Stearate-derived very long-chain fatty acids are indispensable to tumor growth.

Reprogramming of lipid metabolism is emerging as a hallmark of cancer, yet involvement of specific fatty acids (FA) species and related enzymes in tumorigenesis remains unclear. While previous studies have focused on involvement of long-chain fatty acids (LCFAs) including palmitate in cancer, little attention has been paid to the role of very long-chain fatty acids (VLCFAs). Here, we show that depletion of acetyl-CoA carboxylase (ACC1), a critical enzyme involved in the biosynthesis of fatty acids, inhibits both de novo synthesis and elongation of VLCFAs in human cancer cells. ACC1 depletion markedly reduces cellular VLCFA but only marginally influences LCFA levels, including palmitate that can be nutritionally available. Therefore, tumor growth is specifically susceptible to regulation of VLCFAs. We further demonstrate that VLCFA deficiency results in a significant decrease in ceramides as well as downstream glucosylceramides and sphingomyelins, which impairs mitochondrial morphology and renders cancer cells sensitive to oxidative stress and cell death. Taken together, our study highlights that VLCFAs are selectively required for cancer cell survival and reveals a potential strategy to suppress tumor growth.

Destabilization of ß Cell FIT2 by saturated fatty acids alter lipid droplet numbers and contribute to ER stress and diabetes.

SignificanceWith obesity on the rise, there is a growing appreciation for intracellular lipid droplet (LD) regulation. Here, we show how saturated fatty acids (SFAs) reduce fat storage-inducing transmembrane protein 2 (FIT2)-facilitated, pancreatic ß cell LD biogenesis, which in turn induces ß cell dysfunction and death, leading to diabetes. This mechanism involves direct acylation of FIT2 cysteine residues, which then marks the FIT2 protein for endoplasmic reticulum (ER)-associated degradation. Loss of ß cell FIT2 and LDs reduces insulin secretion, increases intracellular ceramides, stimulates ER stress, and exacerbates diet-induced diabetes in mice. While palmitate and stearate degrade FIT2, unsaturated fatty acids such as palmitoleate and oleate do not, results of which extend to nutrition and diabetes.

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.

Stearate-to-palmitate ratio modulates endoplasmic reticulum stress and cell apoptosis in non-B non-C hepatoma cells.

The increased prevalence of hepatocellular carcinoma (HCC) without viral infection, namely, NHCC, is a major public health issue worldwide. NHCC is frequently derived from non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis, which exhibit dysregulated fatty acid (FA) metabolism. This raises the possibility that NHCC evolves intracellular machineries to adapt to dysregulated FA metabolism. We herein aim to identify NHCC-specifically altered FA and key molecules to achieve the adaptation. To analyze FA, imaging mass spectrometry (IMS) was performed on 15 HCC specimens. The composition of saturated FA (SFA) in NHCC was altered from that in typical HCC. The stearate-to-palmitate ratio (SPR) was significantly increased in NHCC. Associated with the SPR increase, the ELOVL6 protein level was upregulated in NHCC. The knockdown of ELOVL6 reduced SPR, and enhanced endoplasmic reticulum stress, inducing apoptosis of Huh7 and HepG2 cells. In conclusion, NHCC appears to adapt to an FA-rich environment by modulating SPR through ELOVL6.

Good, Bad, or Ugly: the Biological Roles of Bone Marrow Fat.

