Rate Of Fatty Acid Uptake Research Articles

AMPKα2deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle

AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca(2+) concentration ([Ca(2+)]). The purpose of this study was to determine whether increases in intracellular [Ca(2+)] induced by caffeine act solely via AMPKα(2) and whether AMPKα(2) is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPKα(2) dominant-negative (DN) transgene mice were perfused during rest (n = 11), treatment with 3 mM caffeine (n = 10), or muscle contraction (n = 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period (P < 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol (P < 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice (P < 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction (P < 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation (P > 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice (P < 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine- or contraction-mediated changes in the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase I or ERK1/2 (P > 0.05). These data suggest that AMPKα(2) is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment.

Ncb5or Deficiency Increases Fatty Acid Catabolism and Oxidative Stress

The endoplasmic reticulum-associated NADH cytochrome b(5) oxidoreductase (Ncb5or) is widely distributed in animal tissues. Ncb5or(-/-) mice develop diabetes at age 7 weeks and have increased susceptibility to the diabetogenic oxidant streptozotocin. Ncb5or deficiency also results in lipoatrophy and increased hepatocyte sensitivity to cytotoxic effects of saturated fatty acids. Here we investigate the mechanisms of these phenomena in prediabetic Ncb5or(-/-) mice and find that, despite increased rates of fatty acid uptake and synthesis and higher stearoyl-CoA desaturase (SCD) expression, Ncb5or(-/-) liver accumulates less triacylglycerol (TAG) than wild type (WT). Increased fatty acid catabolism and oxidative stress are evident in Ncb5or(-/-) hepatocytes and reflect increased mitochondrial content, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) expression, fatty acid oxidation rates, oxidative stress response gene expression, and oxidized glutathione content. Ncb5or(-/-) hepatocytes readily incorporate exogenous fatty acids into TAG but accumulate more free fatty acids (FFA) and have greater palmitate-induced oxidative stress responses and cell death than WT, all of which are alleviated by co-incubation with oleate via TAG channeling. A high fat diet rich in palmitate and oleate stimulates both lipogenesis and fatty acid catabolism in Ncb5or(-/-) liver, resulting in TAG levels similar to WT but increased intracellular FFA accumulation. Hepatic SCD-specific activity is lower in Ncb5or(-/-) than in WT mice, although Ncb5or(-/-) liver has a greater increase in Scd1 mRNA and protein levels. Together, these findings suggest that increased FFA accumulation and catabolism and oxidative stress are major consequences of Ncb5or deficiency in liver.

Characterization of long-chain fatty acid uptake in Caulobacter crescentus

Studies evaluating the uptake of long-chain fatty acids in Caulobacter crescentus are consistent with a protein-mediated process. Using oleic acid (C18:1) as a substrate, fatty acid uptake was linear for up to 15 min. This process was saturable giving apparent V(max) and K(m) values of 374 pmol oleate transported/min/mg total protein and 61 μM oleate, respectively, consistent with the notion that one or more proteins are likely involved. The rates of fatty acid uptake in C. crescentus were comparable to those defined in Escherichia coli. Uncoupling the electron transport chain inhibited oleic acid uptake, indicating that like the long-chain fatty acid uptake systems defined in other gram-negative bacteria, this process is energy-dependent in C. crescentus. Long-chain acyl CoA synthetase activities were also evaluated to address whether vectorial acylation represented a likely mechanism driving fatty acid uptake in C. crescentus. These gram-negative bacteria have considerable long-chain acyl CoA synthetase activity (940 pmol oleoyl CoA formed/min/mg total protein), consistent with the notion that the formation of acyl CoA is coincident with uptake. These results suggest that long-chain fatty acid uptake in C. crescentus proceeds through a mechanism that is likely to involve one or more proteins.

