Tetraspanin TM4SF5 in hepatocytes negatively modulates SLC27A transporters during acute fatty acid supply.

Transmembrane 4 L six family member 5 (TM4SF5) is involved in nonalcoholic steatosis and further aggravation of liver disease. However, its mechanism for regulating FA accumulation is unknown. We investigated how TM4SF5 in hepatocytes affected FA accumulation during acute FA supply. TM4SF5-expressing hepatocytes and mouse livers accumulated less FAs, compared with those of TM4SF5 deficiency or inactivation. Binding of TM4SF5 to SLC27A2 increased gradually upon acute FA treatment, whereas TM4SF5 constitutively bound SLC27A5. Suppression of either SLC27A2 or SLC27A5 in hepatocytes expressing TM4SF5 differentially modulated initial and maximal FA uptake levels for a fast turnover of fatty acid. Altogether, TM4SF5 negatively modulates FA accumulation into hepatocytes via association with the transporters for an energy homeostasis, when FA are supplied acutely.

Fatty acid transport protein 2 reprograms neutrophils in cancer.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that are crucial for the regulation of immune responses in cancer. These cells contribute to the failure of cancer therapies and are associated with poor clinical outcomes. Despite recent advances in the understanding of PMN-MDSC biology, the mechanisms responsible for the pathological activation of neutrophils are not well defined, and this limits the selective targeting of these cells. Here we report that mouse and human PMN-MDSCs exclusively upregulate fatty acid transport protein 2 (FATP2). Overexpression of FATP2 in PMN-MDSCs was controlled by granulocyte-macrophage colony-stimulating factor, through the activation of the STAT5 transcription factor. Deletion of FATP2 abrogated the suppressive activity of PMN-MDSCs. The main mechanism of FATP2-mediated suppressive activity involved the uptake of arachidonic acid and the synthesis of prostaglandin E2. The selective pharmacological inhibition of FATP2 abrogated the activity of PMN-MDSCs and substantially delayed tumour progression. In combination with checkpoint inhibitors, FATP2 inhibition blocked tumour progression in mice. Thus, FATP2 mediates the acquisition of immunosuppressive activity by PMN-MDSCs and represents a target to inhibit the functions of PMN-MDSCs selectively and to improve the efficiency of cancer therapy.

PPARα and PPARγ activation attenuates total free fatty acid and triglyceride accumulation in macrophages via the inhibition of Fatp1 expression.

Lipid accumulation in macrophages interacts with microenvironment signals and accelerates diabetic atherosclerosis. However, the molecular mechanisms by which macrophage metabolism interacts with microenvironment signals during lipid accumulation are not clearly understood. Accordingly, an untargeted metabolomics approach was employed to characterize the metabolic reprogramming, and to identify potential regulatory targets related to lipid accumulation in macrophages treated with oleate, an important nutrient. The metabolomics approach revealed that multiple metabolic pathways were significantly disturbed in oleate-treated macrophages. We discovered that amino acids, nucleosides, lactate, monoacylglycerols, total free fatty acids (FFAs), and triglycerides (TGs) accumulated in oleate-treated macrophages, but these effects were effectively attenuated or even abolished by resveratrol. Notably, 1-monooleoylglycerol and 2-monooleoylglycerol showed the largest fold changes in the levels among the differential metabolites. Subsequently, we found that oleate triggered total FFA and TG accumulation in macrophages by accelerating FFA influx through the activation of Fatp1 expression, but this effect was attenuated by resveratrol via the activation of PPARα and PPARγ signaling. We verified that the activation of PPARα and PPARγ by WY14643 and pioglitazone, respectively, attenuated oleate triggered total FFA and TG accumulation in macrophages by repressing FFA import via the suppression of Fatp1 expression. Furthermore, the inhibition of Fatp1 by tumor necrosis factor α alleviated oleate-induced total FFA and TG accumulation in macrophages. This study provided the first demonstration that accumulation of amino acids, nucleosides, lactate, monoacylglycerols, total FFAs, and TGs in oleate-treated macrophages is effectively attenuated or even abolished by resveratrol, and that the activation of PPARα and PPARγ attenuates oleate-induced total FFA and TG accumulation via suppression of Fatp1 expression in macrophages. Therapeutic strategies aim to activate PPAR signaling, and to repress FFA import and triglyceride synthesis are promising approaches to reduce the risk of obesity, diabetes and atherosclerosis.

