Differentiation-related changes in lipid classes with long-chain and very long-chain polyenoic fatty acids in rat spermatogenic cells

In rat seminiferous tubules (ST), cells that contain polar and neutral lipids with long-chain polyenoic fatty acids (PUFA) and sphingomyelins (SM) and ceramides (Cer) with very long chain (VLC) PUFA of the n-6 series coexist. In this study, pachytene spermatocytes and round spermatids were isolated to determine how these lipids change during spermatogenesis. As the amount per cell of PUFA-rich glycerophospholipids (GPL) decreased with cell size, the 22:5/20:4 ratio increased with cell differentiation. The elovl2 and elovl5 genes, required for 22:5 formation, were expressed (mRNA) in both cell types. Residual bodies- particles with compacted organelles and materials discarded from late spermatids-concentrated cholesterol, 22:5-rich triacylglycerols, and GPL, including plasmalogens and phosphatidylserine. Species of SM and Cer with nonhydroxylated (n-) VLCPUFA (28:4, 30:5, and 32:5) predominated in pachytene spermatocytes, whereas species with the corresponding 2-hydroxy (2-OH) VLCPUFA prevailed in round spermatids. Thus, a dramatic increase in the 2-OH/n-VLCPUFA ratio in SM and Cer was a hallmark of differentiation. A substantial decrease of 2-OH SM occurred between spermatids and mature spermatozoa and 2-OH SM species were collected in residual bodies “en route” to Sertoli cells. Notably, spermatids and spermatozoa gained a significant amount of ceramides devoid of n-VLCPUFA but having 2-OH VLCPUFA as their main fatty acids.     

Peroxisomes attenuate cytotoxicity of very long-chain fatty acids

One of the major functions of peroxisomes in mammals is oxidation of very long-chain fatty acids (VLCFAs). Genetic defects in peroxisomal β-oxidation result in the accumulation of VLCFAs and lead to a variety of health problems, such as demyelination of nervous tissues. However, the mechanisms by which VLCFAs cause tissue degeneration have not been fully elucidated. Recently, we found that the addition of small amounts of isopropanol can enhance the solubility of saturated VLCFAs in an aqueous medium. In this study, we characterized the biological effect of extracellular VLCFAs in peroxisome-deficient Chinese hamster ovary (CHO) cells, neural crest-derived pheochromocytoma cells (PC12), and immortalized adult Fischer rat Schwann cells (IFRS1) using this solubilizing technique. C20:0 FA was the most toxic of the C16–C26 FAs tested in all cells. The basis of the toxicity of C20:0 FA was apoptosis and was observed at 5 μM and 30 μM in peroxisome-deficient and wild-type CHO cells, respectively. The sensitivity of wild-type CHO cells to cytotoxic C20:0 FA was enhanced in the presence of a peroxisomal β-oxidation inhibitor. Further, a positive correlation was evident between cell toxicity and the extent of intracellular accumulation of toxic FA. These results suggest that peroxisomes are pivotal in the detoxification of apoptotic VLCFAs by preventing their accumulation.     

Interactions of very long-chain saturated fatty acids with serum albumin

The remarkable binding properties of serum albumin have been investigated extensively, but little is known about an important class of fatty acids, the very long-chain saturated fatty acids (VLCFA; >18 carbons). Although VLCFA are metabolized efficiently in normal individuals, they are markers for and possibly causative agents of several peroxisomal disorders. We studied the binding of [(13)C]carboxyl-enriched arachidic (C20:0), behenic (C22:0), lignoceric (C24:0), and hexacosanoic (C26:0) acids to bovine serum albumin (BSA) by (13)C-NMR spectroscopy. For each VLCFA, the NMR spectra showed multiple signals at chemical shifts previously identified for long-chain fatty acids (12-18 carbons), suggesting stabilization of binding by similar, if not identical, interactions of the fatty acid carboxyl anion with basic amino acid residues. The maximal binding (mol of VLCFA/mol of BSA) and the number of observed binding sites decreased with increasing chain length, from 4-5 for C20:0, 3-4 for C22:0, and 2 for C24:0; we validated our previous conclusion that BSA has only one site for C26:0 (Ho, J. K., H. Moser, Y. Kishimoto, and J. A. Hamilton. 1995. J. Clin. Invest. 96: 1455-1463). Analysis of chemical shifts suggested that the highest affinity sites for VLCFA are low affinity sites for long-chain fatty acids. In competition experiments with (13)C-labeled C22:0 (3 mol/mol of BSA) and unlabeled oleic acid, C22:0 bound to BSA in the presence of up to 4 mol of oleic acid/mol of BSA, but 1 mol was shifted into a different site. Our studies suggest that albumin has adequate binding capacity for the low plasma levels of VLCFA with 20 to 26 carbons, but the protein may not be able to bind longer chain VLCFA.     