PURPOSE OF REVIEW: Bone marrow fat expresses mixed characteristics, which could correspond to white, brown, and beige types of fat. Marrow fat could act as either energy storing and adipokine secreting white fat or as a source of energy for hematopoiesis and bone metabolism, thus acting as brown fat. However, there is also a negative interaction between marrow fat and other elements of the bone marrow milieu, which is known as lipotoxicity. In this review, we will describe the good and bad roles of marrow fat in the bone, while focusing on the specific components of the negative effect of marrow fat on bone metabolism. RECENT FINDINGS: Lipotoxicity in the bone is exerted by bone marrow fat through the secretion of adipokines and free fatty acids (FFA) (predominantly palmitate). High levels of FFA found in the bone marrow of aged and osteoporotic bone are associated with decreased osteoblastogenesis and bone formation, decreased hematopoiesis, and increased osteoclastogenesis. In addition, FFA such as palmitate and stearate induce apoptosis and dysfunctional autophagy in the osteoblasts, thus affecting their differentiation and function. Regulation of marrow fat could become a therapeutic target for osteoporosis. Inhibition of the synthesis of FFA by marrow fat could facilitate osteoblastogenesis and bone formation while affecting osteoclastogenesis. However, further studies testing this hypothesis are still required.

Site-specific S-acylation of influenza virus hemagglutinin: the location of the acylation site relative to the membrane border is the decisive factor for attachment of stearate.

S-Acylation of hemagglutinin (HA), the main glycoprotein of influenza viruses, is an essential modification required for virus replication. Using mass spectrometry, we have previously demonstrated specific attachment of acyl chains to individual acylation sites. Whereas the two cysteines in the cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to a cysteine positioned at the end of the transmembrane region (TMR). Here we analyzed recombinant viruses containing HA with exchange of conserved amino acids adjacent to acylation sites or with a TMR cysteine shifted to a cytoplasmic location to identify the molecular signal that determines preferential attachment of stearate. We first developed a new protocol for sample preparation that requires less material and might thus also be suitable to analyze cellular proteins. We observed cell type-specific differences in the fatty acid pattern of HA: more stearate was attached if human viruses were grown in mammalian compared with avian cells. No underacylated peptides were detected in the mass spectra, and even mutations that prevented generation of infectious virus particles did not abolish acylation of expressed HA as demonstrated by metabolic labeling experiments with [(3)H]palmitate. Exchange of conserved amino acids in the vicinity of an acylation site had a moderate effect on the stearate content. In contrast, shifting the TMR cysteine to a cytoplasmic location virtually eliminated attachment of stearate. Thus, the location of an acylation site relative to the transmembrane span is the main signal for stearate attachment, but the sequence context and the cell type modulate the fatty acid pattern.

Elaidate, an 18-carbon trans-monoenoic fatty acid, inhibits ß-oxidation in human peripheral blood macrophages.

Consumption of trans-unsaturated fatty acids promotes atherosclerosis, but whether degradation of fats in macrophages is altered by trans-unsaturated fatty acids is unknown. We compared the metabolism of oleate (C18:1Δ9-10 cis; (Z)-octadec-9-enoate), elaidate (C18:Δ9-10 trans; (E)-octadec-9-enoate), and stearate (C18:0, octadecanoate) in adherent peripheral human macrophages. Metabolism was followed by measurement of acylcarnitines in cell supernatants by MS/MS, determination of cellular fatty acid content by GC/MS, and assessment of ß-oxidation rates using radiolabeled fatty acids. Cells incubated for 44 h in 100 µM elaidate accumulated more unsaturated fatty acids, including both longer- and shorter-chain, and had reduced C18:0 relative to those incubated with oleate or stearate. Both C12:1 and C18:1 acylcarnitines accumulated in supernatants of macrophages exposed to trans fats. These results suggested ß-oxidation inhibition one reaction proximal to the trans bond. Comparison of [1-(14)C]oleate to [1-(14)C]elaidate catabolism showed that elaidate completed the first round of fatty acid ß-oxidation at rates comparable to oleate. Yet, in competitive ß-oxidation assays with [9,10-(3)H]oleate, tritium release rate decreased when unlabeled oleate was replaced by the same quantity of elaidate. These data show specific inhibition of monoenoic fat catabolism by elaidate that is not shared by other atherogenic fats.

Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis.

Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH(2)-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.

Highly regioselective enzymatic synthesis of 5'-O-stearate of 1-beta-D-arabinofuranosylcytosine in binary organic solvent mixtures.

