARalpha leads to the development of liver tumors in rats and mice. The assertion that synthetic PPARalpha ligands pose negligible carcinogenic risk to humans is attributable, in part, to the failure to observe peroxisome proliferation in human hepatocytes. To explore the mechanism(s) of species-specific differences in response to PPARalpha ligands, we determined the functional competency of human PPARalpha in vivo and compared its potency with that of mouse PPARalpha. Recombinant adenovirus that expresses human or mouse PPARalpha was produced and administered intravenously to PPARalpha-deficient mice. Human as well as mouse PPARalpha fully restored the development of peroxisome proliferator-induced immediate pleiotropic responses, including peroxisome proliferation and enhanced expression of genes involved in lipid metabolism as well as nonperoxisomal genes, such as CD36, Ly-6D, Rbp7, monoglyceride lipase, pyruvate dehydrogenase kinase-4, and C3f, that have been identified recently to be up-regulated in livers with peroxisome proliferation. These studies establish that human PPARalpha is functionally competent and is equally as dose-sensitive as mouse PPARalpha in inducing peroxisome proliferation within the context of mouse liver environment and that it can heterodimerize with mouse retinoid X receptor, and this human PPARalpha-mouse retinoid X receptor chimeric heterodimer transcriptionally activates mouse PPARalpha target genes in a manner qualitatively similar to that of mouse PPARalpha.

Role of peroxisome proliferator-activated receptor gamma and its ligands in non-neoplastic and neoplastic human urothelial cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and is expressed in several types of tissue. Although PPARgamma reportedly is expressed in normal urothelium, its function is unknown. We examined the expression of PPARgamma in normal urothelium and bladder cancer in an attempt to assess its functional role. Immunohistochemical staining revealed normal urothelium to express PPARgamma uniformly. All low-grade carcinomas were positive either diffusely or focally, whereas staining was primarily focal or absent in high-grade carcinomas. A nonneoplastic urothelial cell line (1T-1), a low-grade (RT4) carcinoma cell line, and two high-grade (T24 and 253J) carcinoma cell lines in culture expressed PPARgamma mRNA and protein. Luciferase assay indicated that PPARgamma was functional. PPARgamma ligands (15-deoxy-Delta(12,14)-prostaglandin J(2), troglitazone and pioglitazone) suppressed the growth of nonneoplastic and neoplastic urothelial cells in a dose-dependent manner. However, neoplastic cells were more resistant than were nonneoplastic cells. Failure to express PPARgamma or ineffective transcriptional activity may be some of the mechanisms responsible for resistance to the inhibitory action of PPARgamma ligands.

Peroxisome proliferator-activated receptor-alpha mice show enhanced hepatocyte proliferation in response to the hepatomitogen 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene, a ligand of constitutive androstane receptor.

Previously, we have suggested that liver cell proliferation induced by certain mitogens is dependent on their binding and activation of nuclear receptors of the steroid/thyroid superfamily. More recently, it was shown that absence of the nuclear receptors peroxisome proliferator-activated receptor-alpha (PPARalpha) and constitutive androstane receptor (CAR) completely abolishes the proliferative response of hepatocytes to the mitogenic stimulus exerted by their specific ligands, peroxisome proliferators (PPs) and 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), respectively. Here we show that deletion of the PPARalpha gene accelerates and enhances the proliferative response evoked by the xenobiotic 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a powerful mouse-liver mitogen and a ligand of the nuclear receptor CAR. Indeed, the number of hepatocytes entering S phase 24 hours after mitogen treatment was much greater in PPARalpha(-/-) mice compared with that of wild type mice (labeling indices 21.4% and 7.5%, respectively). Labeling index of hepatocytes from PPARalpha(-/-) mice was found to be higher than that of wild type mice up to 36 hours after treatment, indicating that lack of PPARalpha not only accelerated but also enhanced the overall proliferative response of the liver. The accelerated entry into S phase observed in hepatocytes from PPARalpha(-/-) mice was associated with a very rapid induction of cyclin D1. No major differences between TCPOBOP-treated PPARalpha(-/-) and wild type mice were observed in the expression of the 2 inhibitors of cyclin/CDKs complexes, p27 and p21. The results suggest that PPARalpha may play a role in modulating CAR-signaling pathways in the cell, in particular those leading to hepatocyte proliferation.