Insulin Sensitivity is Lowest in Obese Women with High Rates of Fatty Acid Uptake

Insulin Sensitivity is Lowest in Obese Women with High Rates of Fatty Acid Uptake

Experimental evaluation of decrease in the activities of polyphosphate/glycogen‐accumulating organisms due to cell death and activity decay in activated sludge

Decrease in bacterial activity (biomass decay) in activated sludge can result from cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The goal of this study was to experimentally differentiate between cell death and activity decay as the cause of decrease in bacterial activity. By means of measuring maximal anaerobic phosphate release rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in situ hybridization (FISH), the decay rates and death rates of polyphosphate-accumulating organisms (PAOs) in a biological nutrient removal (BNR) system and a laboratory phosphate removing sequencing batch reactor (SBR) system were determined, respectively, under famine conditions. In addition, the decay rate and death rate of glycogen-accumulating organisms (GAOs) in a SBR system with an enrichment culture of GAOs were also measured under famine conditions. Hereto the maximal anaerobic volatile fatty acid uptake rates, live/dead staining, and FISH were used. The experiments revealed that in the BNR and enriched PAO-SBR systems, activity decay contributed 58% and 80% to the decreased activities of PAOs, and that cell death was responsible for 42% and 20% of decreases in their respective activities. In the enriched GAOs system, activity decay constituted a proportion of 74% of the decreased activity of GAOs, and cell death only accounted for 26% of the decrease of their activity.

A Coarse Grained Molecular Dynamics Study of Self-Reproduction of Fatty Acid Vesicles

The formation of self-reproducing fatty acid vesicles is believed to be a quintessential building block required for (the origin of) life. In literature a number of vesicle reproduction experiments have been reported [1]. Of particular interest is the so called matrix effect where the final size distribution of vesicles formed upon addition of new surfactants is strongly biased toward the size distribution of pre-existing vesicles. However, the mechanism behind this is poorly understood. Here, we regard this mechanism using coarse grained molecular dynamics simulations with a parameter set based on a previously published force field [2]. These simulations show fission of a vesicle on continuous addition of new fatty acid molecules to the vesicle exterior. The new molecules are incorporated in the outer leaflet of the vesicle's membrane. As the rate of fatty acid redistribution among the leaflets by means of flip-flop is lower than the rate of fatty acid uptake in the outer leaflet, an excess of molecules in the outer leaflet compared to the inner leaflet is formed. This results in spontaneous membrane curvature, causing the vesicle to deform into twin-vesicles. Eventually, the built-up spontaneous curvature induces the formation of a narrow neck, that finally breaks, resulting in full fission. Importantly, no leakage from the interior of the vesicles was observed. The reproduction pathway shown in these simulations agrees with published experimental data, as well as with new data from our group (see abstract Nicole Pfleger). Our coarse grained molecular dynamics simulations offer further insight at the molecular level of the self-reproduction pathway of fatty acid vesicles.[1] Luisi et al. (2008) J. Phys. Chem. B 112, 14655-14664.[2] Markvoort et al. (2005) J. Phys. Chem. B 109, 22649-22654.

Inhibition of glucose uptake by Silybin and Dehydrosilybin

Type 2 diabetes (T2D) is a common disease in industrialized countries. Pathological changes in cellular glucose and fatty acid uptake are hallmarks of T2D. Medical plants from traditional medical systems of China and India are considered as a promising source for new anti-diabetic drugs. Here, we screened a panel of plant extracts and single chemical compounds from traditional medical plants with reported anti-diabetic effects for their ability to modulate cellular glucose and fatty acid uptake rates. Cellular glucose uptake, but not cellular fatty acid uptake, was significantly reduced by several candidate drugs. Of those drugs, we chose Silybin (SIL) and its derivative Dehydrosilybin (DHS), two main flavonoids of Silybum marianum , for further investigations. SIL is reported to improve blood sugar control in several clinical trials and also possesses strong anti-cancer effects. We could show that SIL and DHS inhibit glucose transport in 3T3-L1 fibroblasts and adipocytes. Both flavonoids reduce basal glucose uptake in a dose-dependent manner (52% and 36% of basal uptake for 40 μM SIL, 5% and 20% of basal uptake for 40 μM DHS in fibroblasts and adipocytes). Insulin-stimulated glucose uptake is inhibited by SIL and DHS at concentrations of 20 and 10 μM, respectively. Using time course experiments, immunofluorescence microscopy and subcellular fractionation/immunoblotting, we demonstrated that both substances do not interfere with insulin-triggered translocation of GLUT4 to the plasma membrane. Onward metabolism seems unaffected since there are no inhibitory effects of SIL and DHS on hexokinase activity in 3T3-L1 adipocytes. To determine whether SIL and DHS may affect intrinsic transport activity of GLUT4, we heterologously expressed this isoform in CHO-K1 cells. CHO-K1 expressing GLUT4 shows a 2-fold increase in basal glucose uptake compared to control cells. This increase in glucose transport is completely abolished by 40 μM SIL or DHS. SIL and DHS therefore inhibit glucose uptake by interfering with glucose transporters including GLUT4. Stable expression of GLUT4 in CHO-K1 significantly reduces the cytotoxic effect of both SIL and DHS as determined by MTT assay, indicating that restriction of glucose uptake is an important mechanism for the anti-cancer effect of the flavonoids. Our results show that inhibition of glucose transporters is a potential mechanism by which anti-diabetics from medical plants could work.