In this paper, highly regioselective enzymatic acylations of 1-beta-D-arabinofuranosylcytosine (ara-C) with vinyl stearate (VS) in binary organic solvents were explored for the preparation of 5'-O-stearate of ara-C with potential antitumor activity. Twelve kinds of hydrolases were tested for the regioselective acylation reaction and the immobilized Candida antarctica lipase B (Novozym 435) showed the highest regioselectivity (>99.9%) towards the 5'-OH of ara-C. A comparative study showed that the lipase had much higher catalytic activity in the binary mixture of hexane and pyridine than in other tested co-solvent systems. To better understand lipase-mediated acylation conducted in the best binary organic solvent system, the effects of hydrophobic solvent content, molar ratio of VS to ara-C, initial water activity, and reaction temperature on the acylation reaction were studied. It was found that the most suitable hexane content, VS-ara-C molar ratio, initial water activity, and reaction temperature were shown to be 25% (v/v), 20:1, 0.07, and 50 degrees C, respectively. Under these reaction conditions, the initial reaction rate, the maximum substrate conversion, and regioselectivity were as high as 86.0 mmol.L(-1)h(-1), 96.6%, and >99.9%, respectively. The product of Novozym 435-catalyzed acylation was characterized by Carbon-13((13)C) NMR and confirmed to be 5'-O-stearate of ara-C.

[Stearic acid methyl ether: a new extracellular metabolite of the obligate methylotrophic bacterium Methylophilus quaylei].

Methyl esters of fatty acids, free fatty acids, and hydrocarbons were found in the culture liquid and in the cellular lipids of the obligate methylotrophic bacterium Methylophilus quaylei under optimal growth conditions and osmotic stress. The main extracellular hydrophobic metabolite was methyl stearate. Exogenous free fatty acids C16-C18 and their methyl esters stimulated the M. quaylei growth and survivability, as well as production of exopolysaccharide under osmotic and oxidative stress, playing the role of growth factors and adaptogens. The order of hydrophobic supplements according to the ability to stimulate bacterial growth is C18 : 1 > C18 : 0 > C16 : 0 > methyl oleate > methyl stearate > no supplements > C14: 0 > C12 : 0. The mechanism underlying the protective action of fatty acids and their methyl esters is discussed.

Early hepatic insulin resistance in mice: a metabolomics analysis.

When fed with a high-fat safflower oil diet for 3 wk, wild-type mice develop hepatic insulin resistance, whereas mice lacking glycerol-3-phosphate acyltransferase-1 retain insulin sensitivity. We examined early changes in the development of insulin resistance via liver and plasma metabolome analyses that compared wild-type and glycerol-3-phosphate acyltransferase-deficient mice fed with either a low-fat or the safflower oil diet for 3 wk. We reasoned that diet-induced changes in metabolites that occurred only in the wild-type mice would reflect those metabolites that were specifically related to hepatic insulin resistance. Of the identifiable metabolites (from 322 metabolites) in liver, wild-type mice fed with the high-fat diet had increases in urea cycle intermediates, consistent with increased deamination of amino acids used for gluconeogenesis. Also increased were stearoylglycerol, gluconate, glucarate, 2-deoxyuridine, and pantothenate. Decreases were observed in S-adenosylhomocysteine, lactate, the bile acid taurocholate, and 1,5-anhydroglucitol, a previously identified marker of short-term glycemic control. Of the identifiable metabolites (from 258 metabolites) in plasma, wild-type mice fed with the high-fat diet had increases in plasma stearate and two pyrimidine-related metabolites, whereas decreases were found in plasma bradykinin, alpha-ketoglutarate, taurocholate, and the tryptophan metabolite, kynurenine. This study identified metabolites previously not known to be associated with insulin resistance and points to the utility of metabolomics analysis in identifying unrecognized biochemical pathways that may be important in understanding the pathophysiology of diabetes.

Site-specific attachment of palmitate or stearate to cytoplasmic versus transmembrane cysteines is a common feature of viral spike proteins.

