Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice

The transmembrane protein CD36 has been identified in isolated cell studies as a putative transporter of long chain fatty acids. In humans, an association between CD36 deficiency and defective myocardial uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R, S)-methyl pentadecanoic acid (BMIPP) has been reported. To determine whether this association represents a causal link and to assess the physiological role of CD36, we compared tissue uptake and metabolism of two iodinated fatty acid analogs BMIPP and 15-(p-iodophenyl) pentadecanoic acid (IPPA) in CD36 null and wild type mice. We also investigated the uptake and lipid incorporation of palmitate by adipocytes isolated from both groups. Compared with wild type, uptake of BMIPP and IPPA was reduced in heart (50-80%), skeletal muscle (40-75%), and adipose tissues (60-70%) of null mice. The reduction was associated with a 50-68% decrease in label incorporation into triglycerides and in 2-3-fold accumulation of label in diglycerides. Identical results were obtained from studies of [(3)H]palmitate uptake in isolated adipocytes. The block in diglyceride to triglyceride conversion could not be explained by changes in specific activities of the key enzymes long chain acyl-CoA synthetase and diacylglycerol acyltransferase, which were similar in tissues from wild type and null mice. It is concluded that CD36 facilitates a large fraction of fatty acid uptake by heart, skeletal muscle, and adipose tissues and that CD36 deficiency in humans is the cause of the reported defect in myocardial BMIPP uptake. In CD36-expressing tissues, uptake regulates fatty acid esterification at the level of diacylglycerol acyltransferase by determining fatty acyl-CoA supply. The membrane transport step may represent an important control site for fatty acid metabolism in vivo.     

Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake

Sulfo-N-succinimidyl esters of LCFAs are a powerful tool to investigate the functional significance of plasmalemmal proteins in the LCFA uptake process. This notion is based on the following observations. First, sulfo-N-succinimidyl oleate (SSO) was found to inhibit the bulk of LCFA uptake into various cell types, i.e. rat adipocytes, type II pneumocytes and cardiac myocytes. Second, using cardiac giant membrane vesicles, in which LCFA uptake can be investigated in the absence of mitochondrial -oxidation, SSO retained the ability to largely inhibit LCFA uptake, indicating that inhibition of LCFA transsarcolemmal transport is its primary action. Third, SSO has no inhibitory effect on glucose and octanoate uptake into giant membrane vesicles derived from heart and skeletal muscle, indicating that its action is specific for LCFA uptake. Finally, SSO specifically binds to the 88 kDa plasmalemmal fatty acid transporter FAT, a rat homologue of human CD36, resulting in an arrest of the transport function of this protein.In addition to its inhibitory action at the plasma membrane level, evidence is presented for the lack of a direct inhibitory effect on subsequent LCFA metabolism. First, the relative contribution of oxidation and esterification to LCFA uptake is not altered in the presence of SSO. Second, isoproterenol-mediated channeling of LCFAs into oxidative pathways is not affected by sulfo-N-succinimidyl palmitate (SSP). As an example of its application we used SSP to study the role of FAT/CD36 in contraction- and insulin-stimulated LCFA uptake by cardiac myocytes , showing that this transporter is a primary site of regulation of cellular LCFA utilization.     

Mechanisms of cellular uptake of long chain free fatty acids

Cells take up long chain free fatty acids (FFA) in vivo from the non-protein bound ligand pools in extracellular fluid and plasma, which contain ~100 and 600 M albumin, respectively. The physiologic range of unbound FFA concentrations in such fluids has traditionally been calculated at < 1 M. Studies of [3H]-oleate uptake by hepatocytes, adipocytes, cardiac myocytes and other cell types demonstrate that FFA uptake within this range is saturable, and exhibits many other kinetic properties indicative of facilitated transport. Within this range, the uptake kinetics of the acidic (pKa = 0.5) FFA analog 2,2,3- heptafluorostearate are similar to those of stearate. Thus, uptake of physiologic concentrations of FFA involves facilitated transport of the FFA anion (FA-). Over a much wider range of unbound FFA concentrations hepatocellular [3H]-oleate uptake exhibits both saturable and non-saturable components. Oleate binding to liver plasma membranes (LPM) also demonstrates such components. Comparing the two components of FFA uptake to the corresponding components of binding permits estimates of trans-membrane transport rates. T1/2 for saturable uptake (~ 1 sec) is less than for non-saturable uptake (~ 14 sec). Others have determined the flip-flop rates of protonated FFA (FAH) across small and large unilamellar vesicles (SUV, LUV) and across cellular plasma membranes. These reported flip-flop rates, measured by the decrease in pH resulting from the accompanying proton flux, exhibit a highly significant inverse correlation with cell and vesicle diameter (r = 0.99). Although T1/2's in vesicles are in the msec range, those in cells are > 10 sec, and thus comparable to the rates of non-saturable uptake we determined. Thus, under physiologic conditions, the predominant mechanism of cellular FFA uptake is facilitated transport of FA-; at much higher, non- physiologic FFA concentrations, passive flip-flop of FAH predominates. Several plasma membrane proteins have been identified as potential mediators of facilitated FFA transport. Studies in animal models of obesity and non-insulin dependent diabetes mellitus demonstrate that tissue-specific regulation of facilitated FFA transport has important pathophysiologic consequences.     