Cloning and characterization of PIMT, a protein with a methyltransferase domain, which interacts with and enhances nuclear receptor coactivator PRIP function.

The nuclear receptor coactivators participate in the transcriptional activation of specific genes by nuclear receptors. In this study, we report the isolation of a nuclear receptor coactivator-interacting protein from a human liver cDNA library by using the coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) (ASC2/AIB3/RAP250/NRC/TRBP) as bait in a yeast two-hybrid screen. Human PRIP-interacting protein cDNA has an ORF of 2,556 nucleotides, encodes a protein with 852 amino acids, and contains a 9-aa VVDAFCGVG methyltransferase motif I and an invariant GXXGXXI segment found in K-homology motifs of many RNA-binding proteins. The gene encoding this protein, designated PRIP-interacting protein with methyltransferase domain (PIMT), is localized on chromosome 8q11 and spans more than 40 kb. PIMT mRNA is ubiquitously expressed, with a high level of expression in heart, skeletal muscle, kidney, liver, and placenta. Using the immunofluorescence localization method, we found that PIMT and PRIP proteins appear colocalized in the nucleus. PIMT strongly interacts with PRIP under in vitro and in vivo conditions, and the PIMT-binding site on PRIP is in the region encompassing amino acids 773-927. PIMT binds S-adenosyl-l-methionine, the methyl donor for methyltransfer reaction, and it also binds RNA, suggesting that it is a putative RNA methyltransferase. PIMT enhances the transcriptional activity of peroxisome proliferator-activated receptor gamma and retinoid-X-receptor alpha, which is further stimulated by coexpression of PRIP, implying that PIMT is a component of nuclear receptor signal transduction apparatus acting through PRIP. Definitive identification of the specific substrate of PIMT and the role of this RNA-binding protein in transcriptional regulation remain to be determined.

Peroxisome proliferator-activated receptor alpha-responsive genes induced in the newborn but not prenatal liver of peroxisomal fatty acyl-CoA oxidase null mice.

Mice deficient in fatty acyl-CoA oxidase (AOX(-/-)), the first enzyme of the peroxisomal beta-oxidation system, develop specific morphological and molecular changes in the liver characterized by microvesicular fatty change, increased mitosis, spontaneous peroxisome proliferation, increased mRNA and protein levels of genes regulated by peroxisome proliferator-activated receptor alpha (PPARalpha), and hepatocellular carcinoma. Based on these findings it is proposed that substrates for AOX function as ligands for PPARalpha. In this study we examined the sequential changes in morphology and gene expression in the liver of wild-type and AOX(-/-) mice at Embryonic Day 17.5, and during postnatal development up to 2 months of age. In AOX(-/-) mice high levels of expression of PPARalpha-responsive genes in the liver commenced on the day of birth and persisted throughout the postnatal period. We found no indication of PPARalpha activation in the livers of AOX(-/-) mice at embryonic age E17.5. In AOX(-/-) mice microvesicular fatty change in liver cells was evident at 7 days. At 2 months of age livers showed extensive steatosis and the presence in the periportal areas of clusters of hepatocytes with abundant granular eosinophilic cytoplasm rich in peroxisomes. These results suggest that the biological ligands for PPARalpha vis a vis substrates for AOX either are not functional in fetal liver or do not cross the placental barrier during the fetal development and that postnatally they are likely derived from milk and diet.

Peroxisome proliferator-activated receptor alpha-dependent induction of cell surface antigen Ly-6D gene in the mouse liver.

Spontaneous peroxisome proliferation-related pleiotropic responses occurring in the liver of mice lacking peroxisomal fatty acyl-CoA oxidase (AOX-/-) are attributed to sustained activation of peroxisome proliferator-activated receptor alpha (PPARalpha) by its putative natural ligands that require AOX for their metabolism. In this study, using a gene expression screen, we show that Ly-6 (lymphocyte antigen 6 complex, locus D; mouse ThB), which belongs to a distinctive family of low molecular weight phosphatidyl inositol anchored cell surface glycoproteins, is upregulated in mouse liver with peroxisome proliferation. Increases in Ly-6D mRNA levels are observed in AOX-/- mouse liver with spontaneous peroxisome proliferation and also in the liver of wild-type mice treated with synthetic peroxisome proliferators. Peroxisome proliferators failed to increase hepatic Ly-6D mRNA levels in mice lacking PPARalpha (PPARalpha-/-), suggesting a regulatory role for PPARalpha in the induction of Ly-6D. These observations suggest that changes in certain cell surface proteins also form part of the pleiotropic responses associated with peroxisome proliferation.

Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system.

beta-Oxidation occurs in both mitochondria and peroxisomes. Mitochondria catalyze the beta-oxidation of the bulk of short-, medium-, and long-chain fatty acids derived from diet, and this pathway constitutes the major process by which fatty acids are oxidized to generate energy. Peroxisomes are involved in the beta-oxidation chain shortening of long-chain and very-long-chain fatty acyl-coenzyme (CoAs), long-chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs, and the CoA esters of the bile acid intermediates di- and trihydroxycoprostanoic acids, and in the process they generate H2O2. Long-chain and very-long-chain fatty acids (VLCFAs) are also metabolized by the cytochrome P450 CYP4A omega-oxidation system to dicarboxylic acids that serve as substrates for peroxisomal beta-oxidation. The peroxisomal beta-oxidation system consists of (a) a classical peroxisome proliferator-inducible pathway capable of catalyzing straight-chain acyl-CoAs by fatty acyl-CoA oxidase, L-bifunctional protein, and thiolase, and (b) a second noninducible pathway catalyzing the oxidation of 2-methyl-branched fatty acyl-CoAs by branched-chain acyl-CoA oxidase (pristanoyl-CoA oxidase/trihydroxycoprostanoyl-CoA oxidase), D-bifunctional protein, and sterol carrier protein (SCP)x. The genes encoding the classical beta-oxidation pathway in liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPAR alpha). Evidence derived from mice deficient in PPAR alpha, peroxisomal fatty acyl-CoA oxidase, and some of the other enzymes of the two peroxisomal beta-oxidation pathways points to the critical importance of PPAR alpha and of the classical peroxisomal fatty acyl-CoA oxidase in energy metabolism, and in the development of hepatic steatosis, steatohepatitis, and liver cancer.

Peroxisomal beta-oxidation and steatohepatitis.

Fatty acid beta-oxidation occurs in both mitochondria and peroxisomes. Mitochondria catalyze the beta-oxidation of the bulk of short-, medium-, and long-chain fatty acids derived from diet, and this pathway constitutes the major process by which fatty acids are oxidized to generate energy. Peroxisomes are involved, preferentially, in the beta-oxidation chain shortening of very long chain fatty acids (VLCFAs) and in the process produce H2O2. Long-chain fatty acids and VLCFAs are also metabolized by the cytochrome P450 CYP4A omega-oxidation system to toxic dicarboxylic acids (DCAs) that serve as substrates for peroxisomal beta-oxidation, and this process also leads to the production of superoxide and H2O2. The genes encoding peroxisomal, microsomal, and certain mitochondrial fatty acid metabolizing enzymes in liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPAR alpha). Deficiencies of the enzymes of peroxisomal beta-oxidation have been recognized as important causes of disease. Evidence from mice deficient in PPAR alpha (PPAR alpha-/-), deficient in peroxisomal fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the classical beta-oxidation system, and deficient in both PPAR alpha and AOX (PPAR alpha-/-AOX-/-) points to the critical importance of PPAR alpha-inducible peroxisomal and microsomal oxidation systems that metabolize LCFAs and VLCFAs in the pathogenesis of nonalcoholic microvesicular hepatic steatosis and steatohepatitis. These and other mouse models should provide greater understanding of the molecular mechanism responsible for hepatic steatosis and steatohepatitis. Deficiency of AOX disrupts the oxidation of VLCFAs, DCAs, and other substrates leading to extensive microvesicular steatosis and steatohepatitis. Loss of this enzyme also causes sustained hyperactivation of PPAR alpha, leading to transcriptional up-regulation of PPAR alpha-regulated genes, indicating that unmetabolized substrates of AOX function as ligands of PPAR alpha. beta-Oxidation is the major process by which fatty acids are oxidized to generate energy, especially when glucose availability is low during periods of starvation. Mice deficient in PPAR alpha and those nullizygous for both PPAR alpha and AOX show a minimal steatotic phenotype under fed conditions but manifest an exaggerated steatotic response to fasting, indicating that defects in PPAR alpha-inducible fatty acid oxidation determine the severity of fatty liver phenotype to conditions reflecting energy-related stress.

