Inhibition of fatty acid binding protein-5 in the basolateral amygdala induces anxiolytic effects and accelerates fear memory extinction.

RATIONALE: The endocannabinoid (eCB) system critically controls anxiety and fear-related behaviours. Anandamide (AEA), a prominent eCB ligand, is a hydrophobic lipid that requires chaperone proteins such as Fatty Acid Binding Proteins (FABPs) for intracellular transport. Intracellular AEA transport is necessary for degradation, so blocking FABP activity increases AEA neurotransmission. OBJECTIVE: To investigate the effects of a novel FABP5 inhibitor (SBFI-103) in the basolateral amygdala (BLA) on anxiety and fear memory. METHODS: We infused SBFI-103 (0.5 µg-5 µg) to the BLA of adult male Sprague Dawley rats and ran various anxiety and fear memory behavioural assays, neurophysiological recordings, and localized molecular signaling analyses. We also co-infused SBFI-103 with the AEA inhibitor, LEI-401 (3 µg and 10 µg) to investigate the potential role of AEA in these phenomena. RESULTS: Acute intra-BLA administration of SBFI-103 produced strong anxiolytic effects across multiple behavioural tests. Furthermore, animals exhibited acute and long-term accelerated associative fear memory extinction following intra-BLA FABP5 inhibition. In addition, BLA FABP5 inhibition induced strong modulatory effects on putative PFC pyramidal neurons along with significantly increased gamma oscillation power. Finally, we observed local BLA changes in the phosphorylation activity of various anxiety- and fear memory-related molecular biomarkers in the PI3K/Akt and MAPK/Erk signaling pathways. At all three levels of analyses, we found the functional effects of SBFI-103 depend on availability of the AEA ligand. CONCLUSIONS: These findings demonstrate a novel intra-BLA FABP5 signaling mechanism regulating anxiety and fear memory behaviours, neuronal activity states, local anxiety-related molecular pathways, and functional AEA modulation.

FABP5 is important for cognitive function and is an important regulator of the physiological effects and pharmacokinetics of acute Δ9 tetrahydrocannabinol inhalation in mice.

Fatty acid binding protein 5 (FABP5) interacts with the endocannabinoid system in the brain via intracellular transport of anandamide, as well as Δ9-tetrahydrocannabinol (THC), the main psychoactive component of cannabis. Previous work has established the behavioral effects of genetic deletion of FABP5, but not in the presence of THC. The present study sought to further elucidate the role of FABP5 on the pharmacokinetic and behavioral response to THC through global deletion. Adult FABP5+/+ and FABP5-/- mice were tested for behavioral response to THC using Open Field (OF), Novel Object Recognition (NOR), T-Maze, Morris Water Maze (MWM), and Elevated Plus Maze (EPM). An additional cohort of mice was used to harvest blood, brains, and liver samples to measure THC and metabolites after acute administration of THC. Behavioral tests showed that some cognitive deficits from FABP5 deletion, particularly in MWM, were blocked by THC administration, while this was not observed in other measures of memory and anxiety (such as T-Maze and EPM). Measurement of THC and metabolites in blood serum and brain tissue through UPLC-MS/MS analysis showed that the pharmacokinetics of THC was altered by FABP5. The present study shows further evidence of the importance of FABP5 in cognitive function. Additionally, results showed that FABP5 is an important regulator of the physiological effects and pharmacokinetics of THC.

FABP4 Regulates Cell Proliferation, Stemness, Apoptosis, and Glycolysis in Colorectal Cancer via Modulating ROS/ERK/mTOR Pathway.