Ceramides and sphingomyelins with high proportions of very long-chain polyunsaturated fatty acids in mammalian germ cells

Very long-chain polyunsaturated fatty acids (VLCPUFA) have previously been shown to be components of sphingomyelin (SM) of mammalian testis and spermatozoa. Here we examined the fatty acids of testicular ceramide (Cer) in comparison with those of SM in some mammals with a special focus on the rat testis. In bull, cat, dog, rabbit, mouse, and rat, VLCPUFA were found in both testicular lipids, Cer having a higher percentage of VLCPUFA than SM. Rat testis had the highest percentage of VLCPUFA in both lipids, the major ones being 28:4n-6 and 30:5n-6. VLCPUFA-containing SM and Cer occurred in cells located in the seminiferous tubules, where germ cells had a higher percentage of these species than Sertoli cells. Seminiferous tubule fractionation showed that SM and Cer of mitochondria and lysosomes had mostly saturates and negligible VLCPUFA, the latter being important in the SM and Cer of microsomes and other membrane fractions. VLCPUFA were absent from the SM and Cer of rat prepuberal testis, increased with the onset of spermatogenesis to account for nearly 15 and 40% of the total fatty acids of testicular SM and Cer, respectively, remained at those levels throughout the adult life of fertile rats and tended to decrease at advanced ages. Four conditions that lead to selective death of germ cells in vivo, namely experimental cryptorchidism, post-ischemic reperfusion, focalized x-ray irradiation and treatments with the antineoplasic drug doxorubicin, caused the VLCPUFA to disappear from the testicular SM and Cer of adult fertile rats, showing that these lipids are specific traits of spermatogenic cells.     

High contents of very long-chain polyunsaturated fatty acids in different moss species

Key message

Mosses have high contents of polyunsaturated fatty acids. Tissue-specific differences in fatty acid contents and fatty acid desaturase (FADS)-encoding gene expression exist. The arachidonic acid-synthesizing FADS operate in the ER.

Abstract

Polyunsaturated fatty acids (PUFAs) are important cellular compounds with manifold biological functions. Many PUFAs are essential for the human diet and beneficial for human health. In this study, we report on the high amounts of very long-chain (vl) PUFAs (≥C₂₀) such as arachidonic acid (AA) in seven moss species. These species were established in axenic in vitro culture, as a prerequisite for comparative metabolic studies under highly standardized laboratory conditions. In the model organism Physcomitrella patens, tissue-specific differences in the fatty acid compositions between the filamentous protonema and the leafy gametophores were observed. These metabolic differences correspond with differential gene expression of fatty acid desaturase (FADS)-encoding genes in both developmental stages, as determined via microarray analyses. Depending on the developmental stage and the species, AA amounts for 6–31 %, respectively, of the total fatty acids. Subcellular localization of the corresponding FADS revealed the endoplasmic reticulum as the cellular compartment for AA synthesis. Our results show that vlPUFAs are highly abundant metabolites in mosses. Standardized cultivation techniques using photobioreactors along with the availability of the P. patens genome sequence and the high rate of homologous recombination are the basis for targeted metabolic engineering in moss. The potential of producing vlPUFAs of interest from mosses will be highlighted as a promising area in plant biotechnology.     

Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration

Retinal long-chain PUFAs (LC-PUFAs, C₁₂-C₂₂) play important roles in normal human retinal function and visual development, and some epidemiological studies of LC-PUFA intake suggest a protective role against the incidence of advanced age-related macular degeneration (AMD). On the other hand, retinal very long-chain PUFAs (VLC-PUFAs, C_n>22) have received much less attention since their identification decades ago, due to their minor abundance and more difficult assays, but recent discoveries that defects in VLC-PUFA synthetic enzymes are associated with rare forms of inherited macular degenerations have refocused attention on their potential roles in retinal health and disease. We thus developed improved GC-MS methods to detect LC-PUFAs and VLC-PUFAs, and we then applied them to the study of their changes in ocular aging and AMD. With ocular aging, some VLC-PUFAs in retina and retinal pigment epithelium (RPE)/choroid peaked in middle age. Compared with age-matched normal donors, docosahexaenoic acid, adrenic acid, and some VLC-PUFAs in AMD retina and RPE/choroid were significantly decreased, whereas the ratio of n-6/n-3 PUFAs was significantly increased. All these findings suggest that deficiency of LC-PUFAs and VLC-PUFAs, and/or an imbalance of n-6/n-3 PUFAs, may be involved in AMD pathology.     