Identification of novel peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in mouse liver using cDNA microarray analysis.

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists, are a group of structurally diverse nongenotoxic hepatocarcinogens including the fibrate class of hypolipidemic drugs that induce peroxisome proliferation in liver parenchymal cells. Sustained activation of PPARalpha by these agents leads to the development of liver tumors in rats and mice. To understand the molecular mechanisms responsible for the pleiotropic effects of these agents, we have utilized the cDNA microarray to generate a molecular portrait of gene expression in the liver of mice treated for 2 weeks with Wy-14,643, a potent peroxisome proliferator. PPARalpha activation resulted in the stimulation of expression (fourfold or greater) of 36 genes and decreased the expression (fourfold or more decrease) of 671 genes. Enhanced expression of several genes involved in lipid and glucose metabolism and many other genes associated with peroxisome biogenesis, cell surface function, transcription, cell cycle, and apoptosis has been observed. These include: CYP2B9, CYP2B10, monoglyceride lipase, pyruvate dehydrogenase-kinase-4, cell death-inducing DNA-fragmentation factor-alpha, peroxisomal biogenesis factor 11beta, as well as several cell recognition surface proteins including annexin A2, CD24, CD39, lymphocyte antigen 6, and retinoic acid early transcript-gamma, among others. Northern blotting of total RNA extracted from the livers of PPARalpha-/- mice and from mice lacking both PPARalpha and peroxisomal fatty acyl-CoA oxidase (AOX), that were fed control and Wy-14,643-containing diets for 2 weeks, as well as time course of induction following a single dose of Wy-14,643, revealed that upregulation of genes identified by microarray procedure is dependent upon peroxisome proliferation vis-à-vis PPARalpha. However, cell death-inducing DNA-fragmentation factor-alpha mRNA, which is increased in the livers of wild-type mice treated with peroxisome proliferators, was not enhanced in AOX-/- mice with spontaneous peroxisome proliferation. These observations indicate that the activation of PPARalpha leads to increased and decreased expression of many genes not associated with peroxisomes, and that delayed onset of enhanced expression of some genes may be the result of metabolic events occurring secondary to PPARalpha activation and alterations in lipid metabolism.

Less extrahepatic induction of fatty acid beta-oxidation enzymes by PPAR alpha.

The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear receptor that transcriptionally regulates mitochondrial and peroxisomal fatty acid beta-oxidation enzymes in the liver. Ligands include synthetic peroxisome proliferators and some fatty acids. PPARalpha activation leads to predictable pleiotropic responses in liver including peroxisome proliferation, increased fatty acid oxidation, and hepatocellular carcinoma. In the current study, the response to PPAR alpha-activation was compared in the heart, kidney, and liver since the role of PPAR alpha in extrahepatic fatty acid-oxidizing organs has not been fully explored. Basal expression of mitochondrial beta-oxidation enzymes was comparable in the three tissues, but peroxisomal beta-oxidation enzymes were most abundant in the liver and less so in the kidney and especially in the heart. After PPAR alpha activation with ciprofibrate, both mitochondrial and peroxisomal beta-oxidation enzymes were induced, with the strongest response seen in the liver, a moderate response in the kidney, and no significant response in the heart. PPAR alpha mRNA analysis suggested that the differential response may be related to PPAR alpha expression.

The effect of microvesicular fatty change on liver regeneration after partial hepatectomy.

