Peroxisomes of the midgut epithelium, malpighian tubules and fat body of larvae of the dragonfly, Aeshna cyanea

The enterocytes of the midgut epithelium of Aeshna cyanea larvae are rich in peroxisomes while the nidal regenerative and endocrine cells contain only a few. Most of the enterocytic peroxisomes are microperoxisomes lacking a crystalloid nucleoid, but peroxisomes with well developed nucleoid are also present. The peroxisomes are usually concentrated in the basal region of the cells but may also spread into the apical region and closely intermingle with absorptive lipid droplets. They significantly increase in number, when the larvae are regularly fed lipid-rich natural food or long-chain monounsaturated fatty acids that are unusual dietary components of these animals. This observation seems to indicate that the enterocytic peroxisomes are involved in chain shortening and degradation of fatty acids absorbed from the gut lumen. Numerous microperoxisomes are also present in the lipid-storing cells of the Malpighian tubules and fat body.     

Localization of nonspecific lipid transfer protein (nsLTP = sterol carrier protein 2) and acyl-CoA oxidase in peroxisomes of pigment epithelial cells of rat retina.

We investigated the localization of nonspecific lipid transfer protein (nsLTP) in rat retina, especially in the pigment epithelial (RPE) cells, by the avidin-biotin-peroxidase complex method on cryosections for light microscopy and by the cryoimmunogold method for electron microscopy. Light microscopic observation revealed that the RPE, inner segment layer, nerve fiber layer, and Müller cells contain nsLTP. In the RPE cells gold particles were exclusively concentrated in the small peroxisomes (microperoxisomes; 0.1-0.3 micron in diameter), which were identified by double staining using anti-nsLTP and anti-catalase antibodies. In the peroxisomes gold particles were distributed homogeneously in the matrices and no preferential binding to the limiting membrane was observed. Acyl-CoA oxidase was also localized in the matrices of the peroxisomes. We suggest that the peroxisomes in RPE cells play important roles in the metabolism of lipids of the outer segment disk membranes, especially in the beta-oxidation of polyunsaturated long-chain and very long-chain fatty acids, such as docosahexaenoic acid which is composed of approximately one third of fatty acids in the disk membranes.     

Peroxisomes in the digestive diverticula of the bivalve mollusc Nucula sulcata

Summary Electron microscopy of the digestive diverticula of the protobranch bivalve, Nucula sulcata, revealed the presence of peroxisomes in the basal regions of the epithelial cells lining the main and secondary ducts, and in the digestive and secretory (basophil) cells of the tubules. Those in the secretory cells are elongate and somewhat flattened, while those of the other cell types have a spherical form. Two distinct types of nucleoid are normally present within the secretory cell peroxisomes, one compact, crystalline, and finely polytubular, the other comprising isolated secondary tubules arranged in a linear series across the width of the organelle. The peroxisomes of the digestive and duct cells contain coarsely polytubular cores arranged in two clusters orientated more or less at right angles; the duct cell peroxisomes may also contain a second nucleoid in the form of a compact finely polytubular core. Sections incubated in a medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide reveal an electron dense reaction product within the peroxisomes of all the cell types. Catalase is considered to be responsible for the reaction.     

Development of peroxisomes in amphibians. III. Study on liver, kidney, and intestine during thyroxine-induced metamorphosis

This investigation was undertaken to study the ontogeny of hepatic, renal, and intestinal peroxisomes and/or microperoxisomes during thyroxine-induced anuran metamorphosis. Catalase activity was localized cytochemically after incubation in DAB medium, and studied biochemically by a spectrophotometric method. Our morphological and biochemical investigations suggest the formation of a new population of peroxisomes during the hormonal treatment. This is obvious especially for microperoxisomes of the intestinal epithelium since the larval tissue is completely replaced by a new layer during thyroxine-induced metamorphosis. For the peroxisomes of hepatocytes and kidney proximal tubule cells, our assumption is based on the following observations: 1) The number of peroxisomes increases in liver and kidney during thyroxine treatment; 2) this proliferation is accompanied by an enlargement of renal peroxisomes; and 3) 16 days after the beginning of the hormonal treatment, 5.4- and 2.4-fold increases are found for the specific activities of hepatic and renal catalase, respectively. A temporal coordination exists between the structure and the metabolism of peroxisomes and mitochondria during thyroxine-induced metamorphosis.     