Fatty acid transport protein 1 enhances the macrophage inflammatory response by coupling with ceramide and c-Jun N-terminal kinase signaling.

Macrophages are important cells that need to be controlled at the site of inflammation. Several factors are involved in chronic inflammation and its timely resolution. Free fatty acids drive the inflammatory response in macrophages and contribute to the vicious cycle of the inflammatory response. However, the identity of the uptake pathways of fatty acids is not fully clear in macrophages and how the inflammatory responses are regulated by the uptake of fatty acids remain poorly understood. We investigated the relationship between fatty acid transport protein (FATP) and the inflammatory response signaling pathway in macrophages as the first report. The FATP family has composed six isoforms, FATP1-6. We found that FATP1 is the most highly expressed isoform in macrophages. Forced expression of FATP1 enhanced production of inflammatory cytokines, such as TNFα and IL-6 concomitant with the increased uptake of fatty acids, increased level of ceramide, and increased phosphorylation of c-Jun N-terminal kinase (JNK). The enhancement by FATP1 was abolished by treatment with a JNK inhibitor, NF-κB inhibitor, or ceramide synthesis inhibitor. siRNA-mediated knockdown of FATP1 strongly inhibited the production of TNFα and IL-6. Similarly, an inhibitor of FATP1 inhibited the production of TNFα and IL-6. Finally, an inhibitor of FATP1 attenuated the production of inflammatory cytokines in bronchoalveolar lavage fluid in an LPS-induced acute lung injury in vivo mouse model. In summary, we propose that FATP1 is an important regulator of inflammatory response signaling in macrophages. Our findings suggest that ceramide-JNK signaling is important to terminate or sustain inflammation.

MicroRNA-199a Targets the Fatty Acid Transport Protein 1 Gene and Inhibits the Adipogenic Trans-Differentiation of C2C12 Myoblasts.

BACKGROUND/AIMS: Muscle cells are able to trans-differentiate into adipocytes with adipogenesis induction. MicroRNAs (miRNAs), a class of small non-coding RNAs, widely participate in the regulation of growth and development of cells. However, the expression and regulatory role of miRNAs in the trans-differentiation of muscle cell are largely unknown. METHODS: C2C12 myoblasts were inducted to adipogenesis trans-differentiation and microarrays were used to assay the changes of expression profile of miRNAs. MiR-199a, a miRNA showed significant change in the trans-differentiation, was selected for the subsequent function study via over- expression and knock down. RESULTS: Dozens of miRNAs showed different changes followed the adipogenesis trans-differentiation of C2C12 cells. In which, miR-199a was decreased in the adipogenic cells and miR-199a over-expression inhibited the trans-differentiation and decreased lipid accumulation in the cells. Moreover, Fatty acid transport protein 1 (Fatp1), a major regulator of trans-membrane transportation and the oxidative metabolism of free fatty acids, was showed to be a target of miR-199a by computational and luciferase reporter assays. Additionally, Fatp1 knock-down by small interfering RNA had similar inhibitory effects on the trans-differentiation in C2C12 cells. CONCLUSION: Our study reveals an important role for miR-199a in the regulation of adipogenic trans-differentiation in muscle cells via suppression of Fatp1 gene.

Lactobacillus gasseri NT decreased visceral fat through enhancement of lipid excretion in feces of KK-A(y) mice.

Dietary supplementation with Lactobacillus gasseri NT significantly decreased visceral fat weight and triglyceride (TG) in the liver in KK-A(y) mice on a high-fat diet, but increased fecal TG. A decrease in lipase activity and down regulation of fatty acid transport proteins in the small intestine was involved in fat accumulation by L. gasseri NT.

Arylpiperazines as fatty acid transport protein 1 (FATP1) inhibitors with improved potency and pharmacokinetic properties.

The discovery and optimization of a novel series of FATP1 inhibitors are described. Through the derivatization process, arylpiperazine derivatives 5k and 12a were identified as possessing potent in vitro activity against human and mouse FATP1s as well as excellent pharmacokinetic properties. In vivo evaluation of triglyceride accumulation in the liver, white gastrocnemius muscle and soleus is also described.