Genetic loci associated with circulating levels of very long-chain saturated fatty acids

Very long-chain saturated fatty acids (VLSFAs) are saturated fatty acids with 20 or more carbons. In contrast to the more abundant saturated fatty acids, such as palmitic acid, there is growing evidence that circulating VLSFAs may have beneficial biological properties. Whether genetic factors influence circulating levels of VLSFAs is not known. We investigated the association of common genetic variation with plasma phospholipid/erythrocyte levels of three VLSFAs by performing genome-wide association studies in seven population-based cohorts comprising 10,129 subjects of European ancestry. We observed associations of circulating VLSFA concentrations with common variants in two genes, serine palmitoyl-transferase long-chain base subunit 3 (SPTLC3), a gene involved in the rate-limiting step of de novo sphingolipid synthesis, and ceramide synthase 4 (CERS4). The SPTLC3 variant at rs680379 was associated with higher arachidic acid (20:0 , P = 5.81 × 10⁻¹³). The CERS4 variant at rs2100944 was associated with higher levels of 20:0 (P = 2.65 × 10⁻⁴⁰) and in analyses that adjusted for 20:0, with lower levels of behenic acid (P = 4.22 × 10⁻²⁶) and lignoceric acid (P = 3.20 × 10⁻²¹). These novel associations suggest an inter-relationship of circulating VLSFAs and sphingolipid synthesis.     

Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids

Very long-chain fatty acids (VLCFAs), fatty acids with chain-length greater than 20 carbons, possess a wide range of biological functions. However, their roles at the molecular level remain largely unknown. In the present study, we screened for multicopy suppressors that rescued temperature-sensitive growth of VLCFA-limited yeast cells, and we identified the VPS21 gene, encoding a Rab GTPase, as such a suppressor. When the vps21Δ mutation was introduced into a deletion mutant of the SUR4 gene, which encodes a VLCFA elongase, a synthetic growth defect was observed. Endosome-mediated vesicular trafficking pathways, including endocytosis and the carboxypeptidase Y (CPY) pathway, were severely impaired in sur4Δ vps21Δ double mutants, while the AP-3 pathway that bypasses the endosome was unaffected. In addition, the sur4Δ mutant also exhibited a synthetic growth defect when combined with the deletion of VPS3, which encodes a subunit of the class C core vacuole/endosome tethering (CORVET) complex that tethers transport vesicles to the late endosome/multivesicular body (MVB). These results suggest that, of all the intracellular trafficking pathways, requirement of VLCFAs is especially high in the endosomal pathways.     

The metabolism of long-chain fatty acids and alcohols byCandida tropicalis andSaccharomyces cerevisiae

The factors affecting the growth ofCandida tropicalis andSaccharomyces cerevisiae on medium- and long-chain fatty acids and alcohols in batch culture were investigated. Growth on solid acids and alcohols dispersed in the medium is a maximum for tetradecanoic acid and tetradecanol. The poorer growth observed on shorter chain lengths can be ascribed to their toxicity to the yeasts, whilst the fall off in growth on the higher members is explained by their increasing insolubility in the medium. When the longer-chain-length acids are dissolved in a non-metabolisable hydrocarbon, the growth ofC. tropicalis is improved, but that ofS. cerevisiae is unaffected. This suggests that acids can enter the cells of the former organism by direct contact with the hydrocarbon droplets. The surface ofS. cerevisiae is too hydrophilic for this transfer mechanism to be possible. Fatty acids dissolved in gas oil are utilized as substrates for the growth ofCandida tropicalis in competition with then-paraffins contained in the gas oil. Each fatty acid contributes to a constant proportion of yeast produced, but this proportion decreases as the chain is lengthened. Thus, in mixtures of gas oil with dodecanoic acid, 65% of the yeast is produced from metabolism of the acid, while with octadecanoic acid only 15% is produced. The log specific rates of utilization of the fatty acids within this range diminish linearly with increasing chain length.     