Relationship between the fatty acids of fat body triacylglycerol and lipophorin diacylglycerol of non-diapause and diapause larvae of the southwestern corn borer,Diatraea grandiosella

The fatty acids of the triacylglycerol reserves in the fat body and of the diacylglycerol of lipophorin in the hemolymph of non-diapause and diapause larvae of D. grandiosella were compared. For both non-diapause and diapause larvae palmitate, palmitoleate, oleate, and linoleate were the predominant fatty acids present in fatty body triacylglycerol, and palmitate, oleate, and linoleate were the predominant fatty acids present in lipophorin diacylglycerol. However, differences were detected in the relative amounts of oleate and linoleate present in lipophorin diacylglycerol of non-diapause and diapause larvae. The relative amount of linoleate in lipophorin diacylglycerol declined during diapause, whereas that of oleate remained relatively high during diapause. The fatty acid profile of lipophorin diacylglycerol from non-diapause larvae treated with a juvenile hormone analog to induce a diapause-like state more closely matched that of diapause larvae than that of non-diapause larvae. The differences detected in the fatty acid composition of lipophorin diacylglycerol in non-diapause and diapause larvae appear to be due mainly to the different physiological states rather than to the different rearing temperatures employed. The results are discussed in relation to the essential role fatty acids, especially oleate, play in the survival of diapause larvae.     

Peroxisomal enzymes in the honey bee midgut

Peroxisomes are ubiquitous organelles that contain catalase (CAT) and an array of inducible enzymes that regulate aspects of lipid, purine, xenobiotic, eicosanoid, and phospholipid metabolism. Although peroxisomes are recognized as essential components in the cellular economy of microorganisms, plants, and mammals, little is known about their specialized functions in insect metabolism. Peroxisomal acyl-CoA oxidase (ACO) is a flavin-linked, H₂O₂-producing enzyme that regulates the β-oxidation of long chain fatty acids. We measured ACO and CAT activity in midgut tissues from worker honey bees, Apis mellifera, of known ages from free-flying colonies. The ACO activity peaked in young worker bees that digest and assimilate nutrients from pollen and from trophallaxis. As the bees aged, ACO activity declined. Conversely, CAT activity increased as the bees aged and reached its highest level in the oldest bees that were assayed. Isolated honey bee midguts were then fractionated using sucrose and Metrizamide (MET) density gradient centrifugation. Organelle-bound CAT activity equilibrated in sucrose at densities between 1.19 and 1.22, which are typical of spherical 1 μm peroxisomes. In the MET gradients, the organelle-bound CAT separated into two distinct fractions. The heavier fraction equilibrated at 1.21 and the lighter fraction at 1.15, a density commonly associated with microperoxisomes. These results support our ultrastructural and cytochemical data and suggest that the diverse functions of regionally specialized midgut epithelial cells lead to a heterogeneous population of peroxisomal organelles. ACO activity confirms the role of midgut peroxisomes in the intermediary metabolism of lipids and the increasing CAT activity suggests that the midgut epithelium may metabolize deleterious pro-oxidants of aerobic metabolism associated with foraging and senescence. © 1996 Wiley-Liss, Inc.     

The significance of peroxisomes in secondary metabolite biosynthesis in filamentous fungi

Peroxisomes are ubiquitous organelles characterized by a protein-rich matrix surrounded by a single membrane. In filamentous fungi, peroxisomes are crucial for the primary metabolism of several unusual carbon sources used for growth (e.g. fatty acids), but increasing evidence is presented that emphasize the crucial role of these organelles in the formation of a variety of secondary metabolites. In filamentous fungi, peroxisomes also play a role in development and differentiation whereas specialized peroxisomes, the Woronin bodies, play a structural role in plugging septal pores. The biogenesis of peroxisomes in filamentous fungi involves the function of conserved PEX genes, as well as genes that are unique for these organisms. Peroxisomes are also subject to autophagic degradation, a process that involves ATG genes. The interplay between organelle biogenesis and degradation may serve a quality control function, thereby allowing a continuous rejuvenation of the organelle population in the cells.     

Peroxisomes in the absorptive cells of normal,rachitic, and vitamin-D replete chick intestine: Ultrastructure and histochemistry

Using routine transmission electron microscopy and light and electron microscopic techniques for the histologic demonstration (localization) of catalase (a peroxisomal enzyme), peroxisomes in chick duodenal epithelial cells were identified and studied. In these cells, peroxisomes were seen to be small, ovoid structures, delimited by a single unit membrane. They were concentrated in the supranuclear cytoplasm in initimate association with the smooth endoplasmic reticulum. As demonstrated histochemically, the heterogeneous matrix of these organelles was catalase positive. In addition, most of the larger peroxisomes revealed central nucleoids; however, the smaller peroxisomes were generally anucleoid. It thus appears that two classes of peroxisomes exist in chick intestinal absorptive cells: (1) small, anucleoid microperoxisomes, and (2) larger, nucleoid-containing peroxisomes. In addition to the above morphological characteristics, both peroxisome types were numerous in normal and vitamin-D-replete tissues, but were conspicuously decreased or absent from the apical cytoplasm of rachitic epithelial cells. From these observations it is hypothesized that these organelles may be involved in the overall vitamin-D response of the small intestine.     