Many glycoproteins of enveloped viruses are known to be "palmitoylated" at cysteines located either in the transmembrane region or in the cytoplasmic tail. Although it was recognized early on that "palmitoylation" is not specific for this carbon chain, the exact fatty acid composition of S-acylated proteins has been difficult to determine. Advancements in mass-spectrometry (MS) now allow one to quantify the fatty acids linked to single acylation sites. We report that G of Vesicular Stomatitis virus contains palmitate at a cytoplasmic cysteine, whereas F of Newcastle Disease virus and E1 of Semliki Forest virus (SFV) are stoichiometrically acylated with stearate at a transmembrane cysteine. E2 of SFV contains three molecules of palmitate and one molecule of stearate, the latter probably attached to a transmembrane cysteine. Thus, site-specific attachment of palmitate or stearate, previously described only for HA of influenza virus, is a common feature of viral spike proteins.

Lipase catalyzed interesterification of palm stearin and rice bran oil blends for preparation of zero trans shortening with bioactive phytochemicals.

Palm stearin (PS) and rice bran oil (RBO) blends of varying proportions were subjected to enzymatic interesterification (EIE) using a 1,3 specific immobilized lipase. The interesterified blends were evaluated for their physicochemical characteristics and bioactive phytochemical contents using differential scanning calorimeter (DSC), X-ray diffraction (XRD), gas chromatography (GC) and high performance liquid chromatography (HPLC). The blends of PS and RBO in different proportions (40:60, 50:50, 60:40 and 70:30) had saturated fatty acid content and unsaturated fatty acid content in the range of 37.6-52.0% and 48.0-62.4%, respectively. The blends 40:60, 50:50 and 60:40 showed a considerable reduction in their highest melt peak temperature (TP) and solid fat content (SFC) on EIE. The interesterified blends retained bioactive phytochemicals like tocols (839-1172 ppm), sterols (4318-9647 ppm), oryzanols (3000-6800ppm) and carotene (121-180 ppm). XRD studies demonstrated that the interesterified blends contained beta and beta' polymorphic forms.

Characterization of long-chain fatty-acid-degrading syntrophic associations from a biodegraded oil reservoir.

Molecular methods were used to characterize stearate- and heptadecanoate-degrading methanogenic consortia enriched from a low-temperature biodegraded oil field. Stearate- and heptadecanoate-degrading cultures formed acetate. Growth on heptadecanoate was also accompanied by the production of propionate. These fermentation products were transiently accumulated at the beginning of the exponential phase and were further consumed with the concomitant production of methane. Clone libraries of bacterial and archaeal 16S rRNA genes were generated for each stable enrichment. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant microorganisms in the associations were affiliated with a clone cluster close to the genus Syntrophus in the class "Deltaproteobacteria" and with the methanogenic genera Methanocalculus and Methanosaeta. Confocal scanning laser microscopy showed that the bacterial and archaeal cells formed compact aggregates around the insoluble substrates. No layered structure was observed in the aggregate organization. This study reports the presence of new fatty-acid-degrading syntrophic consortia in oil fields and our results suggest that such associations may have an important ecological role in oil fields under methanogenic conditions.

Long-chain saturated fatty acids induce annexin II translocation to detergent-resistant membranes.

DRM (detergent-resistant membranes), which are resistant to solublization by non-ionic detergents, have been demonstrated to be involved in many key cell functions such as signal transduction, endocytosis and cholesterol trafficking. Covalent modification of proteins by fatty acylation has been proposed to be an important protein-targeting mechanism for DRM association. However, little is known concerning the effects of LCSFA (long-chain saturated fatty acids) on protein composition of DRM in human cancer cells. In the present study, we found that, in Hs578T human breast cancer cells, the major protein increased in DRM in response to the LCSFA stearate (C18:0) was annexin II. Our results demonstrated that annexin II accumulated in DRM specifically in response to physiological concentrations of stearate and palmitate (C16:0), but not long-chain unsaturated fatty acids, in a time- and concentration-dependent manner. This process was reversible and dependent on cholesterol and intracellular calcium. Although calcium was necessary for this translocation, it was not sufficient to induce the annexin II translocation to DRM. We also demonstrate that stearate induced the acylation of caveolin but not that of annexin II. Association of annexin II with caveolin, although not necessarily direct, specifically occurs in DRM in response to stearate. Finally, bromostearate, a stearate analogue that effectively blocks protein acylation, does not induce annexin II translocation to DRM. We conclude that exogenously added LCSFA strongly induces the translocation of annexin II to DRM in Hs578T human breast cancer cells at least partially by association with acylated caveolin.