BACKGROUND: Colorectal cancer is a common digestive tract malignancy. This study aimed to expound the functional role of fatty-acid-binding protein 4 (FABP4) and the potential underlying mechanisms in the development of colorectal cancer. METHODS: Several techniques were utilized to investigate the role of FABP4 in colorectal cancer. FABP4 mRNA expression was quantified using Real time-quantitative PCR (RT-qPCR). Cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), sphere formation assays and flow cytometry evaluated cell growth, stemness, and apoptosis in SW480 and HT29 cells. Glycolysis was assessed via extracellular acidification rate (ECAR) , lactate production, glucose uptake, adenosine triphosphate (ATP)/adenosine 5'-diphosphate (ADP) ratio, and Glut1 and Elevated lactate dehydrogenase A (LDHA) protein expression. Reactive oxygen species (ROS) levels were analyzed by flow cytometry. Western blot measured the protein expression of FABP4, Proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, Glut1, LDHA, stemness makers (Sox2, Oct4, and ALDHA1), and extracellular regulated protein kinase (ERK)/mammalian target of rapamycin (mTOR) pathway proteins. In vivo experiments, BALB/c nude mice (n = 12) were inoculated with 200 µL HT29 cells (5 × 106 cells) transfected with sh-FABP4 or short hairpin (sh)-negative control (NC), forming two groups with 6 mice each. The in vivo mice tumor model allowed for evaluating FABP4's impact on tumor growth. RESULTS: FABP4 was significantly upregulated in colorectal cancer tissues and cells (p < 0.05). FABP4 knockdown markedly inhibited cell proliferation, stemness, and glycolysis, while promoting apoptosis in these cells (p < 0.05). Additionally, FABP4 depletion led to a significant increase in ROS level (p < 0.05). However, N-acetyl-L-cysteine (NAC) (p < 0.05), a ROS scavenger, mitigates these effects. Furthermore, the effects of FABP4 depletion on cell growth, stemness, glycolysis, and apoptosis in colorectal cancer cells were also retarded by NAC (p < 0.05). Notably, FABP4 knockdown also suppressed the ERK/mTOR pathway, suggesting its regulation via ROS (p < 0.05). In vivo study results showed, FABP4 depletion significantly curbed tumor growth in colorectal cancer (p < 0.05). CONCLUSIONS: These results suggest that FABP4 depletion inhibits colorectal cancer progression by modulating cell growth, stemness, glycolysis and apoptosis. This regulation occurs through the ROS/ERK/mTOR pathway.

FABP5 regulates lipid metabolism to facilitate pancreatic neuroendocrine neoplasms progression via FASN mediated Wnt/ß-catenin pathway.

Pancreatic neuroendocrine neoplasms (pNENs) are among the most frequently occurring neuroendocrine neoplasms (NENs) and require targeted therapy. High levels of fatty acid binding protein 5 (FABP5) are involved in tumor progression, but its role in pNENs remains unclear. We investigated the mRNA and protein levels of FABP5 in pNEN tissues and cell lines and found them to be upregulated. We evaluated changes in cell proliferation using CCK-8, colony formation, and 5-ethynyl-2'-deoxyuridine assays and examined the effects on cell migration and invasion using transwell assays. We found that knockdown of FABP5 suppressed the proliferation, migration, and invasion of pNEN cell lines, while overexpression of FABP5 had the opposite effect. Co-immunoprecipitation experiments were performed to clarify the interaction between FABP5 and fatty acid synthase (FASN). We further showed that FABP5 regulates the expression of FASN via the ubiquitin proteasome pathway and both proteins facilitate the progression of pNENs. Our study demonstrated that FABP5 acts as an oncogene by promoting lipid droplet deposition and activating the WNT/ß-catenin signaling pathway. Moreover, the carcinogenic effects of FABP5 can be reversed by orlistat, providing a novel therapeutic intervention option.

Identification of Andrographolide as a novel FABP4 inhibitor for osteoarthritis treatment.

BACKGROUND AND PURPOSE: Fatty acid binding protein 4 (FABP4) has been identified as a contributor to cartilage degradation in osteoarthritis (OA) patients, and inhibiting FABP4 using small molecules has emerged as a promising approach for developing OA drugs. Our previous research showed that Andrographis paniculata, a medicinal plant, strongly inhibits FABP4 activity. This led us to hypothesize that Andrographis paniculata ingredients might have protective effects on OA cartilage through FABP4 inhibition. METHODS: We analyzed scRNA-seq data from joint tissue of OA patients (GSE152805; GSE145286) using Scanpy 1.9.1 and Single Cell Portal. We conducted docking analysis of FABP4 inhibitors using Autodock Vina v.1.0.2. We evaluated the anti-FABP4 activity using a fluorescence displacement assay and measured the fatty acid oxidation (FAO) activity using the FAOBlue assay. We used H2DCF-DA to measure reactive oxygen species (ROS) levels. We studied signaling pathways using bulk RNA sequencing and western blot analysis in human C28/I2 chondrocytes. We evaluated anti-OA activity in monosodium iodoacetate (MIA)-induced rats. RESULTS: We identified Andrographolide (AP) as a novel FABP4 inhibitor. Bulk RNA-sequencing analysis revealed that FABP4 upregulated FAO and ROS in chondrocytes, which was inhibited by AP. ROS generation activated the NF-κB pathway, leading to overexpression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), which is a responsible factor for cartilage degradation in OA patients. AP inhibited FABP4, thereby reducing the overexpression of ADAMTS4 by inhibiting the NF-κB pathway. In MIA rats, AP treatment reduced the overexpression of ADAMTS4, repaired cartilage and subchondral bone, and promoted cartilage regeneration. CONCLUSION: Our results indicate that the inhibition of FABP4 activity by AP explains the anti-OA properties of Andrographis paniculata by protecting against cartilage degradation in OA patients. Additionally, our findings suggest that AP may be a promising therapeutic agent for OA treatment due to its ability to alleviate cartilage damage and bone erosion.