Seasonal upregulation of fatty acid transporters in flight muscles of migratory white-throated sparrows (Zonotrichia albicollis)

Endurance flights of birds, some known to last several days, can only be sustained by high rates of fatty acid uptake by flight muscles. Previous research in migratory shorebirds indicates that this is made possible in part by very high concentrations of cytosolic heart-type fatty acid binding protein (H-FABP), which is substantially upregulated during migratory seasons. We investigated if H-FABP and other components of muscle fatty acid transport also increase during these seasons in a passerine species, the white-throated sparrow (Zonotrichia albicollis). Fatty acid translocase (FAT/CD36) and plasma-membrane fatty acid binding protein (FABPpm) are well characterized mammalian proteins that facilitate transport of fatty acid through the muscle membrane, and in this study they were identified for the first time in birds. We used quantitative PCR to measure mRNA of FAT/CD36, FABPpm and H-FABP and immunoblotting to measure protein expression of FABPpm and H-FABP in the pectoralis muscles of sparrows captured in migratory (spring, fall) and non-migratory (winter) seasons. During migratory seasons, mRNA expression of these genes increased 70-1000% above wintering levels, while protein expression of H-FABP and FABPpm increased 43% and 110% above wintering levels. Activities of key metabolic enzymes, 3-hydroxyacyl-CoA-dehydrogenase (HOAD), carnitine palmitoyl transferase II (CPT II), and citrate synthase (CS) also increased (90-110%) in pectoralis muscles of migrant birds. These results support the hypothesis that enhanced protein-mediated transport of fatty acids from the circulation into muscle is a key component of the changes in muscle biochemistry required for migration in birds.

Endogenous Peroxisome Proliferator-Activated Receptor-γ Augments Fatty Acid Uptake in Oxidative Muscle

In the setting of insulin resistance, agonists of peroxisome proliferator-activated receptor (PPAR)-gamma restore insulin action in muscle and promote lipid redistribution. Mice with muscle-specific knockout of PPARgamma (MuPPARgammaKO) develop excess adiposity, despite reduced food intake and normal glucose disposal in muscle. To understand the relation between muscle PPARgamma and lipid accumulation, we studied the fuel energetics of MuPPARgammaKO mice. Compared with controls, MuPPARgammaKO mice exhibited significantly increased ambulatory activity, muscle mitochondrial uncoupling, and respiratory quotient. Fitting with this latter finding, MuPPARgammaKO animals compared with control siblings exhibited a 25% reduction in the uptake of the fatty acid tracer 2-bromo-palmitate (P < 0.05) and a 13% increase in serum nonesterified fatty acids (P = 0.05). These abnormalities were associated with no change in AMP kinase (AMPK) phosphorylation, AMPK activity, or phosphorylation of acetyl-CoA carboxylase in muscle and occurred despite increased expression of fatty acid transport protein 1. Palmitate oxidation was not significantly altered in MuPPARgammaKO mice despite the increased expression of several genes promoting lipid oxidation. These data demonstrate that PPARgamma, even in the absence of exogenous activators, is required for normal rates of fatty acid uptake in oxidative skeletal muscle via mechanisms independent of AMPK and fatty acid transport protein 1. Thus, when PPARgamma activity in muscle is absent or reduced, there will be decreased fatty acid disposal leading to diminished energy utilization and ultimately adiposity.