Changes in lipids containing long- and very long-chain polyunsaturated fatty acids in cryptorchid rat testes

The aim of the present study was to examine the effects of experimental cryptorchidism on rat testicular phospholipids and neutral lipids that contain long-chain (C(18)-C(22)) and very long-chain (VLC) (C(24)-C(32)) polyunsaturated fatty acids (PUFA). The weight of the cryptorchid testis was nearly half that of the contralateral control at postsurgical Days 7-10 owing to the depletion of germ cells. Concomitantly, the amounts of major glycerophospholipids (GPL) and sphingomyelin (SM) per testis decreased. Both these lipids lost their characteristic long-chain and very long-chain PUFA, notably 22:5n-6 and 28:4n-6, respectively, which suggests that these species are linked to the membranes of germ cells. In contrast, the amounts and concentrations of triglycerides (TG; triacylglycerols and 1-O-alkyl-2,3-diacylglycerols) and cholesterol esters (CE) increased several fold in the surviving cells (mainly Sertoli cells) in the cryptorchid testis. All these neutral lipids, but especially CE, accumulated large amounts of the major PUFA of the testis, 22:5n-6, as well as pentaenes with longer carbon chains (i.e., 24:5n-6 in TG and 28:5n-6 in CE). This accretion suggests that neutral lipids may store preformed PUFA coming from dying germ cell GPL and also VLCPUFA no longer needed as a source of PUFA destined to assemble new germ cell GPL. The lipid adjustments observed in cryptorchidism suggest a possible role for Sertoli cell CE in the turnover and conservation of PUFA within seminiferous tubules.     

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits. We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important omega-3 fatty acid in oilseed crops.     

Sequential depletion of rat testicular lipids with long-chain and very long-chain polyenoic fatty acids after X-ray-induced interruption of spermatogenesis

When a single dose of X-rays is applied to the adult rat testis, stem spermatogonia are damaged, and spermatogenesis is interrupted. Supported by Sertoli cells, spermatogenic cells that endure irradiation complete their differentiation and gradually leave the testis as spermatozoa. In this study, the in vivo changes taking place a number of weeks after irradiation revealed cell-specific features of testicular lipid classes. A linear drop, taking about six weeks, in testis weight, nonlipid materials, free cholesterol, and 22:5n-6-rich glycerophospholipids took place with germ cell depletion. Sphingomyelins and ceramides with nonhydroxy very long-chain polyenoic fatty acids (n-VLCPUFA) disappeared in four weeks, together with the last spermatocytes, whereas species with 2-hydroxy VLCPUFA lasted for six weeks, disappearing with the last spermatids and spermatozoa. The amount per testis of 22:5n-6-rich triacylglycerols, unchanged for four weeks, fell between weeks 4 and 6, associating these lipids with spermatids and their residual bodies, detected as small, bright lipid droplets. In contrast, 22:5n-6-rich species of cholesterol esters and large lipid droplets increased in seminiferous tubules up to week 6, revealing they are Sertoli cell products. At week 30, the lipid and fatty acid profiles reflected the resulting permanent testicular involution. Our data highlight the importance of Sertoli cells in maintaining lipid homeostasis during normal spermatogenesis.     

Gestational diabetes mellitus decreases placental uptake of long-chain polyunsaturated fatty acids: involvement of long-chain acyl-CoA synthetase

The long-chain polyunsaturated fatty acids (LC-PUFAs) arachidonic (AA) and docosahexaenoic (DHA) acids are essential for fetal development. Gestational diabetes mellitus (GDM) is a pregnancy disorder associated with perinatal and lifelong risk complications for both the mother and the newborn. Our aim was to investigate the influence of GDM, and some of its associated conditions, upon the placental uptake of AA and DHA. Uptake of ¹⁴C-AA and ¹⁴C-DHA by human trophoblasts obtained from normal pregnancies (NTB cells) was mediated by both saturable (for lower substrate concentrations) and non-saturable (for higher substrate concentrations) mechanisms. Uptake of both fatty acids was inhibited by other LC-PUFAs and, markedly, by the long-chain acyl-CoA synthetase (ACSL) inhibitor, triacsin C. Human trophoblasts obtained from GDM pregnancies (DTB cells) showed a significantly lower ¹⁴C-AA and ¹⁴C-DHA accumulation, through a decrease in both the saturable and the non-saturable components of uptake, which was associated with a decrease in ACSL1 mRNA levels. Uptake of LC-PUFAs by NTB cells increased (by 20–25%) after short-term exposure to TNF-α (¹⁴C-AA and ¹⁴C-DHA) and insulin (¹⁴C-DHA). In conclusion, GDM, distinctly from its associated conditions, markedly decreases placental uptake of LC-PUFAs, which probably contributes to the deleterious effects of this disease for the newborn.     