Arabidopsis mutants affected in the transcriptional control of allene oxide synthase, the enzyme catalyzing the entrance step in octadecanoid biosynthesis.

Allene oxide synthase (AOS) catalyzes the entrance reaction in the biosynthesis of the octadecanoids 12-oxophytodienoic acid (OPDA) and jasmonic acid (JA). The enzyme is feedback-regulated by JA and thus a target of the JA-signalling pathway. A fusion genetic approach was used to isolate mutants in this signalling pathway. Seeds from transgenic Arabidopsis thaliana plants expressing the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) under the control of the AOS promoter were mutagenized with ethylmethane sulfonate and the progeny was screened for individuals exhibiting constitutive expression of uidA in the absence of an added octadecanoid. From 21,000 mutagenized plants, 8 lines showing constitutive AOS expression were obtained. The mutant lines were characterized further and fell into four classes, I to IV. All showed signs of growth inhibition encompassing both shoot and root systems, and accumulated higher than normal levels of OPDA. Mutants belonging to classes I and IV failed to set seeds due to defects in flower development which prevented self-pollination. One mutant, designated cas1, was characterized in more detail and showed, in addition to elevated levels of AOS mRNA, AOS polypeptide, OPDA, and JA, constitutive expression of JA-responsive genes ( VSP2, PDF1.2). The cas1 mutation is recessive and affects a single locus. Using cleaved amplified polymorphic sequences (CAPS) and simple sequence length polymorphisms (SSLP), the mutated gene was mapped to chromosome IV next to the SSLP marker CIW7.

Long-chain fatty acid uptake and oxidation in the perfused liver of Walker-256 tumour-bearing rats.

AIMS/BACKGROUND: The effect of the Walker-256 tumour on uptake and oxidation of long-chain fatty acids was investigated in perfused livers of rats. METHODS: Isolated livers were perfused in a non-recirculating system. Fatty acid uptake, ketogenesis, oxygen uptake and 14CO2-production were measured as well as the activities of the acyl carnitine transferases I and II (CAT I and CAT II). RESULTS: Basal oxygen uptake of livers from tumour-bearing rats was lower. Ketone bodies production derived from the long-chain fatty acids in livers from starved tumour-bearing rats was lower relative to the controls, but 14CO2 production was similar in both groups. The oxygen uptake increment and the mitochondrial NADH/NAD+ redox ratio were also decreased in tumour-bearing rats. The extent of these differences was dependent on the chain length and structure of the fatty acid, the following decreasing sequence of differences between control and tumour-bearing animals being valid: palmitate > oleate > stearate. The CAT I activity of the enzyme isolated from livers of tumour-bearing rats was half that from normal rats when palmitoyl-CoA and oleoyl-CoA were the substrates. CONCLUSIONS: Ketogenesis from exogenous fatty acids is decreased in the livers of Walker-256 tumour-bearing rats in consequence of the diminished activity of the mitochondrial CAT I. The lower rates of oxygen uptake indicate a decreased ATP synthesis, which is consistent with the in vivo lower phosphorylation potential.

The peroxisomal proliferator clofibrate enhances the hepatic cytoplasmic movement of fatty acids in rats.