Mechanisms and Regulation of Protein-Mediated Cellular Fatty Acid Uptake: Molecular, Biochemical, and Physiological Evidence

![Figure][1] Arend Bonen Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada abonen{at}uoguelph.ca ![Figure][1] Adrian Chabowski Department of Physiology, Medical University of Bialystok, Bialystok, Poland ![

Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

POINT/COUNTERPOINTIs Membrane Transport of FFA Mediated by Lipid, Protein, or Both?Published Online:01 Feb 2007https://doi.org/10.1152/physiologyonline.2007.22.1.29MoreSectionsPDF (68 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Points of Agreement The movement of long-chain fatty acids across the plasma membrane is likely facilitated by membrane-associated proteins. The exact molecular mechanisms of this protein-facilitated movement across the plasma membrane are not fully understood. A number of membrane-associated proteins involved in facilitating the uptake of fatty acids into metabolically important tissues have been identified (i.e., FAT/CD36, FABPpm, FATP1–6). However, at this point, it is not clear as to 1) why there are so many different proteins, 2) whether they directly interact with each other, 3) whether some or all of these proteins enhance uptake through means (e.g., esterification or oxidation) other than transport, or 4) whether they associate with other proteins to channel fatty acids to different metabolic fates.In heart and muscle, physiological stimuli (insulin, contraction) induce the translocation of FABPpm and/or FAT/CD36 from an endosomal pool to the plasma membrane, resulting in an increased rate of fatty acid uptake. Evidence for endocrine (insulin) regulation of adipocyte fatty acid uptake remains contradictory. It cannot be discounted that other proteins or groups of proteins participate in the transmembrane transport of fatty acids, particularly in adipose tissue where the roles of known fatty acid transport proteins remain elusive. Given the very different metabolic roles of muscle tissues and adipose tissue, it would not be surprising if, in these tissues, the fatty acid transport system and its regulation differ considerably. Points of Disagreement The role of FAT/CD36 and FABPpm in mediating transport of long-chain fatty acids across membranes. Bonen et al. find that FAT/CD36 and FABPpm mediate fatty acid transport into giant sarcolemmal vesicles, whereas Kampf and Kleinfeld conclude that neither FAT/CD36 nor FABPpm mediate membrane transport of long-chain fatty acids in adipocyte cell lines.The role of lipid phase (diffusion) and protein-mediated mechanisms in the membrane transport of long-chain fatty acids. Bonen et al. find evidence for a small contribution by a lipid phase (diffusion) mechanism and a quantitatively larger protein-mediated mechanism in giant sarcolemmal vesicles, whereas Kampf and Kleinfeld conclude that fatty acids transport in adipocytes occurs solely through a protein-mediated mechanism.Whether inhibitors of fatty acid transport have been identified. Kampf and Kleinfeld have not observed transport inhibition in adipocytes, whereas Bonen et al find that specific reagents inhibit fatty acid transport across the plasma membrane of giant sarcolemmal vesicles.Whether cells can be washed without removing the bulk of the unesterified fatty acids. Kampf and Kleinfeld have found that washing cells removes virtually all unesterified fatty acid from adipocytes, whereas Bonen et al. find that the bulk of the transported fatty acids observed after washing remains unesterified. Fatty Acid Nomenclature: A Source of Confusion.In papers dealing with fatty acids and their derivatives, as in the present Point/ Counterpoint, there is a considerable lack of uniformity in the terms used to designate these molecules (Glatz and van der Vusse. Trends Biochem Sci 13: 167–168, 1988). For example, the term “free fatty acid” is often used to refer to nonesterified fatty acids, which is itself an ambiguous term because a fatty acid is by definition not esterified. A more accurate term for the covalently modified fatty acid found in, for example, triacylglycerols is “fatty ester,” as recommended by the IUPACIUB. An additional problem is that the fatty acid may be solubilized in the aqueous phase or bound to a macromolecule such as a protein or a membrane. Glatz and van der Vusse have proposed the designation “fatty acid” (FA) for the aqueous phase form (also known as unbound FA) and protein (macromolecule)-bound FA for the nonaqueous form. Bonen et al. have used this nomenclature. To emphasize the direct measurement of the aqueous form of the FA, Kampf and Kleinfeld have designated the aqueous FA as FFAu and have used the term FFA to designate the sum of the noncovalently bound and unbound FA. Another complication is that the aqueous FA at neutral pH is largely ionized (and therefore not an acid), whereas the macromolecular bound FA may be either ionized or protonated. Both sides of this Point/Counterpoint discussion agree that the nomenclature proposed by Glatz and van der Vusse represents a consistent and more accurate terminology than is currently in general use.This article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 22Issue 1February 2007Pages 29-29 Copyright & Permissions© 2007 Int. Union Physiol. Sci./Am. Physiol. Soc.https://doi.org/10.1152/physiologyonline.2007.22.1.29History Published online 1 February 2007 Published in print 1 February 2007 Metrics