Hydroxy long-chain fatty acids in fungi

Hydroxy long-chain fatty acids occur widely in animals and plants and have important physiological activities in these eukaryotes. There are indications that these compounds are also common and important in fungi. The occurrence of hydroxy-polyunsaturated fatty acids (hydroxy-PUFAs) is of biotechnological importance, because these compounds are potentially high-value lipid products with medical applications. This review pays particular attention to the production of hydroxy-PUFAs by yeasts and other fungi. Hydroxy-PUFAs derived from lipoxygenase activity appear to be present in most fungi, while hydroxy-PUFAs from cyclooxygenase activity (i.e. prostaglandins) have mainly been implicated in the Oomycota and in yeasts from the genus Dipodascopsis. The occurrence of other hydroxy long-chain fatty acids in fungi is also discussed briefly; these include hydroxy fatty acids that are generally associated with cytochrome P-450 monooxygenase activity (i.e. terminal and sub-terminal hydroxy acids and diols derived from the corresponding epoxides) as well as 2-hydroxy-fatty acids and 3-hydroxy-fatty acids.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein, 9300, South Africa     

Increased levels of policosanol and very long-chain fatty acids in potato pulp fermented with Rhizopus oryzae

Significant amounts of policosanol and very long-chain fatty acids (VLFAs) ranging in carbon length from 22 to 30 were found in the lipophilic fraction obtained from potato pulp fermented with Rhizopus oryzae. It is believed that these compounds would have originally been present as suberin-related compounds, but not as wax, in the periderm of potato tubers and concentrated into potato pulp during the process of starch production. Moreover, the policosanol and VLFAs extracted from potato pulp with organic solvents were found to have increased after fermentation.     

Characterization of four mammalian 3-hydroxyacyl-CoA dehydratases involved in very long-chain fatty acid synthesis

Very long-chain fatty acids are produced through a four-step cycle. However, the 3-hydroxyacyl-CoA dehydratase catalyzing the third step in mammals has remained unidentified. Mammals have four candidates, HACD1-4, based on sequence similarities to the recently identified yeast Phs1, although HACD3 and HACD4 share relatively weak similarity. We demonstrate that all four of these human proteins are indeed 3-hydroxyacyl-CoA dehydratases, in growth suppression experiments using a PHS1-shut off yeast strain and/or in vitro 3-hydroxypalmitoyl-CoA dehydratase assays. HACD proteins exhibit distinct tissue-expression patterns. We also establish that HACD proteins interact with the condensation enzymes ELOVL1-7, with some preferences.     

Omega-oxidation of very long-chain fatty acids in human liver microsomes. Implications for X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder biochemically characterized by elevated levels of very long-chain fatty acids (VLCFA). Excess levels of VLCFAs are thought to play an important role in the pathogenesis of X-ALD. Therefore, therapeutic approaches for X-ALD are focused on the reduction or normalization of VLCFAs. In this study, we investigated an alternative oxidation route for VLCFAs, namely omega-oxidation. The results described in this study show that VLCFAs are substrates for the omega-oxidation system in human liver microsomes. Moreover, VLCFAs were not only converted into omega-hydroxy fatty acids, but they were also further oxidized to dicarboxylic acids via cytochrome P450-mediated reactions. High sensitivity toward the specific P450 inhibitor 17-octadecynoic acid suggested that omega-hydroxylation of VLCFAs is catalyzed by P450 enzymes belonging to the CYP4A/F subfamilies. Studies with individually expressed human recombinant P450 enzymes revealed that two P450 enzymes, i.e. CYP4F2 and CYP4F3B, participate in the omega-hydroxylation of VLCFAs. Both enzymes belong to the cytochrome P450 4F subfamily and have a high affinity for VLCFAs. In summary, this study demonstrates that VLCFAs are substrates for the human omega-oxidation system, and for this reason, stimulation of the in vivo VLCFA omega-oxidation pathway may provide an alternative mode of treatment to reduce the levels of VLCFAs in patients with X-ALD.