BACKGROUND/AIMS: Hepatic resection for primary and secondary liver tumors and transplantation of segments of liver into patients with severe liver disease has become a common practice. However, the information on the regenerative potential of liver with various underlying diseases, particularly, the common disorder fatty change, is sparse. METHODOLOGY: In the present study we have evaluated the regenerative potential of liver in mice lacking fatty acyl-CoA oxidase (AOX-/-) after partial hepatectomy. Liver regeneration was assessed by measuring mitotic activity and 5-bromo-2'-deoxyuridine (BrdU) labeling using immunohistochemistry. RESULTS: BrdU labeling index in AOX-/- mice was 99 +/- 32, 156 +/- 28, 54 +/- 13, and 120 +/- 33 per 1000 cells at 48, 60, 72 and 84 hours, respectively after partial hepatectomy. Comparable BrdU labeling index was observed in AOX+/+ mice. Similarly, mitotic index in both groups was comparable. CONCLUSIONS: These results suggest that DNA synthesis after partial hepatectomy in genetically altered mice with microvesicular fatty change is unaffected.

Defect in peroxisome proliferator-activated receptor alpha-inducible fatty acid oxidation determines the severity of hepatic steatosis in response to fasting.

Fasting causes lipolysis in adipose tissue leading to the release of large quantities of free fatty acids into circulation that reach the liver where they are metabolized to generate ketone bodies to serve as fuels for other tissues. Since fatty acid-metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPARalpha), we investigated the role of PPARalpha in the induction of these enzymes in response to fasting and their relationship to the development of hepatic steatosis in mice deficient in PPARalpha (PPARalpha(-/-)), peroxisomal fatty acyl-CoA oxidase (AOX(-/-)), and in both PPARalpha and AOX (double knock-out (DKO)). Fasting for 48-72 h caused profound impairment of fatty acid oxidation in both PPARalpha(-/-) and DKO mice, and DKO mice revealed a greater degree of hepatic steatosis when compared with PPARalpha(-/-) mice. The absence of PPARalpha in both PPARalpha(-/-) and DKO mice impairs the induction of mitochondrial beta-oxidation in liver following fasting which contributes to hypoketonemia and hepatic steatosis. Pronounced steatosis in DKO mouse livers is due to the added deficiency of peroxisomal beta-oxidation system in these animals due to the absence of AOX. In mice deficient in AOX alone, the sustained hyperactivation of PPARalpha and up-regulation of mitochondrial beta-oxidation and microsomal omega-oxidation systems as well as the regenerative nature of a majority of hepatocytes containing numerous spontaneously proliferated peroxisomes, which appear refractory to store triglycerides, blunt the steatotic response to fasting. Starvation for 72 h caused a decrease in PPARalpha hepatic mRNA levels in wild type mice, with no perceptible compensatory increases in PPARgamma and PPARdelta mRNA levels. PPARgamma and PPARdelta hepatic mRNA levels were lower in fed PPARalpha(-/-) and DKO mice when compared with wild type mice, and fasting caused a slight increase only in PPARgamma levels and a decrease in PPARdelta levels. Fasting did not change the PPAR isoform levels in AOX(-/-) mouse liver. These observations point to the critical importance of PPARalpha in the transcriptional regulatory responses to fasting and in determining the severity of hepatic steatosis.

Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR.

We previously isolated and identified steroid receptor coactivator-1 (SRC-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/PPARBP) as coactivators for PPAR, using the ligand-binding domain of PPARgamma as bait in a yeast two-hybrid screening. As part of our continuing effort to identify cofactors that influence the transcriptional activity of PPARs, we now report the isolation of a novel coactivator from mouse, designated PRIP (peroxisome proliferator-activated receptor interacting protein), a nuclear protein with 2068 amino acids and encoded by 13 exons. Northern analysis showed that PRIP mRNA is ubiquitously expressed in many tissues of adult mice. PRIP contains two LXXLL signature motifs. The amino-terminal LXXLL motif (amino acid position 892 to 896) of PRIP was found to be necessary for nuclear receptor interaction, but the second LXXLL motif (amino acid position 1496 to 1500) appeared unable to bind PPARgamma. Deletion of the last 12 amino acids from the carboxyl terminus of PPARgamma resulted in the abolition of the interaction between PRIP and PPARgamma. PRIP also binds to PPARalpha, RARalpha, RXRalpha, ER, and TRbeta1, and this binding is increased in the presence of specific ligands. PRIP acts as a strong coactivator for PPARgamma in the yeast and also potentiates the transcriptional activities of PPARgamma and RXRalpha in mammalian cells. A truncated form of PRIP (amino acids 786-1132) acts as a dominant-negative repressor, suggesting that PRIP is a genuine coactivator.