Discovery and optimization of novel fatty acid transport protein 1 (FATP1) inhibitors.

The discovery, optimization and structure-activity relationship of novel FATP1 inhibitors have been described. The detailed SAR studies of each moiety of the inhibitors combined with metabolite analysis led to the identification of the potent inhibitors 11p and 11q with improved blood stability.

Development and validation of a high-throughput screening assay for human long-chain fatty acid transport proteins 4 and 5.

Dietary long-chain fatty acid (LCFA) uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). Six subtypes of this transporter are differentially expressed throughout the human and rodent body. To facilitate drugs discovery against FATP subtypes, the authors used mammalian cell lines stably expressing the recombinant human FATP4 and 5 and developed a high-throughput screening (HTS) assay using a 96-well fluorometric imaging plate reader (FLIPR). LCFA uptake signal-to-background ratios were between 3- and 5-fold. Two 4-aryl-dihydropyrimidinones, j3 and j5, produced inhibition of FATP4 with a half-maximal inhibitory concentration (IC(50)) of 0.21 and 0.63 microM, respectively, and displayed approximately 100-fold selectivity over FATP5. The US Drug Collection library was screened against the FATP5. A hit rate of around 0.4% was observed with a Z' factor of 0.6 +/- 0.2. Two confirmed hits are bile acids, chenodiol and ursodiol with an IC(50) of 2.4 and 0.22 microM, respectively. To increase throughput, a single time point measurement in a 384-well format was developed using the Analyst HT, and the results are comparable with the 96-well format. In conclusion, the FATP4 and 5 cell-based fluorescence assays are suitable for a primary drug screen, whereas differentiated cell lines are useful for a secondary drug screen.

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(50)s 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.

Regulation of cardiolipin biosynthesis by fatty acid transport protein-1 IN HEK 293 cells.

Cardiolipin (CL) is a major phospholipid involved in energy metabolism mammalian mitochondria and fatty acid transport protein-1 (FATP-1) is a fatty acid transport protein that may regulate the intracellular level of fatty acyl-Coenzyme A's. Since fatty acids are required for oxidative phosphorylation via mitochondrial oxidation, we examined the effect of altering FATP-1 levels on CL biosynthesis. HEK-293 mock- and FATP-1 siRNA transfected cells or mock and FATP-1 expressing cells were incubated for 24 h with 0.1 mM oleic acid bound to albumin (1:1 molar ratio) then incubated for 24 h with 0.1 mM [1,3-(3)H]glycerol and radioactivity incorporated into CL determined. FATP-1 siRNA transfected cells exhibited reduced FATP-1 mRNA and increased incorporation of [1,3-(3)H]glycerol into CL (2-fold, p<0.05) compared to controls indicating elevation in de novo CL biosynthesis. The reason for this was an increase in [1,3-(3)H]glycerol uptake and increase in activity and mRNA expression of the CL biosynthetic enzymes. In contrast, expression of FATP-1 resulted a reduction in incorporation of [1,3-(3)H]glycerol into CL (65%, p<0.05) indicating reduced CL synthesis. [1,3-(3)H]Glycerol uptake was unaltered whereas activity of cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol synthetase (CDS) and CDS-2 mRNA expression were reduced in FATP-1 expressing cells compared to control. In addition, in vitro CDS activity was reduced by exogenous addition of oleoyl-Coenzyme A. The data indicate that CL de novo biosynthesis may be regulated by FATP-1 through CDS-2 expression in HEK 293 cells.

High-throughput screening for fatty acid uptake inhibitors in humanized yeast identifies atypical antipsychotic drugs that cause dyslipidemias.

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C(1)-BODIPY-C(12)). The library used consisted of 2,080 compounds with known biological activities. Of these, approximately 1.8% reduced cell-associated C(1)-BODIPY-C(12) fluorescence and were selected as potential inhibitors of human FATP2-mediated fatty acid uptake. Based on secondary screens, 28 compounds were selected as potential fatty acid uptake inhibitors. Some compounds fell into four groups with similar structural features. The largest group was structurally related to a family of tricyclic, phenothiazine-derived drugs used to treat schizophrenia and related psychiatric disorders, which are also known to cause metabolic side effects, including hypertriglyceridemia. Potential hit compounds were studied for specificity of interaction with human FATP and efficacy in human Caco-2 cells. This study validates this screening system as useful to assess the impact of drugs in preclinical screening for fatty acid uptake.