Subcellular localization of long-chain alcohol dehydrogenase and aldehyde dehydrogenase in n-alkane-grown Candida tropicalis

Long-chain alcohol dehydrogenase and longchain aldehyde dehydrogenase were induced in the cells of Candida tropicalis grown on n-alkanes. Subcellular localization of these dehydrogenases, together with that of acyl-CoA synthetase and glycerol-3-phosphate acyltransferase, was studied in terms of the metabolism of fatty acids derived from n-alkane substrates. Both longchain alcohol and aldehyde dehydrogenases distributed in the fractions of microsomes, mitochondria and peroxisomes obtained from the alkane-grown cells of C. tropicalis. Acyl-CoA synthetase was also located in these three fractions. Glycerol-3-phosphate acyltransferase was found in microsomes and mitochondria, in contrast to fatty acid -oxidation system localized exclusively in peroxisomes. Similar results of the enzyme localization were also obtained with C. lipolytica grown on n-alkanes. These results suggest strongly that microsomal and mitochondrial dehydrogenases provide long-chain fatty acids to be utilized for lipid synthesis, whereas those in peroxisomes supply fatty acids to be degraded via -oxidation to yield energy and cell constituents.     

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.     

Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.     

Structural and cytochemical characterization of three specialized peroxisome types in soybean

Structural and cytochemical comparisons were made between three peroxisome types in soybean [ Glycine max (L.) Merr. cv. Centennial]. Leaf peroxisomes were densely granular organelles with an amorphous nucleoid and were generally located in close proximity to the chloroplasts. Catalase (EC 1.11.1.6) and glycolate oxidase (EC 1.1.3.1) were localized in these peroxisomes although glycolate oxidase was absent from the nucleoid region. Glyoxysomes, present in the etiolated cotyledons, were coarsely granular organelles that were generally in close proximity to lipid bodies. Malate synthase (EC 4.1.3.2), catalase, and glycolate oxidase were present throughout the matrix. Although peroxisomes were found in both infected and uninfected nodule tissue, uninfected interstitial cell peroxisomes were the most developed. These organelles contained a core surrounded by a less electron-opaque periphery that frequently was in close association with (but distinct from) a network of smooth endoplasmic reticulum. Of the enzymes studied, only catalase and urate oxidase (EC 1.7.3.3) were detected in the nodule peroxisomes. Neither enzyme was detected in the peripheral area of the peroxisome. These data indicate that peroxisomes in the three tissue types have organelle associations, internal structures, enzyme constitutions and packaging that reflect their metabolic differences.     

Microsomal and cytosolic epoxide hydrolases, the peroxisomal fatty acid beta-oxidation system and catalase. Activities, distribution and induction in rat liver parenchymal and non-parenchymal cells

A number of structurally unrelated hypolipidaemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in rodent liver, but there is relatively limited data regarding the specific effects of these drugs on liver non-parenchymal cells. In the present study, liver parenchymal, Kupffer and endothelial cells from untreated and fenofibrate-fed rats were isolated and the activities of two enzymes associated with peroxisomes (catalase and the peroxisomal fatty acid beta-oxidation system) as well as cytosolic and microsomal epoxide hydrolase were measured. Microsomal epoxide hydrolase, cytosolic epoxide hydrolase and catalase activities were 7-12-fold higher in parenchymal cells than in Kupffer or endothelial cells from untreated rats; the peroxisomal fatty acid beta-oxidation activity was only detected in parenchymal cells. Fenofibrate increased catalase, cytosolic epoxide hydrolase and peroxisomal fatty acid beta-oxidation activities in parenchymal cells by about 1.5-, 3.5- and 20-fold, respectively. The induction of catalase (2-3-fold) and cytosolic epoxide hydrolase (3-5-fold) was also observed in Kupffer and endothelial cells; furthermore, a low peroxisomal fatty acid beta-oxidation activity was detected in endothelial cells. Morphological examination by electron microscopy showed that peroxisomes were confined to liver parenchymal cells in untreated animals, but could also be observed in endothelial cells after administration of fenofibrate.     

Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi

Many microorganisms secrete surface‐active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis‐targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross‐talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.     

