Cellular Fatty Acid Transport Research Articles

Effects of castration on the adiposity and expression of lipid metabolism genes in various fat depots of Korean cattle

Castration increases intramuscular (IM) fat deposition in the musculus longissimus dorsi, a highly desirable trait in Korean beef cattle. However, castration may also affect accumulation in other fat depots. We examined whether castration affects adipose cellularity and lipid metabolism gene expression in various fat depots, including subcutaneous (SC), abdominal (AB), perirenal (PR), and IM fat. First, frozen sections taken from fat depots were stained with hematoxylin and eosin and the mean fat cell size was determined. Steers showed larger cell sizes in AB (P=0.01), SC (P=0.05), and PR (P=0.01) fat compared to bulls. Next, lipid metabolism gene expression in AB fat was compared between bulls and steers. In AB fat, steers showed increased mRNA expression of CCAAT/enhancer binding protein alpha (C/EBPα), a gene related to adipogenesis, compared to bulls (P<0.01). In contrast, steers showed decreased protein expression of medium-chain acyl-CoA dehydrogenase (MCAD), an enzyme involved in fatty acid β-oxidation, compared to bulls (P<0.05). Our results demonstrate that castration induces hypertrophy in body fat cells, and that the up-regulation of adipogenesis and down-regulation of fatty acid β-oxidation may in part contribute to this effect. We compared fat cell sizes among the various fat depots in Korean cattle steers. The order of fat cell size was AB>PR>SC>IM. The expression levels of lipid metabolism genes were compared among various fat depots. The mRNA levels of C/EBPα and peroxisome proliferator-activated receptor-γ were highest (P<0.05) in AB fat and lowest in IM fat. The mRNA levels of fatty acid binding protein-4 and lipogenic acetyl-coenzyme A carboxylase and fatty acid synthase genes were highest (P<0.05) in AB fat, and lowest in IM fat. Expression levels of lipolytic hormone-sensitive lipase and fatty oxidation MCAD genes were also highest (P<0.05) in AB fat and lowest in IM fat. Our results suggest that combined effects of higher adipogenesis, lipogenesis, and cellular fatty acid transport are responsible for the largest AB and smallest IM fat cells among the fat depots examined.

Identification and characterization of small compound inhibitors of human FATP2

Fatty acid transport proteins (FATPs) are bifunctional proteins, which transport long chain fatty acids into cells and activate very long chain fatty acids by esterification with coenzyme A. In an effort to understand the linkage between cellular fatty acid transport and the pathology associated with excessive accumulation of exogenous fatty acids, we targeted FATP-mediated fatty acid transport in a high throughput screen of more than 100,000 small diverse chemical compounds in yeast expressing human FATP2 (hsFATP2). Compounds were selected for their ability to depress the transport of the fluorescent long chain fatty acid analogue, C 1-BODIPY-C 12. Among 234 hits identified in the primary screen, 5 compounds, each representative of a structural class, were further characterized in the human Caco-2 and HepG2 cell lines, each of which normally expresses FATP2, and in 3T3-L1 adipocytes, which do not. These compounds were effective in inhibiting uptake with IC 50s in the low micromolar range in both Caco-2 and HepG2 cells. Inhibition of transport was highly specific for fatty acids and there were no effects of these compounds on cell viability, trans-epithelial electrical resistance, glucose transport, or long chain acyl-CoA synthetase activity. The compounds were less effective when tested in 3T3-L1 adipocytes suggesting selectivity of inhibition. These results suggest fatty acid transport can be inhibited in a FATP-specific manner without causing cellular toxicity.

Nuclear receptors PPARβ/δ and PPARα direct distinct metabolic regulatory programs in the mouse heart