Intestinal peroxisome proliferator-activated receptor α-fatty acid-binding protein 1 axis modulates nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor α (PPARα) regulates fatty acid transport and catabolism in liver. However, the role of intestinal PPARα in lipid homeostasis is largely unknown. Here, intestinal PPARα was examined for its modulation of obesity and NASH. APPROACH AND RESULTS: Intestinal PPARα was activated and fatty acid-binding protein 1 (FABP1) up-regulated in humans with obesity and high-fat diet (HFD)-fed mice as revealed by using human intestine specimens or HFD/high-fat, high-cholesterol, and high-fructose diet (HFCFD)-fed C57BL/6N mice and PPARA -humanized, peroxisome proliferator response element-luciferase mice. Intestine-specific Ppara or Fabp1 disruption in mice fed a HFD or HFCFD decreased obesity-associated metabolic disorders and NASH. Molecular analyses by luciferase reporter assays and chromatin immunoprecipitation assays in combination with fatty acid uptake assays in primary intestinal organoids revealed that intestinal PPARα induced the expression of FABP1 that in turn mediated the effects of intestinal PPARα in modulating fatty acid uptake. The PPARα antagonist GW6471 improved obesity and NASH, dependent on intestinal PPARα or FABP1. Double-knockout ( Ppara/Fabp1ΔIE ) mice demonstrated that intestinal Ppara disruption failed to further decrease obesity and NASH in the absence of intestinal FABP1. Translationally, GW6471 reduced human PPARA-driven intestinal fatty acid uptake and improved obesity-related metabolic dysfunctions in PPARA -humanized, but not Ppara -null, mice. CONCLUSIONS: Intestinal PPARα signaling promotes NASH progression through regulating dietary fatty acid uptake through modulation of FABP1, which provides a compelling therapeutic target for NASH treatment.

Inhibitory Effects of Hydrolysable Tannins on Lipid Accumulation in 3T3-L1 Cells.

Obesity is currently the most common cause of metabolic diseases including type 2 diabetes and hyperlipidemia. Obesity results from excess lipid accumulation in adipose tissue. Several studies have investigated the inhibitory effects of natural plant-derived products on adipocyte differentiation and lipid accumulation. In this study, we examined the effect of hydrolysable tannins composed of gallic acid and glucose on adipocyte differentiation in 3T3-L1 cells. 1,2,3,4,6-Penta-O-galloyl-ß-D-glucose (PGG) (1), a representative gallotannin, inhibited lipid accumulation in 3T3-L1 cells, whereas ellagitannins (tellimagrandin I, eugeniin and casuarictin) did not. The expression of adipocyte differentiation-related genes, including peroxisome proliferator activator γ2 (Pparγ2), CCAAT/enhancer binding protein α (C/EBPα) and adipocyte fatty acid binding protein (aP2), was significantly suppressed in PGG (1)-treated 3T3-L1 cells beginning at day 2 after induction of differentiation. While PGG (1) did not directly reduce Pparγ2 expression, it reduced the expression of its target genes in mature adipocytes. In addition, PGG (1) treatment inhibited mitotic clonal expansion, one of earliest events of adipocyte differentiation. These findings indicate that PGG (1) has an inhibitory effect on adipocyte differentiation through the suppression of mitotic clonal expansion.