Fatty Acid Transport Protein 1 Is Required for Nonshivering Thermogenesis in Brown Adipose Tissue

Nonshivering thermogenesis in brown adipose tissue (BAT) generates heat through the uncoupling of mitochondrial beta-oxidation from ATP production. The principal energy source for this process is fatty acids that are either synthesized de novo in BAT or are imported from circulation. How uptake of fatty acids is mediated and regulated has remained unclear. Here, we show that fatty acid transport protein (FATP)1 is expressed on the plasma membrane of BAT and is upregulated in response to cold stimuli, concomitant with an increase in the rate of fatty acid uptake. In FATP1-null animals, basal fatty acid uptake is reduced and remains unchanged following cold exposure. As a consequence, FATP1 knockout (KO) animals display smaller lipid droplets in BAT and fail to defend their core body temperature at 4 degrees C, despite elevated serum free fatty acid levels. Similarly, FATP1 is expressed by the BAT-derived cell line HIB-1B upon differentiation, and both fatty acid uptake and FATP1 protein levels are rapidly elevated following isoproterenol stimulation. Stimulation of fatty uptake by isoproterenol required both protein kinase A and mitogen-activated kinase signaling and is completely dependent on FATP1 expression, as small-hairpin RNA-mediated knock down of FATP1 abrogated the effect.

Rat Long Chain Acyl-CoA Synthetase 5 Increases Fatty Acid Uptake and Partitioning to Cellular Triacylglycerol in McArdle-RH7777 Cells

Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 microM [1-(14)C]oleic acid. Metabolism of [1-(14)C]oleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of [1-(14)C]oleate and [1,2,3-(3)H]glycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, [1-(14)C]acetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage.

Differences in transport of fatty acids and expression of fatty acid transporting proteins in adipose tissue of obese black and white women

We have reported that the rate of de novo triglyceride (TG) synthesis by omental, but not subcutaneous, adipose tissue was higher in African-American women (AAW) than in Caucasian women (CAW). The purpose of this study was to explore the potential mechanisms underlying this increase. Toward that end, we determined the activities of key enzymes in the pathway of TG synthesis, the rates of uptake of fatty acids by adipocytes, mRNA and protein levels of the fatty acid-transporting proteins FAT/CD36 and FATP, and mRNA and protein levels of PPARgamma in omental fat of AAW and CAW. The results showed 1) no difference in the activity of phosphofructokinase, glycerol-3-phosphate dehydrogenase, or diacylglycerol acyltransferase; 2) a higher rate of fatty acid uptake by adipocytes of the AAW; 3) an increase in the mRNA and protein levels of CD36 and FATP4 in the fat of the AAW; and 4) an increase in the mRNA and protein levels of PPARgamma, which can stimulate the expression of CD36 and FATP. These results suggest that the increase in the transport of fatty acid, which is mediated by the overexpression of the transport proteins in the omental adipose tissue of the AAW, might contribute to the higher prevalence of obesity in AAW.

FoxO1 Stimulates Fatty Acid Uptake and Oxidation in Muscle Cells through CD36-dependent and -independent Mechanisms

Emerging evidence documents a key function for the forkhead transcription factor FoxO1 in cellular metabolism. Here, we investigate the role of FoxO1 in the regulation of fatty acid (FA) metabolism in muscle cells. C2C12 cells expressing an inducible construct with either wild type FoxO1 or a mutant form (FoxO1/TSS) refractory to the protein kinase B inhibitory effects were generated. FoxO1 activation after myotube formation altered the expression of several genes of FA metabolism. Acyl-CoA oxidase and peroxisome proliferator-activated receptor delta mRNA levels increased 2.2-fold and 1.4-fold, respectively, whereas mRNA for acetyl-CoA carboxylase decreased by 50%. Membrane uptake of oleate increased 3-fold, and oleate oxidation increased 2-fold. Cellular triglyceride content was also increased. The enhanced FA utilization induced by FoxO1 was mediated by a severalfold increase in plasma membrane level of the fatty acid translocase FAT/CD36 and eliminated by cell treatment with the CD36 inhibitor sulfo-N-succinimidyl-oleate. We conclude that FoxO1 activation induces coordinate increases in FA uptake and oxidation and that these effects are mediated, at least in part, by membrane enrichment in CD36. The data suggest that FoxO1 contributes to preparing the muscle cell for the increased reliance on FA metabolism that is characteristic of fasting. Dysregulation of FoxO1 in muscle could contribute to intramuscular lipid accumulation and insulin resistance by maintaining activation of FA uptake.