In the diabetic heart, chronic activation of the PPARalpha pathway drives excessive fatty acid (FA) oxidation, lipid accumulation, reduced glucose utilization, and cardiomyopathy. The related nuclear receptor, PPARbeta/delta, is also highly expressed in the heart, yet its function has not been fully delineated. To address its role in myocardial metabolism, we generated transgenic mice with cardiac-specific expression of PPARbeta/delta, driven by the myosin heavy chain (MHC-PPARbeta/delta mice). In striking contrast to MHC-PPARalpha mice, MHC-PPARbeta/delta mice had increased myocardial glucose utilization, did not accumulate myocardial lipid, and had normal cardiac function. Consistent with these observed metabolic phenotypes, we found that expression of genes involved in cellular FA transport were activated by PPARalpha but not by PPARbeta/delta. Conversely, cardiac glucose transport and glycolytic genes were activated in MHC-PPARbeta/delta mice, but repressed in MHC-PPARalpha mice. In reporter assays, we showed that PPARbeta/delta and PPARalpha exerted differential transcriptional control of the GLUT4 promoter, which may explain the observed isotype-specific effects on glucose uptake. Furthermore, myocardial injury due to ischemia/reperfusion injury was significantly reduced in the MHC-PPARbeta/delta mice compared with control or MHC-PPARalpha mice, consistent with an increased capacity for myocardial glucose utilization. These results demonstrate that PPARalpha and PPARbeta/delta drive distinct cardiac metabolic regulatory programs and identify PPARbeta/delta as a potential target for metabolic modulation therapy aimed at cardiac dysfunction caused by diabetes and ischemia.

Metabolic Adaptations to Resistance Training in Skeletal Muscle From Young and Older Women

Resistance training (RT) is well-characterized for its ability to improve muscle strength and function and is widely recommended to protect against muscle loss that occurs with aging. However, a failure to maintain or increase the cellular factors involved in regulating energy metabolism in response to RT could compromise metabolic health and functional capacity. PURPOSE: To examine the influence of 12 weeks of progressive RT on the expression of skeletal muscle proteins involved in cellular transport of fatty acids (FAT/CD36) and glucose (GLUT4), mitochondrial oxidative phosphorylation (COX1), and transcription factors involved in regulating energy metabolism (peroxisome proliferators activated receptor γ - co activator-1 [PGC-1 α] and myocyte enhancer factor [Mef2]). METHODS: Nineteen women (n=9 older [O], 74±1 yr; n=10 younger [Y], 25±1 yr) underwent 12 wk of progressive RT (3d/week) targeting the knee and hip extensor muscles. Muscle biopsies were obtained from the vastus lateralis muscle before and after RT and protein content of FAT/CD36, GLUT4, COX1, PGC-1Δ, and Mef2 were determined by immunoblot analysis. RESULTS: Before training, COX1 content was ∼2-fold higher (P<0.05) in O compared to Y while no age-related differences were found for PGC-1 Δ, Mef2, FAT/CD36, or GLUT4 before training. In response to RT, COX1 content increased (P<0.05) 37±15% in Y only. Additionally, although RT decreased the abundance of full-length (91 kDa) PGC-1α (P<0.05) in O, the short variant (32 kDa) of PGC-1 α remained unchanged. No change in either PGC-1 Δ variant was found in Y after training. Abundance of Mef2, FAT/CD36, and GLUT4 were maintained after training in both groups. CONCLUSION: These findings indicate that RT does not impact markers for substrate transport and oxidative metabolism in young and older women.

Fatty acid transport protein 1 and long-chain acyl coenzyme A synthetase 1 interact in adipocytes

The fatty acid transport proteins (FATP) and long-chain acyl coenzyme A synthetase (ACSL) proteins have been shown to play a role in facilitating long-chain fatty acid (LCFA) transport in mammalian cells under physiologic conditions. The involvement of both FATP and ACSL proteins is consistent with the model of vectorial acylation, in which fatty acid transport is coupled to esterification. This study was undertaken to determine whether the functions of these proteins are coordinated through a protein-protein interaction that might serve as a point of regulation for cellular fatty acid transport. We demonstrate for the first time that FATP1 and ACSL1 coimmunoprecipitate in 3T3-L1 adipocytes, indicating that these proteins form an oligomeric complex. The efficiency of FATP1 and ACSL1 coimmunoprecipitation is unaltered by acute insulin treatment, which stimulates fatty acid uptake, or by treatment with isoproterenol, which decreases fatty acid uptake and stimulates lipolysis. Moreover, inhibition of ACSL1 activity in adipocytes impairs fatty acid uptake, suggesting that esterification is essential for fatty acid transport. Together, our findings suggest that a constitutive interaction between FATP1 and ACSL1 contributes to the efficient cellular uptake of LCFAs in adipocytes through vectorial acylation.