Transcriptome-based biomarker gene screening and evaluation of the extracellular fatty acid-binding protein (Ex-FABP) on immune and angiogenesis-related genes in chicken erythrocytes of tibial dyschondroplasia.

BACKGROUND: Tibial dyschondroplasia (TD) is a bone disorder in which dead chondrocytes accumulate as a result of apoptosis and non-vascularization in the tibial bone of broiler chickens. The pathogenicity of TD is under extensive research but is yet not fully understood. Several studies have linked it to apoptosis and non-vascularization in the tibial growth plate (GP). We conceived the idea to find the differentially expressed genes (DEGs) in chicken erythrocytes which vary in expression over time using a likelihood-ratio test (LRT). Thiram was used to induce TD in chickens, and then injected Ex-FABP protein at 0, 20, and 50 µg.kg-1 to evaluate its therapeutic effect on 30 screened immunity and angiogenesis-related genes using quantitative PCR (qPCR). The histopathology was also performed in TD chickens to explore the shape, circularity, arrangements of chondrocytes and blood vessels. RESULTS: Clinical lameness was observed in TD chickens, which decreased with the injection of Ex-FABP. Histopathological findings support Ex-FABP as a therapeutic agent for the morphology and vascularization of affected chondrocytes in TD chickens. qPCR results of 10 immunity (TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, IL-7, MyD88, MHCII, and TRAF6) and 20 angiogenesis-related genes (ITGAV, ITGA2, ITGB2, ITGB3, ITGA5, IL1R1, TBXA2R, RPL17, F13A1, CLU, RAC2, RAP1B, GIT1, FYN, IQGAP2, PTCH1, NCOR2, VAV-like, PTPN11, MAML3) regulated when Ex-FABP is injected to TD chickens. CONCLUSION: Immunity and angiogenesis-related genes can be responsible for apoptosis of chondrocytes and vascularization in tibial GP. Injection of Ex-FABP protein to thiram induced TD chickens decrease the chondrocytes damage and improves vascularization.

Human Neural Stem Cell Secretome Inhibits Lipopolysaccharide-Induced Neuroinflammation Through Modulating Microglia Polarization by Activating Peroxisome Proliferator-Activated Receptor Gamma.

Neuroinflammation is one of the typical events in multiple neurodegenerative diseases, whereas microglia are the critical participants in the pathogenesis of neuroinflammation. Several studies suggest that neural stem cells (NSCs) present immunomodulatory benefits due to their paracrine products, which contain mounting trophic factors. In the current study, the anti-inflammatory effects of NSC secretome (NSC-S) on lipopolysaccharide (LPS)-induced neuroinflammatory models were evaluated in vivo and the underlying mechanism was further investigated in vitro. It was revealed that NSC-S significantly attenuated the severity of LPS-induced behavior disorders and inflammatory response in mice. In vitro studies found that NSC-S significantly promoted the polarization of microglia from proinflammatory M1 to anti-inflammatory M2 phenotype, and reduced the production of proinflammatory cytokines, whereas elevated anti-inflammatory cytokines in BV2 cells. NSC-S promoted peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway activation. However, these effects of NSC-S were abrogated by PPAR-γ inhibitor GW9662. Notably, the fatty acid-binding protein 5 (FABP5) in NSC-S may mediate PPAR-γ activation and inflammation remission. In summary, NSC-S promotes the regression of LPS-induced microglia-mediated inflammation through the PPAR-γ pathway. This function might be achieved through FABP5.

Cytochrome P450 2E1-dependent hepatic ethanol metabolism induces fatty acid-binding protein 4 and steatosis.