The cellular localization of long chain fatty acids in sponges

Examination of fractionated sponge tissue shows that long chain fatty acids (LCFAs) occur in high proportions in cell membranes. This conclusion refutes a recent suggestion made by other workers that sponge membranes would contain conventional fatty acids similar to those found in membranes from other organisms.     

Cellular uptake and intracellular trafficking of long chain fatty acids.

While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues.     

The function of very long chain polyunsaturated fatty acids in the pineal gland

The mammalian pineal gland is a prominent secretory organ with a high metabolic activity. Melatonin (N-acetyl-5-methoxytryptamine), the main secretory product of the pineal gland, efficiently scavenges both the hydroxyl and peroxyl radicals counteracting lipid peroxidation in biological membranes. Approximately 25% of the total fatty acids present in the rat pineal lipids are represented by arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3). These very long chain polyunsaturated fatty acids play important roles in the pineal gland. In addition to the production of melatonin, the mammalian pineal gland is able of convert these polyunsaturated fatty acids into bioactive lipid mediators. Lipoxygenation is the principal lipoxygenase (LOX) activity observed in the rat pineal gland. Lipoxygenation in the pineal gland is exceptional because no other brain regions express significant LOX activities under normal physiological conditions. The rat pineal gland expresses both 12- and 15-lipoxygenase (LOX) activities, producing 12- and 15-hydroperoxyeicosatetraenoic acid (12- and 15-HpETE) from arachidonic acid and 14- and 17-hydroxydocosahexaenoic acid (14- and 17-HdoHE) from docosahexaenoic acid, respectively. The rat pineal also produces hepoxilins via LOX pathways. The hepoxilins are bioactive epoxy-hydroxy products of the arachidonic acid metabolism via the 12S-lipoxygenase (12S-LOX) pathway. The two key pineal biochemical functions, lipoxygenation and melatonin synthesis, may be synergistically regulated by the status of n-3 essential fatty acids.     

Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].     

Synthesis of very long chain (up to 36 carbon) tetra, penta and hexaenoic fatty acids in retina.

The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) fatty acids (VLCPUFA) is investigated in bovine retina using [14C]acetate. Saturates on the one hand (mainly palmitate), and polyenes on the other (mainly VLCPUFA), incorporate most of the label found in lipids. Phosphatidylcholine (PC) is the most highly labelled lipid class, since both types of 14C-labelled fatty acids, but especially this novel series of VLCPUFA, are concentrated in this phospholipid. Radioactivity from [14C]acetate is found in very long chain tetra, penta and hexaenoic fatty acids of PC. The labelling of 20:4(n - 6), 20:5(n - 3), 22:5(n - 6) and 22:6(n - 3) is much lower than that of longer polyenes of each of these series, indicating that VLCPUFA are synthesized in situ by successive elongations of the above polyenes, pre-existing in retina lipids. In various subcellular fractions isolated from retinas after incubations with [14C]acetate (including cytosol, microsomes, mitochondria and photoreceptor membranes), the labelling of the VLCPUFA of PC is very high, even at relatively short intervals of incubation. The results suggest that not only the synthesis but also the intracellular traffic among membranes of VLCPUFA-containing species of PC are very active processes in the retina.     

Yeast sphingolipids do not need to contain very long chain fatty acids

Synthesis of VLCFAs (very long chain fatty acids) and biosynthesis of DHS (dihydrosphingosine) both are of vital importance for Saccharomyces cerevisiae. The bulk of VLCFAs and DHS are used for ceramide synthesis by the Lag1p (longevity-assurance gene 1)/Lac1p (longevity-assurance gene cognate 1)/Lip1p (Lag1p/Lac1p interacting protein) ceramide synthase. LAG1 and LAC1 are redundant but LIP1 is essential. Here we show that 4Delta (lag1Deltalac1Deltaypc1Deltaydc1Delta) cells devoid of all known endogenous ceramide synthesis pathways are unviable but can be rescued by the expression of Lass5, a mouse LAG1 homologue. Ceramide synthase activity of 4Delta.Lass5 cells only utilizes C16 and C18 fatty acids and does not require the help of Lip1p, an essential cofactor of Lag1p/Lac1p. HPLC-electrospray ionization-MS/MS analysis demonstrated that in IPCs (inositolphosphorylceramides) of 4Delta.Lass5, the very long chain fatty acids (C26 and C24) account for <1% instead of the normal >97%. Notwithstanding, IPCs incorporated into glycosylphosphatidylinositol anchors of 4Delta.Lass5 show normal mobility on TLC and the ceramide- and raft-dependent traffic of Gas1p (glycophospholipid-anchored surface protein) from endoplasmic reticulum to Golgi remains almost normal. Moreover, the biosynthesis of C24:0 fatty acids remains essential. Thus, C(24:0) and dihydrosphingosine are both necessary for survival of yeast cells even if they utilize C16 and C18 fatty acids for sphingolipid biosynthesis.     