The pH-dependent unfolding mechanism of P2 myelin protein: An experimental and computational study

The P2 protein is a small, extrinsic protein of the myelin membrane in the peripheral nervous system that structurally belongs to the fatty acid binding proteins (FABPs) family, sharing with them a 10 strands β-barrel structure. FABPs appear to be involved in cellular fatty acid transport, but very little is known about the role of P2 in the metabolism of peripheral myelin lipids. Study of protein conformation at different pHs is a useful tool for the characterization of the unfolding mechanisms and the intrinsic conformational properties of the protein, and may give insight into factors that guide protein folding pathways. In particular, low pH conditions have been shown to induce partially folded states in several proteins. In this paper, the acidic unfolding of purified P2 protein was studied with both spectroscopic techniques and molecular dynamics simulation. Both experimental and computational results indicate the presence of a partly folded state at low pH, which shows structural changes mainly involving the lid that is formed by the helix-turn-helix domain. The opening of the lid, together with a barrel relaxation, could regulate the ligand exchanges near the cell membrane, supporting the hypothesis that the P2 protein may transport fatty acids between Schwann cells and peripheral myelin.

Estrogen-related receptor alpha directs peroxisome proliferator-activated receptor alpha signaling in the transcriptional control of energy metabolism in cardiac and skeletal muscle.

Estrogen-related receptors (ERRs) are orphan nuclear receptors activated by the transcriptional coactivator peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha), a critical regulator of cellular energy metabolism. However, metabolic target genes downstream of ERRalpha have not been well defined. To identify ERRalpha-regulated pathways in tissues with high energy demand such as the heart, gene expression profiling was performed with primary neonatal cardiac myocytes overexpressing ERRalpha. ERRalpha upregulated a subset of PGC-1alpha target genes involved in multiple energy production pathways, including cellular fatty acid transport, mitochondrial and peroxisomal fatty acid oxidation, and mitochondrial respiration. These results were validated by independent analyses in cardiac myocytes, C2C12 myotubes, and cardiac and skeletal muscle of ERRalpha-/- mice. Consistent with the gene expression results, ERRalpha increased myocyte lipid accumulation and fatty acid oxidation rates. Many of the genes regulated by ERRalpha are known targets for the nuclear receptor PPARalpha, and therefore, the interaction between these regulatory pathways was explored. ERRalpha activated PPARalpha gene expression via direct binding of ERRalpha to the PPARalpha gene promoter. Furthermore, in fibroblasts null for PPARalpha and ERRalpha, the ability of ERRalpha to activate several PPARalpha targets and to increase cellular fatty acid oxidation rates was abolished. PGC-1alpha was also shown to activate ERRalpha gene expression. We conclude that ERRalpha serves as a critical nodal point in the regulatory circuitry downstream of PGC-1alpha to direct the transcription of genes involved in mitochondrial energy-producing pathways in cardiac and skeletal muscle.

Implementation of fatty acid carriers to skin irritation and the epidermal barrier.

Acute perturbations are followed by barrier repair and enhanced lipid synthesis, as well as cellular fatty acid trafficking, yet irritation of the skin may be induced by repeat disturbance of barrier function. Recently, new insights in cellular fatty acid transport and metabolism have evolved with respect to skin irritation and barrier disturbances: (1) Employing sodium dodecyl sulfate, skin irritation is accompanied by the induction of an epidermal (E) cytosolic fatty acid binding protein (FABP) associated with enhanced barrier repair. Whether E-FABP contributes to the water barrier function in normal skin remains to be elucidated; (2) Cutaneous inflammation, as it occurs in irritant contact dermatitis, can be reduced by peroxisome proliferating activated receptor (PPAR) agonists, such as linoleic acid, with clinical effects comparable to that of glucocorticoids; (3) PPARalpha agonists accelerate barrier recovery and enhance lamellar body synthesis, neutral lipid synthesis, in particular that of ceramides and cholesterol; (4) PPARalpha agonists increase the minimal erythema dose in UVB-irradiated human skin. This review provides a brief overview of the current understanding of mammalian fatty acid (FA) metabolism with respect to epidermal barrier abrogation and repair, including new insights into cellular FA transport and metabolism.

Reduced secretion of triacylglycerol in CaCo-2 cells transfected with intestinal fatty acid-binding protein.