BACKGROUND: Hepatic steatosis is an early pathology of alcohol-associated liver disease (ALD). Fatty acid-binding protein-4 (FABP4, a FABP not normally produced in the liver) is secreted by hepatocytes in ALD and stimulates hepatoma proliferation and migration. This study sought to investigate the mechanism[s] by which hepatic ethanol metabolism regulates FABP4 and steatosis. METHODS: Human hepatoma cells (HepG2/HuH7) and cells stably transfected to express cytochrome P450 2E1 (CYP2E1), were exposed to ethanol in the absence or presence of chlormethiazole (a CYP2E1-inhibitor; CMZ) and/or EX-527 (a sirtuin-1 [SIRT1] inhibitor). The culture medium was analyzed for ethanol metabolism and FABP4 protein abundance. Cells were analyzed for FABP4 mRNA expression, SIRT1 protein abundance, and neutral lipid accumulation. In parallel, cells were analyzed for forkhead box O1 [FOXO1], ß-catenin, peroxisome proliferator-activated receptor-α [PPARα], and lipin-1α protein abundance in the absence or presence of ethanol and pharmacological inhibitors of the respective target proteins. RESULTS: CYP2E1-dependent ethanol metabolism inhibited the amount of SIRT1 protein detected, concomitant with increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation, effects abolished by CMZ. Analysis of pathways associated with lipid oxidation revealed increased FOXO1 nuclear localization and decreased ß-catenin, PPARα, and lipin-1α protein levels in CYP2E1-expressing cells in the presence of ethanol. Pharmacological inhibition of SIRT1 mimicked the effects of ethanol, while inhibition of FOXO1 abrogated the effect of ethanol on FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation in CYP2E1-expressing cells. Pharmacological inhibition of ß-catenin, PPARα, or lipin-1α failed to alter the effects of ethanol on FABP4 or neutral lipid accumulation. CONCLUSION: CYP2E1-dependent ethanol metabolism inhibits SIRT1-FOXO1 signaling, which leads to increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation. These data suggest that FABP4 released from steatotic hepatocytes could play a role in promoting tumor cell expansion in the setting of ALD and represents a potential target for therapeutic intervention.

Fasciola hepatica fatty acid binding protein (Fh12) induces apoptosis and tolerogenic properties in murine bone marrow derived dendritic cells.

In a previous study we demonstrated that Fasciola hepatica fatty acid binding protein (Fh12) significantly suppress macrophage function by inhibiting IL-6, IL-1ß, tumor necrosis factor (TNF)-α and IL-12 production in TLR4-stimulated murine macrophages, an effect mediated through the signaling of CD14 co-receptor without affecting the viability of these cells. Given that dendritic cells (DCs) are immune cells that play a central role in the initiation of primary immune responses and that are the only antigen-presenting cells capable of stimulating naïve T-cells, in the present study we investigated the effect of Fh12 on DCs. We found that Fh12 exerts a strong suppressive effect on activation and function of DCs. However, in contrast to the effect observed on macrophages, Fh12 induces early and late apoptosis of DCs being this phenomenon dose-dependent and CD14-coreceptor independent. At low concentration Fh12 modulates the LPS-induced DCs maturation status by suppressing the MHC-II, and co-stimulatory molecules CD40 and CD80 surface expression together with the pro-inflammatory cytokines IL-12p70 and IL-6 production whereas increase the IL-10 levels. Besides, Fh12 decreased the ability of LPS-activated DCs to induce IFN-γ production against allogeneic splenocytes, while increasing IL-4 production. We have described for the first time the ability of Fh12 to modify selectively the viability of DCs by apoptosis induction. The selective diminution in DCs survival could be a F. hepatica strategy in order to prevent a host immune response during the earliest phases of infection.

Design of a Novel Multi-Epitope Vaccine Against Echinococcus granulosus in Immunoinformatics.

All the time, echinococcosis is a global zoonotic disease which seriously endangers public health all over the world. In order to speed up the development process of anti-Echinococcus granulosus vaccine, at the same time, it can also save economic cost. In this study, immunoinformatics tools and molecular docking methods were used to predict and screen the antigen epitopes of Echinococcus granulosus, to design a multi-epitope vaccine containing B- and T-cell epitopes. The multi-epitope vaccine could activate B lymphocytes to produce specific antibodies theoretically, which could protect the human body against Echinococcus granulosus infection. It also could activate T lymphocytes and clear the infected parasites in the body. In this study, four CD8+ T-cell epitopes, three CD4+ T-cell epitopes and four B-cell epitopes of Protein EgTeg were identified by immunoinformatics methods. Meanwhile, three CD8+ T-cell epitopes, two CD4+ T-cell epitopes and four B-cell epitopes of Protein EgFABP1 were identified. We constructed the multi-epitope vaccine using linker proteins. The study based on the traditional methods of antigen epitope prediction, further optimized the prediction results combined with molecular docking technology and improved the precision and accuracy of the results. Finally, in vivo and in vitro experiments had verified that the vaccine designed in this study had good antigenicity and immunogenicity.