Identification and characterization of 4-aryl-3,4-dihydropyrimidin-2(1H)-ones as inhibitors of the fatty acid transporter FATP4.

Several potent, cell permeable 4-aryl-dihydropyrimidinones have been identified as inhibitors of FATP4. Lipophilic ester substituents at the 5-position and substitution at the para-position (optimal groups being -NO(2) and CF(3)) of the 4-aryl group led to active compounds. In two cases racemates were resolved and the S enantiomers shown to have higher potencies.

Oleic acid uptake by in vitro English sparrow skeletal muscle.

Studies of prolonged avian flight have shown it to require large amounts of energy supplied mainly by free fatty acids (FFA). In the present study, the high levels of plasma ketone bodies found in sparrows (2.58 mmol l(-1)) are supportive of the use of fatty acids for flight. To determine the nature of fatty acid (oleic acid, OA) uptake, various pharmacological agents were used. The uptake of OA was examined using the extensor digitorum communis (EDC) muscle of English sparrows incubated in vitro. Initial studies demonstrated that radiolabeled OA uptake decreased in the presence of increasing unlabeled OA, suggesting that uptake occurred by a facilitative transport process. To further characterize OA uptake, EDC muscles were incubated with either: insulin (2 ng ml(-1)), insulin-like growth factor isoform-1 (IGF-I; 48 ng ml(-1)), 5'-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 2 mmol) or caffeine (5 mmol). Insulin, but not IGF-I, significantly increased OA uptake by avian EDC (P < 0.01). Caffeine and AICAR were ineffective at increasing OA uptake. A specific inhibitor of FFA transport by fatty acid transporters (FAT/CD36), sulfo-N-succinimidyl oleate (SSO; 500 micromoles), significantly decreased OA uptake at 2.5 min. The effectiveness of SSO suggests that a FAT/CD36-like protein is expressed in avian tissues. As uptake of OA was not completely blocked by SSO, it is likely that other mechanisms for FFA movement across membranes, such as diffusion, may be present.

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation.

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 muM) palmitate oxidation was decreased by 95% (P<0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P<0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.

Anandamide transport: a critical review.

Anandamide (AEA) uptake has been described over the last decade to occur by facilitated diffusion, but a protein has yet to be isolated. In some cell types, it has recently been suggested that AEA, an uncharged hydrophobic molecule, passively diffuses through the plasma membrane in a process that is not protein-mediated. Since that observation, recent kinetics studies (using varying assay conditions) have both supported and denied the presence of an AEA transporter. In this review, we analyze the current literature exploring the mechanism of AEA uptake and endeavor to explain the reasons for the divergent views. One of the main variables among laboratories is the incubation time of the cells with AEA. Initial kinetics (at time points <1 min depending upon the cell type) isolate events that occur at the plasma membrane and are most useful to study saturability of uptake and effects of purported transport inhibitors upon uptake. Results with longer incubation times reflect events not only at the plasma membrane but also interactions at intracellular sites that may include enzyme(s), other proteins, or specialized lipid-binding domains. Furthermore, at long incubation times, antagonists to AEA receptors reduce AEA uptake. Another complicating factor in AEA transport studies is the nonspecific binding to plastic culture dishes. The magnitude of this effect may exceed AEA uptake into cells. Likewise, AEA may be released from plastic culture dishes (without cells) in such a manner as to mimic efflux from cells. AEA transport protocols using BSA, similar to the method used for fatty acid uptake studies, are gaining acceptance. This may improve AEA solution stability and minimize binding to plastic, although some groups report that BSA interferes with uptake. In response to criticisms that many transport inhibitors also inhibit the fatty acid amide hydrolase (FAAH), new compounds have recently been synthesized. Following their characterization in FAAH+/+ and FAAH-/- cells and transgenic mice, several inhibitors have been shown to have physiological activity in FAAH-/- mice. Their targets are now being characterized with the possibility that a protein transporter for AEA may be characterized.