The fatty acid-binding proteins are hypothesized to be involved in cellular fatty acid transport and trafficking. We established CaCo-2 cells stably transfected with intestinal fatty acid-binding protein (I-FABP) and examined how the expression of this protein may influence fatty acid metabolism. I-FABP expression was detectable in I-FABP-transfected cells, whereas parent CaCo-2 cells as well as mock-transfected cells failed to express detectable levels of I-FABP mRNA or protein at any stage of differentiation. For studies of lipid metabolism, cells were incubated with [14C]oleic acid in taurocholate micelles containing monoolein, and distribution of labeled fatty acid in cellular and secreted lipids was examined. In one transfected cell clone, expressing the highest level of I-FABP, labeled cellular triacylglycerol increased approximately twofold as compared to control cells. The level of intracellular triacylglycerol in two other I-FABP-transfected clones resembled that of control cells. However, secretion of triacylglycerol was markedly reduced in all the I-FABP-expressing cell lines. Our data suggest that increased expression of I-FABP leads to reduced triacylglycerol secretion in intestinal cells.

Cellular fatty acid transport in heart and skeletal muscle as facilitated by proteins.

Despite the importance of long-chain fatty acids (FA) as fuels for heart and skeletal muscles, the mechanism of their cellular uptake has not yet been clarified. There is dispute as to whether FA are taken up by the muscle cells via passive diffusion and/or carrier-mediated transport. Kinetic studies of FA uptake by cardiac myocytes and the use of membrane protein-modifying agents have suggested the bulk of FA uptake is due to a protein component. Three membrane-associated FA-binding proteins were proposed to play a role in FA uptake, a 40-kDa plasma membrane FA-binding protein (FABPpm), an 88-kDa FA translocase (FAT/CD36), and a 60-kDa FA transport protein (FATP). In cardiac and skeletal myocytes the intracellular carrier for FA is cytoplasmic heart-type FA-binding protein (H-FABP), which likely transports FA from the sarcolemma to their intracellular sites of metabolism. A scenario is discussed in which FABPpm, FAT/CD36, and H-FABP, probably assisted by an albumin-binding protein, cooperate in the translocation of FA across the sarcolemma.

Ligand binding alters the backbone mobility of intestinal fatty acid-binding protein as monitored by 15N NMR relaxation and 1H exchange.

The backbone dynamics of the liganded (holo) and unliganded (apo) forms of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) have been characterized and compared using amide 15N relaxation and 1H exchange NMR measurements. The amide 1H/15N resonances for apo and holo I-FABP were assigned at 25 degrees C, and gradient- and sensitivity-enhanced 2D experiments were employed to measure l5N T1, T2, and [1H]15N NOE values and relative 1H saturation transfer rates. The 15N relaxation parameters were analyzed using five different representations of the spectral density function based on the Lipari and Szabo formalism. A majority of the residues in both apo and holo I-FABP were characterized by relatively slow hydrogen exchange rates, high generalized order parameters, and no conformational exchange terms. However, residues V26-N35, S53-R56, and A73-T76 of apo I-FABP were characterized by rapid hydrogen exchange, low order parameters, and significant conformational exchange. These residues are clustered in a single region of the protein where variability and apparent disorder were previously observed in the chemical shift analyses and in the NOE-derived NMR structures of apo I-FABP. The increased mobility and discrete disorder in the backbone of the apo protein may permit the entry of ligand into the binding cavity. We postulate that the bound fatty acid participates in a series of long-range cooperative interactions that cap and stabilize the C-terminal half of helix II and lead to an ordering of the portal region. This ligand-modulated order-disorder transition has implications for the role of I-FABP in cellular fatty acid transport and targeting.

Inhibitory effect of cholephilic anions on fatty acid uptake by the perfused rat liver

The mechanisms of uptake and intracellular transport of plasma free fatty acids by the liver cell are poorly known. A cytosolic protein, termed Z protein, has been identified in the liver, intestine and other tissues; in vitro studies show a high affinity of fatty acid and organic anions for this protein and suggest that it could be involved in the cellular transport of fatty acids in vivo. The present experiments demonstrate an inhibitory effect of various organic cholephilic anions (B.S.P., D.B.S.P., flavaspidic acid, bilirubin, etc) on fatty acid uptake by the isolated perfused rat liver. This inhibition is rapidly reversible when the liver is perfused with a medium lacking cholephilic anions, which together with the absence of any functional or ultrastructural damage to the organ, excludes any hepatotoxic effect. Such an inhibition of fatty acid uptake could be due to a competition for binding either to membrane sites and/or to intracellular carriers common to both fatty acids and cholephilic anions. These data are consistent with the involvement of carrier-mediated processes in fatty acid transport in the liver. Finally, simultaneous uptake of radioactive fatty acid and cholephilic anions does not interfere with further fatty acid utilization by the cell.

Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine.

Fatty acid binding protein (FABP) is a protein of 12,000 mol wt found in cytosol of intestinal mucosa and other tissues, which exhibits high affinity for long chain fatty acids. It has been suggested that FABP (which may comprise a group of closely related proteins of 12,000 mol wt) participates in cellular fatty acid transport and metabolism. Although earlier findings were consistent with this concept, the present studies were designed to examine its physiological function more directly. Everted jejunal sacs were incubated in mixed fatty acid-monoglyceride-bile acid micelles, in the presence or absence of equimolar concentrations of either of two compounds which inhibit oleate binding to FABP:flavaspidic acid-N-methyl-glucaminate and alpha-bromopalmitate. Oleate uptake, mucosal morphology, and oxidation of [14C]acetate remained unaffected by these agents, but oleate incorporation into triglyceride was inhibited by 62-64% after 4 min. The inhibition by flavaspidic acid was reversible with higher oleate concentrations. The effect of these compounds on enzymes of triglyceride biosynthesis was examined in intestinal microsomes. Neither flavaspidic acid nor alpha-bromopalmitate inhibited acyl CoA:monoglyceride acyl-transferase. Fatty acid:coenzyme A ligase activity was significantly enhanced in the presence of partially purified FABP, probably reflecting a physical effect on the fatty acid substrate or on the formation of the enzyme-substrate complex. Activity of the enzyme in the presence of 0.1 mM oleate was only modestly inhibited by equimolar flavaspidic acid and alpha-bromopalmitate, and this effect was blunted or prevented by FABP. We conclude that in everted gut sacs, inhibition of triglyceride synthesis by flavaspidic acid and alpha-bromopalmitate could not be explained as an effect on fatty acid uptake or on esterifying enzymes in the endoplasmic reticulum but rather can be interpreted as reflecting inhibition of fatty acid binding to FABP. These findings lend further support to the concept that FABP participates in cellular fatty acid transport and metabolism. It is also possible that FABP, by effecting an intracellular compartmentalization of fatty acids and acyl CoA, may play a broader role in cellular lipid metabolism.

Fatty acid-binding protein in small intestine. Identification, isolation, and evidence for its role in cellular fatty acid transport.

A soluble fatty acid-binding protein (FABP), mol wt approximately 12,000 is present in intestinal mucosa and other tissues that utilize fatty acids, including liver, myocardium, adipose, and kidney. This protein binds long chain fatty acids both in vivo and in vitro.FABP was isolated from rat intestine by gel filtration and isoelectric focusing. It showed a reaction of complete immunochemical identity with proteins in the 12,000 mol wt fatty acid-binding fractions of liver, myocardium, and adipose tissue supernates. (The presence of immunochemically nonidentical 12,000 mol wt FABP in these tissues is not excluded.) By quantitative radial immunodiffusion, supernatant FABP concentration in mucosa from proximal and middle thirds of jejuno-ileum significantly exceeded that in distal third, duodenum, and liver, expressed as micrograms per milligram soluble protein, micrograms per gram DNA, and micrograms per gram tissue. FABP concentration in villi was approximately three times greater than in crypts. Small quantities of FABP were present in washed nuclei-cell membrane, mitochondrial and microsomal fractions. However, the amount of FABP solubilized per milligram membrane protein was similar for all particulate fractions, and total membrane-associated FABP was only about 16% of supernatant FABP. Intestinal FABP concentration was significantly greater in animals maintained on high fat diets than on low fat; saturated and unsaturated fat diets did not differ greatly in this regard.The preponderance of FABP in villi from proximal and middle intestine, its ability to bind fatty acids in vivo as well as in vitro, and its response to changes in dietary fat intake support the concept that this protein participates in cellular fatty acid transport during fat absorption. Identical or closely related 12,000 mol wt proteins may serve similar functions in other tissues.