Development of FABP4/5 inhibitors with potential therapeutic effect on type 2 Diabetes Mellitus.

Fatty acid-binding protein 4 (FABP4) and fatty acid-binding protein 5 (FABP5) are promising therapeutic targets for the treatment of various metabolic diseases. However, the weak potency, low selectivity over FABP3, or poor pharmacokinetic profiles of currently reported dual FABP4/5 inhibitors impeded further research. Here, we described the characterization of a series of dual FABP4/5 inhibitors with improved metabolic stabilities and physicochemical properties based on our previous studies. Among the compounds, D9 and E1 exhibited good inhibitory activities against FABP4/5 and favorable selectivity over FABP3 in vitro. In cell-based assays, D9 and E1 exerted a decrease of FABP4 secretion, a strong anti-lipolytic effect in mature adipocytes, and suppression of MCP-1 expression in THP-1 macrophages. Moreover, D9 and E1 possessed good metabolic stabilities in mouse hepatic microsomes and acceptable pharmacokinetics profiles in ICR mice. Further in vivo experiments showed that D9 and E1 could potently decrease serum FABP4 levels and ameliorate glucose metabolism disorders in obese diabetic db/db mice. These results demonstrated that D9 and E1 could serve as lead compounds for the development of novel anti-diabetic drugs.

The fatty acid-binding protein (FABP) decreases the clinical signs and modulates immune responses in a mouse model of experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: An increasing body of studies has shown that Fasciola hepatica can affect immune responses. This study explored whether the fatty acid-binding protein (FABP) of F. hepatica can modulate the immune system in a mouse model of experimental autoimmune encephalomyelitis (EAE). METHODS: EAE-induced C57BL/6 mice were treated with vehicle, F. hepatica total extract (TE) or FABP. The clinical signs, body weights, and the expression of IFN-γ, T-bet, IL-4, GATA3, IL-17, RORγ, TGF-ß, FOXP3, IL-10, TNF-α genes and proteins were determined in the isolated CD4+ splenocytes. Besides, the percentage of Treg cells and degree of demyelination were evaluated. RESULTS: We found that TE and FABP treatments decreased the clinical scores, lymphocyte infiltration rate, and demyelinated plaques in EAE mice. The expressions of IL-4 and GATA3 were increased, whereas IL-17 and TNF-α were down-regulated. FABP did not affect the expression of IFN-γ, RORγ, IL-10, and TGF-ß genes or proteins but reduced the expression of T-bet. TE administration did not affect the expression of IL-10 and the Tbet genes, and increased the expression levels of IFN-γ and FOXP3 in CD4+ lymphocytes. Both FABP and TE treatment did not affect the Treg cell percentage. CONCLUSION: This study indicates that F. hepatica FABP and TE can suppress the inflammatory responses in EAE-induced mice and shift the immune system toward Th2 responses. However, FABP exerts stronger anti-inflammatory effects and seems to be more effective than TE for EAE treatment.

Effect of cogon grass root ethanol extract on fatty acid binding protein 4 and oxidative stress markers in a sepsis mouse model.

Background: Sepsis causes several immunological and metabolic alterations that induce oxidative stress. The modulation of fatty acid-binding protein 4 (FABP4) has been shown to worsen this condition. Extract of cogon grass root (ECGR) contains flavonoids and isoeugenol compounds that exhibit anti-inflammatory and antioxidant properties. This study aimed to assess the effects of ECGR on FABP4 and oxidative stress-related factors in a sepsis mouse model. Methods: Twenty-nine male mice ( Mus musculus) of the Deutsche Denken Yoken strain were divided into four groups: group 1, control; group 2, mice treated with 10 µL/kg body weight (BW) lipopolysaccharide (LPS); and groups 3 and 4, mice pre-treated with 90 and 115 mg/kg BW, respectively, and then treated with 10 µL/kg BW LPS for 14 d. Blood, liver, lymph, and cardiac tissue samples were collected and subjected to histological and complete blood examinations. Antioxidant (Glutathione peroxidase 3 (GPx3) and superoxide dismutase), FABP4 levels, and immune system-associated biomarker levels (TNF-α, IL-6 and IL-1ß) were measured. Results: Significant increases in platelet levels (p = 0.03), cardiomyocyte counts (p =0.004), and hepatocyte counts (p = 0.0004) were observed in group 4 compared with those in group 2. Conversely, compared with those in group 2, there were significant decreases in TNF-α expression in group 3 (p = 0.004), white pulp length and width in group 4 (p = 0.001), FABP4 levels in groups 3 and 4 (p = 0.015 and p = 0.012, respectively), lymphocyte counts in group 4 (p = 0.009), and monocyte counts (p = 0.000) and polymorphonuclear cell counts in the livers (p = 0.000) and hearts (p = 0.000) of groups 3 and 4. Gpx3 activity was significantly higher in group 3 than in group 1 (p = 0.04). Conclusions: ECGR reduces FABP4 level and modulating oxidative stress markers in sepsis mouse model.

