MEETING BRIEF

Let Them Eat Fat
Proliferation and Diseases of Peroxisomes

by Stefan Alexson

(Posted July 11, 1997 · Issue 12; archived July 25, 1997)

Abstract

The peroxisome, long a mystery subcellular organelle, is now understood to be a major intracellular site of dietary fat metabolism. Acknowledging the explosion on the topic, The Nobel Assembly devoted its annual meeting to peroxisome structure, function, and biogenesis. The meeting highlighted the synergistic interactions between investigators who have used yeast as a model system for characterizing this organelle and those who have focused on peroxisomes in more complex eukaryotes. The work reported here also provides a foundation for developing new and improved pharmacological agents to treat diseases involving lipid metabolism.

Over forty years ago Johannes Rhodin, a Ph.D. student at Vitrum AB in Stockholm, described a hitherto unknown cellular organelle that he simply called a microbody. In the 1950s and 1960s, biochemical studies from Dr. Christian de Duve and others showed that microbodies contained enzymes producing and degrading hydrogen peroxide, and in 1966 the name peroxisome was coined for this organelle. The physiological role, biogenesis, and origin of the peroxisome was long unclear. But the discovery of lipid-metabolizing enzymes in peroxisomes, as well as the identification of peroxisomal disorders that seriously affect humans, and the development of mutant strains of yeast that are defective in peroxisome biogenesis, have greatly improved the understanding of peroxisomes. Therefore the Nobel Assembly devoted its twenty-ninth conference, held May 31-June 3, 1997 at Saltsjöbaden, near Stockholm, to the "Metabolic Functions, Proliferation, and Diseases of Peroxisomes." The conference, which covered these topics with three days of lectures and an evening poster session, emphasized the very rapid development in the understanding of the peroxisome.

The first day was devoted to the biogenesis of peroxisomes. In his talk entitled "The Riddle of the Microbody," Nobel laureate Dr. Christian de Duve of the International Institute of Cellular and Molecular Pathology in Brussels opened the session by summarizing the historical milestones leading to the present concepts of peroxisome function and biogenesis. Drs. Suresh Subramani, University of California at San Diego, and Wolf-H. Kunau, Ruhr Universität Bochum elegantly demonstrated the power of yeast genetics for studying peroxisome biogenesis. Generating peroxisome assembly mutants has so far identified about twenty genes involved in peroxisomal protein import and biogenesis. The early steps of protein import, involving interaction between peroxisomal targeting signals and corresponding receptors, are being well characterized, but the complexity of the import machinery is becoming apparent. Most of these genes are conserved in evolution, and therefore cloning of the human counterparts has shown that many of these genes are mutated in human disorders. The molecular basis for most of the peroxisomal disorders can therefore be expected to be understood in the near future.

For many years, peroxisomes were thought to arise by budding from the endoplasmic reticulum, but in the current model of peroxisome biogenesis it is believed that peroxisomal proteins are post-translationally imported into preexisting organelles that then divide by fission. Therefore the new data presented by Dr. Richard Rachubinski of the University of Alberta at Edmonton and by Dr. Subramani demonstrating the targeting of some peroxisomal proteins via the endoplasmic reticulum reconnects these two compartments in the biogenesis of peroxisomes. These conclusions were based on their findings that an integral peroxisomal membrane protein was targeted to peroxisomes via the endoplasmic reticulum, where it was found to be O-glycosylated, and the findings that some temperature-sensitive Sec mutants were defective also in formation of normally sized peroxisomes.

Proliferation and induction of peroxisomal proteins is mediated by transcriptional activation of several genes. In higher eukaryotes these events are mediated through the peroxisome proliferator-activated receptor (see further notes on PPARs below). In several yeasts, addition of fatty acids (e.g., oleate) induces the expression of peroxisomal proteins and leads to peroxisome proliferation. Here Dr. Gillian Small from the Mount Sinai School of Medicine, New York City, reported on the isolation and characterization of genes encoding two oleate activation factors (OAF1 and OAF2). OAF1 and OAF2 heterodimerize and mediate not only the transcriptional control of peroxisomal enzymes, but are also crucial for growth and proliferation of the peroxisomes. Peroxisome proliferation and transient appearance of peroxisomal reticuli in eukaryotic cells were discussed by Dr. Dariush Fahimi of the University of Heidelberg on the second day.

The second day's theme was the role of peroxisomes in genetic diseases and carcinogenesis. Dr. Sidney Goldfischer of the Albert Einstein College of Medicine, New York City, started by giving a historical view of peroxisomal diseases. The striking pleiotropism of peroxisomal disorders, which manifest as multiple phenotypic effects due to a single mutant gene as well as genetic heterogeneity where several genetic mechanisms lead to similar phenotypes, was discussed by Dr. Ann Moser of the Kennedy Krieger Institute, Baltimore. Today, six genes have been identified that correspond to six out of the ten complementation groups identified at the Kennedy Krieger Institute. The peroxisomal disorders range from peroxisome biogenesis defects, where functional peroxisomes are lacking, to single enzyme defects.