Regulation of peroxisomal lipid metabolism by catalytic activity of tumor suppressor H-rev107

H-rev107 is a mammalian protein belonging to the HRAS-like suppressor family. Although the protein was originally found as a tumor suppressor, currently it is receiving considerable attention as a regulator of adipocyte lipolysis. We recently revealed that purified recombinant H-rev107 has phospholipase A(1/2) activity, releasing free fatty acids from glycerophospholipids with a preference for esterolysis at the sn-1 position. In the present study, we constitutively expressed H-rev107 in cloned HEK293 cells to examine its biological function in living cells. Initially, the cells accumulated free fatty acids. We also found a remarkable decrease in the levels of ether-type lipids, including plasmalogen and ether-type triglyceride, with a concomitant increase in fatty alcohols, substrates for the biosynthesis of ether-type lipids. Considering that peroxisomes are involved in the ether-type lipid biosynthesis, we next focused on peroxisomes and found that the peroxisomal markers 70-kDa peroxisomal membrane protein and catalase were abnormally distributed in the transfected cells. These biochemical and morphological abnormalities were not seen in HEK293 cells stably expressing a catalytically inactive mutant of H-rev107. When H-rev107 or its fusion protein with enhanced green fluorescence protein was transiently expressed in mammalian cells, both proteins were associated with peroxisomes in some of the observed cells. These results suggest that H-rev107 interferes with the biosynthesis of ether-type lipids and is responsible for the dysfunction of peroxisomes in H-rev107-expressing cells.     

Localization of the pre-squalene segment of the isoprenoid biosynthetic pathway in mammalian peroxisomes

Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunofluorescence and cell permeabilization techniques. Our present findings clearly show and confirm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal β-oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool.     

Studies on microperoxisomes. VII. Pigment epithelial cells and other cell types in the retina of rodents

The pigment epithelial cell of the retina actively participates in two aspects of lipid metabolism: (a) the fatty acid esterification of vitamin A and its storage and transport to the photoreceptors, and (b) the phagocytosis and degradation of the lipoprotein membrane disks shed from the photoreceptor cells. Study of the pigment epithelial cells of adult albino and pigmented rodents has revealed the abundance of an organelle, microperoxisomes, not previously known to exist in this cell type. The metabolism, transport, and storage of lipids are major functions of other cell types which possess large numbers of microperoxisomes associated with a highly developed smooth endoplasmic reticulum. Microperoxisomes were encountered, but relatively rarely, in Müller cells and vascular endothelial cells. A tubular system in photoreceptor terminals is reactive in the cytochemical procedure used to visualize microperoxisomes.     

Peroxisomes of soybean (Glycine max) root nodule vascular parenchyma cells contain a "nodule—specific" urate oxidase

The cortex of soybean ( Glycine max L. cv. Centennial) nodules contain an organellerich layer of vascular parenchyma tissue, which encircles the elaborate vascular tissue of the nodule. Peroxisomes with small, electron-opaque nucleoids are found in the vascular parenchyma cells. Positive cytochemical staining for catalase (EC 1.11.1.6) confirms their morphological identification as peroxisomes. Activities of both glycolate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were detected cytochemically in these peroxisomes. Nodule-specific urate oxidase was localized principally in the nucleoid region of these vascular parenchyma peroxisomes, as indicated by immunogold labelling using antibodies against nodulin-35, the nodule-specific urate oxidase. The density of urate oxidase immunogold labelling in the vascular parenchyma peroxisome nucleoid is similar to that of the more well-characterized interstitial cell peroxisomes of the infected zone. These results show that the induction of nodule-specific urate oxidase may be induced in tissue outside of the infected zone.     

The significance of peroxisome function in chronological aging of Saccharomyces cerevisiae

We studied the chronological lifespan of glucose‐grown Saccharomyces cerevisiae in relation to the function of intact peroxisomes. We analyzed four different peroxisome‐deficient (pex) phenotypes. These included Δpex3 cells that lack peroxisomal membranes and in which all peroxisomal proteins are mislocalized together with Δpex6 in which all matrix proteins are mislocalized to the cytosol, whereas membrane proteins are still correctly sorted to peroxisomal ghosts. In addition, we analyzed two mutants in which the peroxisomal location of the β‐oxidation machinery is in part disturbed. We analyzed Δpex7 cells that contain virtually normal peroxisomes, except that all matrix proteins that contain a peroxisomal targeting signal type 2 (PTS2, also including thiolase), are mislocalized to the cytosol. In Δpex5 cells, peroxisomes only contain matrix proteins with a PTS2 in conjunction with all proteins containing a peroxisomal targeting signal type 1 (PTS1, including all β‐oxidation enzymes except thiolase) are mislocalized to the cytosol. We show that intact peroxisomes are an important factor in yeast chronological aging because all pex mutants showed a reduced chronological lifespan. The strongest reduction was observed in Δpex5 cells. Our data indicate that this is related to the complete inactivation of the peroxisomal β‐oxidation pathway in these cells due to the mislocalization of thiolase. Our studies suggest that during chronological aging, peroxisomal β‐oxidation contributes to energy generation by the oxidation of fatty acids that are released by degradation of storage materials and recycled cellular components during carbon starvation conditions.