Exogenous FABP4 interferes with differentiation, promotes lipolysis and inflammation in adipocytes.

PURPOSE: Fatty acid binding protein 4 (FABP4) has been demonstrated to be secreted from adipocytes in an unconventional pathway associated with lipolysis. Circulating FABP4 is elevated in metabolic disorders and has been shown to affect various peripheral cells such as pancreatic ß-cells, hepatocytes and macrophages, but its effects on adipocytes remains unclear. The aim of this study was to investigate the effects of exogenous FABP4 (eFABP4) on adipocyte differentiation and function. METHODS: 3T3-L1 pre-adipocytes or mature adipocytes were treated with recombinant FABP4 in the absence or presence of FABP4 inhibitor I-9/p38 MAPK inhibitor SB203580; Meanwhile male C57BL/6J mice were subcutaneously injected twice a day with recombinant FABP4 (0.35 mg/kg) with or without I-9 (50 mg/kg) for 2 weeks. The effects of eFABP4 on differentiation, lipolysis and inflammation were determined by triglyceride measurement or lipolysis assay, western blotting, or RT-qPCR analysis. RESULTS: eFABP4 treatment significantly reduced intracellular triglyceride content and decreased expression of adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα), intracellular FABP4, and adiponectin in 3T3-L1 cells. Besides, eFABP4 promoted lipolysis and inflammation in differentiated 3T3-L1 adipocytes as well as in adipose tissue of eFABP4-treated C57BL/6J mice, with elevated gene expression of monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, and elevated protein expression of adipose triglyceride lipase (ATGL), phosphorylation of hormone-sensitive lipase (HSL) (Ser-660), p38, and nuclear factor-kappa B (NF-κB). The pro-inflammatory and pro-lipolytic effects of eFABP4 could be reversed by SB203580/I-9. CONCLUSIONS: These findings indicate that eFABP4 interferes with adipocyte differentiation, induces p38/HSL mediated lipolysis and p38/NF-κB mediated inflammation in adipocytes in vitro and in vivo.

Cellular growth and tube formation of HTR8/SVneo trophoblast: effects of exogenously added fatty acid-binding protein-4 and its inhibitor.

Adequate placental angiogenesis is critical for the establishment of the placental circulation and thus for normal feto-placental growth and development. Fatty acid-binding protein-4 (FABP4) plays a pro-angiogenic role in endothelial cells; however, very little information is available in placental first trimester trophoblast cells. Here we report that exogenously added FABP4 (exo-FABP4) stimulated tube formation (as a measure of in vitro angiogenesis) in HTR8/SVneo trophoblastic cells. HTR-8/SVneo cells were incubated in the presence of exogenously added FABP4 at different concentrations and time points. Cellular growth, proliferation, in vitro tube formation, expression of growth stimulatory-, fatty acid transporters, and angiogenic genes were investigated. Internalization of exo-FABP4 was carried out using immunocytochemistry. Radioactive fatty acid uptake was determined in the presence and absence of FABP4 metabolic inhibitor. Exo-FABP4 (10-100 ng/ml) stimulated proliferation of HTR8/SVneo cells as compared to control. Exo-FABP4 dose dependently increased growth and viability of the cells to the similar extent as done by 50 µM of arachidonic acid. Exo-FABP4-induced tube formation and proliferation were significantly inhibited by FABP4 (BMS309403) inhibitor. Exo-FABP4 stimulated the expression of growth stimulatory genes such as tissue inhibitor of matrix metalloproteinases-1 (TIMP1), insulin-like growth factor 1 (IGF1), and also prokineticin 2 (PROK2), the pro-angiogenic mediators in these cells. In addition, expressions of genes associated with proliferation and differentiation such as sonic hedgehog (SHH) and WNT1 inducible signalling pathway protein 1 (WISP1) were significantly expressed when cells were exposed to exo-FABP4. Our findings reveal a pro-angiogenic role of FABP4 in first trimester placental trophoblast cells and its regulation may have impact in placental physiology.