The biochemistry of peroxisomal disorders was further discussed by Dr. Ronald Wanders, University Hospital of Amsterdam. From a biochemical point of view, peroxisomal disorders can be divided into three groups in which a mutation in a single gene can lead to generalized, multiple, or single loss of enzyme activities. The first group is characterized by a defect in peroxisome assembly in which all matrix proteins are essentially lacking. The second group is characterized by a deficiency in one of the peroxisome targeting signal receptors (the PTS2 receptor) that results in the absence of four (currently known) proteins in peroxisomes. The recently identified phytanoyl-CoA hydroxylase was shown to be a PTS2 protein, and this finding has finally conclusively identified classical Refsum's disease as a peroxisomal disorder, the reason being that the organization of the alpha-oxidation pathway has long remained enigmatic. Adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder, was reviewed by Dr. Patrick Aubourg of the Medical School University of Paris. The disorder is characterized by demyelination within the central nervous system, leading to neurological deterioration followed by a vegetative state or death. The disorder is due to a protein defect that results in oxidation of very-long-chain fatty acids. The gene encodes a protein that is a member of the ATP-binding cassette transporter family, and may be involved in transport of fatty acids across the peroxisomal membrane. Molecular analysis has shown that nearly every ALD family has a different mutation (more than 120 different mutations have been identified in 150 ALD kindred). Targeted mutation of the ALD gene has given access to an animal model that may clarify the disease and possibly help develop new therapeutic approaches.

Peroxisome proliferators represent a specific class of non-genotoxic hepatocarcinogens in certain rodents. The species-selective effects of peroxisome proliferators on enzyme activities and apoptosis, as well as a molecular analysis of peroxisome proliferator-induced rat liver tumors, were discussed by Dr. Cliff Elcombe (University of Dundee), Dr. Cris Corton (Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina) and Dr. Ruth Roberts (Zeneca Pharmaceuticals, Macclesfield). Cliff Elcombe's data showed that administration of peroxisome proliferators resulted in increased DNA synthesis and decreased apoptosis both in vivo and in vitro in responding species (e.g., rats), but not in nonresponders such as guinea pigs and in human hepatocytes. Dr. Corton discussed the highly different regulation of expression of various genes in tumors and surrounding tissues before and after treatment and withdrawal of treatment with peroxisome proliferators. Dr. Roberts discussed the possible mechanisms for the observed suppression of hepatocyte apoptosis by peroxisome proliferators. She showed that there was no difference between responders and nonresponders (hamster and guinea pig) on spontaneous and TGF-beta-1 induced apoptosis. However, only hepatocytes from responders (rat and mouse) showed induction of S-phase in response to nafenopin. She further discussed a novel isoform of human PPAR-alpha that appears to act as a dominant negative repressor. Using this isoform, it was possible to demonstrate that PPAR-alpha is the causative mediator in the suppression of apoptosis by peroxisome proliferators.

On the third day, metabolism and regulation were discussed. First, Dr. Takashi Hashimoto (Shinshu University School of Medicine, Matsumoto) discussed the molecular characterization of peroxisomal fatty acid oxidation enzymes. Notably, two acyl-CoA synthetases, active on long chain and very-long-chain fatty acids, have been cloned and characterized. Peroxisomes also contain two multifunctional enzymes, as part of the beta-oxidation pathway, that are specific for L-3 and D-3 hydroxyacyl-CoAs. The functional organization of the mammalian peroxisomal beta-oxidation system was further discussed by Dr. Guy Mannaerts of the University of Leuven. He described the recent isolation and molecular cloning of different acyl-CoA oxidases, multifunctional enzymes, and 3-keto thiolases that show specificities for different types of lipids. This data conclusively demonstrates that there exist separate peroxisomal beta-oxidation pathways for straight and branched-chain acyl-CoAs. The specificity is exerted at the steps of acyl-CoA oxidase (3 different enzymes), two multifunctional hydratase/dehydrogenases, and two types of 3-ketoacyl-CoA thiolases. Structural and functional aspects of peroxisomal multifunctional enzymes were discussed by Dr. Kalervo Hiltunen of the University of Oulu. By crystallization, structural determination, and site-directed mutagenesis of rat mitochondrial enoyl-CoA hydratase, information was obtained that could be used to explain the broad chain-length specificity of the enzyme as well as the dramatically different catalytic activity towards long-and-short-chain substrates.