The role of cytosolic fatty acid binding protein (FABP) in cellular fatty acid metabolism remains poorly defined. The intracellular movement of fatty acids is thought to be facilitated through codiffusion with FABP. Peroxisomal proliferators like clofibrate induce FABP and may stimulate fatty acid use by increasing cytoplasmic diffusion rates. Our aim was to determine if induction of FABP by clofibrate increases the cytoplasmic transport of a fluorescent fatty acid NBD-stearate. Male rats were fed clofibrate for 9 days to increase the hepatic concentration of FABP. Hepatocytes were isolated using collagenase perfusion. Cytosolic FABP was measured by enzyme-linked immunosorbent assay (ELISA) using a specific antibody to hepatic FABP. Two-dimensional laser photobleaching (FRAP) was used to measure the cytoplasmic movement of NBD-stearate in hepatocytes. Cytoplasmic transport of NBD-stearate occurred by isotropic diffusion with no evidence for convective or directed transport. Treatment with clofibrate increased FABP levels, the fraction of NBD-stearate found in cytosol, and the observed cytoplasmic diffusion rate. The increase in cytoplasmic movement exceeded that predicted from FABP binding changes suggesting that clofibrate also enhances fatty acid diffusion within intracellular membranes. Nonspecific effects of clofibrate on cytoplasmic viscosity or pore size were not observed. These data suggest that clofibrate treatment induces cytosolic FABP and stimulates the intracellular movement of fatty acids by 2 distinct mechanisms. Soluble proteins like FABP promote the cytoplasmic transport of fatty acids by increasing diffusion. These data further support a role for intracellular binding proteins in facilitating the intracellular movements of fatty acids.

Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein.

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid-binding protein (FABP). This facilitation may occur by decreasing binding to immobile membranes, leading to faster cytoplasmic diffusion. The aims of this study were to measure the intracellular transport of 12-N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in villus rat enterocytes and to determine 1) the mechanism of its cytoplasmic transport and 2) if its transport rate correlated with the known variation of FABP binding capacity along the length of the small intestine. Two-dimensional laser photobleaching was used to measure the movement of a fluorescent fatty acid NBD-stearate in enterocytes isolated from different segments of rat intestine. The fraction of NBD-stearate found in the cytostol of enterocytes was determined by differential centrifugation. Cytoplasmic transport of NBD-stearate occurred solely by diffusion and not by convection. Diffusion was homogeneous (nondirectional), consistent with isotropic diffusion. The diffusion rate varied with location along the intestine, correlating with the local FABP concentration and measured cytosolic binding. We conclude that cytoplasmic proteins like FABP promote the intracellular transport of fatty acids by enhancing their diffusive flux. We suggest that facilitation is not specific for a particular cell type but occurs in a variety of cells that transport fatty acids and may contain different types of FABP.

Cytoplasmic codiffusion of fatty acids is not specific for fatty acid binding protein.

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid binding protein (FABP). Previous work suggested that FABP decreases fatty acid binding to immobile membranes, causing faster cytoplasmic diffusion. However, the specificity for binding to FABP has not been addressed. The aim of the current study was to determine whether specific FABP binding is required or whether binding to other proteins will produce the same effect. A model cytoplasm consisted of a fatty acid, proteins, and liposomes to simulate intracellular membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the movement of the fluorescent fatty acid 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazoaminostearate (NBD-stearate) in model cytoplasm, in normal and permeabilized Hep G2 cells, and after incubation of permeabilized cells with bovine serum albumin (BSA) or FABP. Increasing protein in the model cytoplasm increased the diffusion rate in proportion to the extent of protein binding. Cell permeabilization reduced diffusion of NBD-stearate to < 5% of controls. Incubation of permeabilized cells with FABP or BSA resulted in a concentration-dependent increase in the NBD-stearate diffusion rate. BSA was more effective than FABP in binding NBD-stearate and increasing its diffusion rate after permeabilization. Proteins like FABP promote the diffusion of fatty acids. Removal of these proteins drastically reduces cytoplasmic diffusion. Substitution with BSA reestablishes the diffusive flux, suggesting that specific binding to FABP is not required. These data support a role for intracellular binding proteins in facilitating the cytoplasmic movement of fatty acids.