Recombinant Fasciola hepatica fatty acid binding protein suppresses toll-like receptor stimulation in response to multiple bacterial ligands.

Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1ß and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.

Exogenous FABP4 induces endoplasmic reticulum stress in HepG2 liver cells.

BACKGROUND AND AIMS: Fatty acid binding protein 4 (FABP4) is an intracellular fatty acid (FA) carrier protein that is, in part, secreted into circulation. Circulating FABP4 levels are increased in obesity, diabetes and other insulin resistance (IR) diseases. FAs contribute to IR by promoting endoplasmic reticulum stress (ER stress) and altering the insulin signaling pathway. The effect of FABP4 on ER stress in the liver is not known. The aim of this study was to investigate whether exogenous FABP4 (eFABP4) is involved in the lipid-induced ER stress in the liver. METHODS: HepG2 cells were cultured with eFABP4 (40 ng/ml) with or without linoleic acid (LA, 200 µM) for 18 h. The expression of ER stress-related markers was determined by Western blotting (ATF6, EIF2α, IRE1 and ubiquitin) and real-time PCR (ATF6, CHOP, EIF2α and IRE1). Apoptosis was studied by flow cytometry using Annexin V-FITC and propidium iodide staining. RESULTS: eFABP4 increased the ER stress markers ATF6 and IRE1 in HepG2 cells. This effect led to insulin resistance mediated by changes in AKT and JNK phosphorylation. Furthermore, eFABP4 significantly induced both apoptosis, as assessed by flow cytometry, and CHOP expression, without affecting necrosis and ubiquitination. The presence of LA increased the ER stress response induced by eFABP4. CONCLUSIONS: eFABP4, per se, induces ER stress and potentiates the effect of LA in HepG2 cells, suggesting that FABP4 could be a link between obesity-associated metabolic abnormalities and hepatic IR mechanisms.

Local Production of Fatty Acid-Binding Protein 4 in Epicardial/Perivascular Fat and Macrophages Is Linked to Coronary Atherosclerosis.

OBJECTIVE: Fatty acid-binding protein 4 (FABP4) is expressed in adipocytes and macrophages, and elevated circulating FABP4 level is associated with obesity-mediated metabolic phenotype. We systematically investigated roles of FABP4 in the development of coronary artery atherosclerosis. APPROACH AND RESULTS: First, by immunohistochemical analyses, we found that FABP4 was expressed in macrophages within coronary atherosclerotic plaques and epicardial/perivascular fat in autopsy cases and macrophages within thrombi covering ruptured coronary plaques in thrombectomy samples from patients with acute myocardial infarction. Second, we confirmed that FABP4 was secreted from macrophages and adipocytes cultured in vitro. Third, we investigated the effect of exogenous FABP4 on macrophages and human coronary artery-derived smooth muscle cells and endothelial cells in vitro. Treatment of the cells with recombinant FABP4 significantly increased gene expression of inflammatory markers in a dose-dependent manner. Finally, we measured serum FABP4 level in the aortic root (Ao-FABP4) and coronary sinus (CS-FABP4) of 34 patients with suspected or known coronary artery disease. Coronary stenosis score assessed by the modified Gensini score was weakly correlated with CS-FABP4 but was not correlated with Ao-FABP4. A stronger correlation (r=0.59, P<0.01) was observed for the relationship between coronary stenosis score and coronary veno-arterial difference in FABP4 level, (CS-Ao)-FABP4, indicating local production of FABP4 during coronary circulation in the heart. Multivariate analysis indicated that (CS-Ao)-FABP4 was an independent predictor of the severity of coronary stenosis after adjustment of conventional risk factors. CONCLUSIONS: FABP4 locally produced by epicardial/perivascular fat and macrophages in vascular plaques contributes to the development of coronary atherosclerosis.