Dr. Henk van den Bosch of Utrecht University provided new insights into the molecular biology of the ether lipid synthesis in peroxisomes. The peroxisomal involvement in ether lipid biosynthesis was evident from studies in Zellweger syndrome, a severe peroxisomal disorder. The pathway is now elucidated further by the isolation and molecular cloning of alkyl-DHAP synthase, the second enzyme in the process. Interestingly the protein was found to contain an N-terminal peroxisomal targeting signal (PTS2), which explains the previous finding that the enzyme is lacking in the autosomal recessive disorder rhizomelic chondrodysplasia punctata, where the defect was recently found to be at the level of the PTS2 receptor. Isoprenoid metabolism in peroxisomes was discussed by Dr. Gustav Dallner of the Karolinska Institute, Huddinge, Sweden. The complexity of the mevalonate pathway is emphasized by the large number of enzymatic steps with multi-site localizations. Cholesterol is synthesized both in the endoplasmic reticulum and in peroxisomes. Similarly, dolichol synthesis is present in both compartments, whereas ubiquinone is not present in peroxisomes. Paradoxically, ubiquinone content is increased in several rat tissues in response to peroxisome proliferator treatment. Notably, the Niemann-Pick type 3 disease, which has been identified as a lysosomal deficiency, was found to also have a peroxisome deficiency similar to a number of peroxisomal disorders.

The remaining lectures focused on the structure, activity and function of PPARs. First, Dr. Frank Gonzalez of the National Cancer Institute, Bethesda, presented data obtained from the PPAR-alpha null mouse, conclusively demonstrating the crucial role of this PPAR subtype in the mediation of the effects elicited by peroxisome proliferators. All the classical effects of peroxisome proliferators on mouse liver were absent in the PPAR-alpha null mouse.

Dr. Eckardt Treuter of the Karolinska Institute discussed transcriptional cofactors that may mediate ligand-dependent PPAR activation, or act as coactivator and mediator between the receptor and the transcription machinery. Screening with the yeast two-hybrid system has identified a number of candidate factors, among them RIP140. The role of PPARs in the proliferation of peroxisomes in proliferating and differentiating brown adipose tissue during cold acclimatization was discussed by Dr. Stefan Alexson (of the Karolinska Institute). Changes in expression of PPAR-alpha and gamma correlated to the state of proliferation-differentiation of the tissue, suggesting important roles on these processes during adipogenesis. Steven Kliewer, at Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina, discussed PPAR-subtype specific activation by fatty acids, thiazolidinediones (TZDs), and fibrates. Notably, administration of TZDs, which are PPAR-gamma-specific activators, to obese Zucker rats increased brown adipose tissue mass and lowered glucose and insulin levels, suggesting that PPAR-gamma may be the target for the antidiabetic effect of these compounds. Previous work from the last speaker of the conference, Dr. Bruce Spiegelman of the Dana-Farber Cancer Institute at Harvard Medical School, Boston, has documented the crucial role of PPAR-gamma in the differentiation of adipocytes. The new aspects discussed by Dr. Spiegelman were of the role of phosphorylation of PPAR-gamma by protein kinases (in particular the mitogen-activated protein, or MAP, kinase) as a potentially very important step in controlling PPAR activity. He also presented data demonstrating that liposarcoma, the most common sarcoma in adult humans, express high levels of PPAR-gamma. When treated with thiazolidinediones, the cells undergo adipogenesis, express fat-cell-specific genes, and appear to enter a post-mitotic state. Thus PPAR ligands may be used for therapy in the future.

The conference clearly demonstrated the rapid progress in the understanding of peroxisome function and biogenesis. It also becomes more evident that the peroxisome proliferator-activated receptors play very important roles not only in the regulation of expression of peroxisomal enzymes and proliferation of peroxisomes, but also in several other cellular processes such as regulation of expression of many extraperoxisomal enzymes and regulation of cell proliferation-differentiation-apoptosis. Therefore PPAR ligands may be useful future therapeutic tools.

Stefan Alexson is an associate professor in the Division of Clinical Chemistry at the Karolinska Institute, Huddinge, Sweden.

Send us your comments and ideas for future articles.

Endlinks

The Beginnings of Life on Earth - essay by Christian de Duve, 1974 Nobel laureate in medicine with Albert Claude and George E. Palade "for their discoveries concerning the structural and functional organization of the cell," from the September-October 1995 American Scientist.

Celebrating 50 Years of Electron Microscopy and Modern Cell Biology - Historic early cell photomicrographs, plus notes on the contributions of early researchers including de Duve.

Of the following articles on peroxisomes, the first is an abstract that may be read at no charge; the latter two are full-text articles that may be viewed for a fee.

H.R. Waterham and J.M. Cregg. 1997. Peroxisome biogenesis. BioEssays 19:57-66.

W. Wahli, Braissant, O., and Desvergne, B. 1995. Peroxisome proliferator activated receptors: Transcriptional regulators of adipogenesis, lipid metabolism and more. Chem. & Biol. 2:261-266.