Mouse Very Long-chain Acyl-CoA Synthetase 3/Fatty Acid Transport Protein 3 Catalyzes Fatty Acid Activation but Not Fatty Acid Transport in MA-10 Cells

The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both long-chain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.

Palmitic acid metabolism in the soleus muscle in vitro in hypo- and hyperthyroid rats.

The aim of this study was to establish whether the rate of fatty acid (FA) incorporation and its utilization by the isolated soleus muscle is modified under conditions of thyroid hormone deficit or excess. The rate of palmitic acid (PA) uptake, oxidation and incorporation into intramuscular lipids with increasing PA concentration (0.5-1.5 mM) in the incubation medium were determined. In hypothyroid rats intramuscular triacylglycerol (TG) synthesis was increased, while the rate of PA oxidation to CO2 and incorporation into mono- and diacylglycerols (MG/DG) and phospholipids (PL) remained unchanged. In rats with triiodothyronine (T3) excess the rate of all processes studied was enhanced, although the percentage incorporation of PA into different classes of intramuscular lipids was fairly constant and, independently of thyroid state and FA concentration in the medium, was 56-66% for TG, 9-14% for MG/DG and 24-32% for PL. Our results thus indicate that even short-term T3 excess accelerates the rate of FA uptake and metabolism in the oxidative soleus muscle, whereas in hypothyroid rats only intramuscular TG synthesis is affected.

Regulation of fatty acid transport by fatty acid translocase/CD36.

Fatty acid (FA) translocase (FAT)/CD36 is a key protein involved in regulating the uptake of FA across the plasma membrane in heart and skeletal muscle. A null mutation of FAT/CD36 reduces FA uptake rates and metabolism, while its overexpression increases FA uptake rates and metabolism. FA uptake into the myocyte may be regulated (a) by altering the expression of FAT/CD36, thereby increasing the plasmalemmal content of this protein (i.e. streptozotocin-induced diabetes, chronic muscle stimulation), or (b) by relocating this protein to the plasma membrane, without altering its expression (i.e. obese Zucker rats). By repressing FAT/CD36 expression, and thereby lowering the plasmalemmal FAT/CD36 (i.e. leptin-treated animals), the rate of FA transport is reduced. Within minutes of beginning muscle contraction or being exposed to insulin FA transport is increased. This increase is a result of the contraction- and insulin-induced translocation of FAT/CD36 from an intracellular depot to the cell surface. Neither PPAR alpha nor PPAR gamma activation alter FAT/CD36 expression in muscle, despite the fact that PPAR alpha activation increases FAT/CD36 by 80% in liver. A novel observation is that FAT/CD36 also appears to be involved in mitochondrial FA oxidation, as this protein is located on the mitochondrial membrane and seems to be required to participate in moving FA across the mitochondrial membrane. Clearly, FAT/CD36 has an important role in FA homeostasis in skeletal muscle and the heart.

Cellular fatty acid uptake is acutely regulated by membrane-associated fatty acid-binding proteins

Cellular long-chain fatty acid uptake is believed to occur largely by protein-mediated transmembrane transport of fatty acids, and also by passive diffusional uptake. It is postulated that the membrane proteins function in trapping of fatty acids from extracellular sources, whereafter their transmembrane translocation occurs by passive diffusion through the lipid bilayer. The key membrane-associated proteins involved are plasma membrane fatty acid-binding protein (FABP pm) and fatty acid translocase (FAT/CD36). Their plasma membrane contents are positively correlated with rates of fatty acid uptake. In studies with heart and skeletal muscle we observed that FAT/CD36 is regulated acutely, in that both contraction and insulin can translocate FAT/CD36 from an intracellular depot to the sarcolemma, thereby increasing the rate of fatty acid uptake. In addition, from studies with obese Zucker rats, an established rodent model of obesity and insulin resistance, evidence has been obtained that in heart, muscle and adipose tissue FAT/CD36 is permanently relocated from an intracellular pool to the plasma membrane, resulting in increased fatty acid uptake rates in this condition. These combined observations indicate that protein-mediated fatty acid uptake is a key step in cellular fatty acid utilization, and suggest that malfunctioning of the uptake process could be a critical factor in the pathogenesis of insulin resistance.

Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats.

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.