Peroxisomal disorders: complementation analysis using beta-oxidation of very long chain fatty acids

Complementation studies, using fused cell lines from patients with peroxisomal disorders, have shown correction of defective plasmalogen synthesis and phytanic acid oxidation as well as an increase in the number of peroxisomes. At least six complementation groups have been reported. We demonstrate here that complementing cell lines also acquire the ability to oxidize very long chain fatty acids (VLCFA), and that complementation groups defined with this technique are identical to those reported previously when plasmalogen synthesis was used as the criterion for complementation. This VLCFA complementation technique is of particular value in the study of patients in whom defective VLCFA is the only or major enzymatic defect, and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal fatty acid beta-oxidation enzymes.     

Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.     

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.     

Cholesteryl esters as a depot for very long chain fatty acids in human meibum

Human meibomian gland secretions (also known as meibum) were analyzed for the presence of cholesteryl esters (Chl-E) using HPLC in combination with atmospheric pressure chemical ionization mass spectrometry. A special procedure based on detection of the in-source generated ion m/z 369 was developed to monitor all Chl-E simultaneously. The structures of the detected compounds were studied using in-source and postsource fragmentation of the precursor (M+H)(+) ions. In concordance with previous studies, Chl-E were found in all of the tested samples and comprised approximately 31% of the entire lipid pool (w/w, dry weight). There were at least 20 different saturated and unsaturated Chl-E species observed, whose fatty acid residues ranged from C18 to C34. Monounsaturated fatty acids were the most visible components of the Chl-E pool. The eleven most prominent compounds were: C20:0-, C22:1-, C22:0-, C24:1-, C24:0, C25:0-, C26:1-, C26:0-, C28:1-, C28:0-, and C30:1-Chl-E. Other Chl-containing compounds were detected but not identified at the time. Therefore, Chl-E are a depot for very long chain saturated and monounsaturated fatty acids in human meibum.     

Nutritional significance and metabolism of very long chain fatty alcohols and acids from dietary waxes

Very long chain fatty alcohols obtained from plant waxes and beeswax have been reported to lower plasma cholesterol in humans. This review discusses nutritional or regulatory effects produced by wax esters or aliphatic acids and alcohols found in unrefined cereal grains, beeswax, and many plant-derived foods. Reports suggest that 5-20 mg per day of mixed C24-C34 alcohols, including octacosanol and triacontanol, lower low-density lipoprotein (LDL) cholesterol by 21%-29% and raise high-density lipoprotein cholesterol by 8%-15%. Wax esters are hydrolyzed by a bile salt-dependent pancreatic carboxyl esterase, releasing long chain alcohols and fatty acids that are absorbed in the gastrointestinal tract. Studies of fatty alcohol metabolism in fibroblasts suggest that very long chain fatty alcohols, fatty aldehydes, and fatty acids are reversibly inter-converted in a fatty alcohol cycle. The metabolism of these compounds is impaired in several inherited human peroxisomal disorders, including adrenoleukodystrophy and Sj?gren-Larsson syndrome. Reports on dietary management of these diseases confirm that very long chain fatty acids (VLCFA) are normal constituents of the human diet and are synthesized endogenously. Concentrations of VLCFA in blood plasma increase during fasting and when children are placed on ketogenic diets to suppress seizures. Existing data support the hypothesis that VLCFA exert regulatory roles in cholesterol metabolism in the peroxisome and also alter LDL uptake and metabolism.     

Biosynthesis of very long chain monounsaturated fatty acids by subcellular fractions of developing seeds

Subcellular fractions from developing seeds of mustard (Sinapis alba), honesty (Lunaria annua) and nasturtium (Tropaeolum majus) synthesize very long chain cis (n−9) monounsaturated fatty acids, e.g. gadoleic (20:1), erucic (22:1) and nervonic (24:1) acid, from oleoyl-CoA and malonyl-CoA by condensation reactions. The particulate 2000 × g and 15 000 × g fractions exhibit considerably higher elongase activities compared to the microsomal or oil body fractions, whereas the soluble (150 000 × g supernatant) fraction is devoid of such activities.