FABP4 mRNA Research Articles

Anti-Obesity Effects of Petasites japonicus (Meowi) Ethanol Extract on RAW 264.7 Macrophages and 3T3-L1 Adipocytes and Its Characterization of Polyphenolic Compounds.

Koreans have been consuming Petasites Japonicus (PJ) as food. Although the therapeutic effect of PJ on allergic or inflammatory reactions associated with asthma has been proven, its effect on obesity is unclear. Therefore, the present study was aimed to assess the obesity related anti-inflammatory and anti-adipogenic effects of ethanol extract PJ (EPJ) on the inflammatory response in RAW 264.7 macrophages and on differentiation in 3T3-L1 adipocytes. In addition, the polyphenolic compound was quantitatively characterized from the EPJ using ultra performance liquid chromatography coupled with diode array detector, quadrupole time-of-flight-mass spectrometry (UPLC-DAD-QToF-MS). In RAW 264.7 or 3T3-L1, reduction of nitric oxide (in macrophages) production as well as monocyte chemoattractant protein-1 and tumor necrosis factor-α were observed. Treatment of EPJ in adipocyte differentiation showed an improvement in adiponectin and lipid accumulation and a significant reduction of PPARγ and FABP-4 mRNA expression levels. On the other hand, mRNA expression of UCP-1, PPARα, and ACO increased in the EPJ treated group. In addition, a total of 26 polyphenolic compounds were detected and of which 12 are reported for the first time from PJ. The higher content of diverse polyphenolic compounds presented in EPJ might be responsible for the observed anti-inflammatory and anti-adipogenic effect. These results suggest that PJ is valuable in improving obesity-related inflammatory responses.

Assessing the epigenetics effects on the expression of the Fabp3 promoter region of Zinc‐Transporter 7‐deficient mice

Zinc is an essential trace mineral for life. Zinc transporter 7 (ZNT7) transports zinc from the cytosol to the Golgi to maintain cellular zinc homeostasis. Mice with null‐Znt7 gene expression exhibit low zinc status, low body weight, as well as alterations in glucose and triglyceride homeostasis leading to a reduction in fat storage in adipocytes, and subsequently an increase in fat accumulation in muscle. The increase in muscle‐triglycerides in the Znt7KO mice is accompanied by a surge in the expression of Fabp3. Fatty‐Acid binding proteins (FABPs) belong to a family of proteins involved in intracellular lipid transport and status, yet some functions remain unknown. Fabp3 is found to be abundantly expressed in the heart, but it can also be moderately detected in skeletal muscle. Altered Fabp3 expression has been linked to a higher risk of developing metabolic syndrome in humans, hence this area of research is promising as rates of obesity and obesity‐related diseases continue to rise. DNA methylation has been shown to regulate human Fabp3 mRNA expression. Therefore, we aim to discover the methylation status of the mouse Fabp3 gene in a zinc deficient state using Znt7KO mice and determine a relationship between cellular zinc deficiency and the increased lipid accumulation in muscle, a cause of insulin resistance in Znt7KO male mice. Our preliminary results indicate that the cellular zinc level‐related DNA demethylation mainly occur in the area surround the transcriptional start site of Fabp3.Support or Funding InformationUSDA/ARS 2032‐51000‐005‐00D

Role of peroxisome proliferator-activated receptor-α on the synthesis of monounsaturated fatty acids in goat mammary epithelial cells.

A key member of the nuclear receptor superfamily is the peroxisome proliferator-activated receptor alpha (PPARA) isoform, which in nonruminants is closely associated with fatty acid oxidation. Whether PPARA plays a role in milk fatty acid synthesis in ruminants is unknown. The main objective of the present study was to use primary goat mammary epithelial cells (GMEC) to activate PPARA via the agonist WY-14643 (WY) or to silence it via transfection of small-interfering RNA (siRNA). Three copies of the peroxisome proliferator-activated receptor response element (PPRE) contained in a luciferase reporter vector were transfected into GMEC followed by incubation with WY at 0, 10, 20, 30, 50, or 100 µM. A dose of 50 µM WY was most effective at activating PPRE without influencing PPARA mRNA abundance. Transfecting siRNA targeting PPARA decreased its mRNA abundance to 20% and protein level to 50% of basal levels. Use of WY upregulated FASN, SCD1, ACSL1, DGAT1, FABP4, and CD36 (1.1-, 1.5-, 2-, 1.4-, 1.5-, and 5-fold, respectively), but downregulated DGAT2 and PGC1A (-20% and -40%, respectively) abundance. In contrast, triacylglycerol concentration decreased and the content and desaturation index of C16:1 and C18:1 increased. Thus, activation of PPARA via WY appeared to channel fatty acids away from esterification. Knockdown of PPARA via siRNA downregulated ACACA, SCD1, AGPAT6, CD36, HSL, and SREBF1 (-43%, -67%, -16%, -56%, -26%, and -29%, respectively), but upregulated ACSL1, DGAT2, FABP3, and PGC1A (2-, 1.4-, 1.3-, and 2.5-fold, respectively) mRNA abundance. A decrease in the content and desaturation index of C16:1 and C18:1 coupled with an increase in triacylglycerol content accompanied those effects at the mRNA level. Overall, data suggest that PPARA could promote the synthesis of MUFA in GMEC through its effects on mRNA abundance of genes related to fatty acid synthesis, oxidation, transport, and triacylglycerol synthesis.

Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

ABSTRACT Adipocyte differentiation of bovine adipose-derived stem cells (ASC) was induced by foetal bovine serum (FBS), biotin, pantothenic acid, insulin, rosiglitazone, dexamethasone and 3-isobutyl-1-methylxanthine, followed by incubation in different media to test the influence of ascorbic acid (AsA), bovine serum lipids (BSL), FBS, glucose and acetic acid on transdifferentiation into functional adipocytes. Moreover, different culture plate coatings (collagen-A, gelatin-A or poly-L-lysine) were tested. The differentiated ASC were subjected to Nile red staining, DAPI staining, immunocytochemistry and quantitative reverse transcription PCR (for NT5E, THY1, ENG, PDGFRα, FABP4, PPARγ, LPL, FAS, GLUT4). Nile red quantification showed a significant increase in the development of lipid droplets in treatments with AsA and BSL without FBS. The presence of BSL induced a prominent increase in FABP4 mRNA abundance and in FABP4 immunofluorescence signals in coincubation with AsA. The abundance of NT5E, ENG and THY1 mRNA decreased or tended to decrease in the absence of FBS, and ENG was additionally suppressed by AsA. DAPI fluorescence was higher in cells cultured in poly-L-lysine or gelatin-A coated wells. In additional experiments, the multi-lineage differentiation potential to osteoblasts was verified in medium containing ß-glycerophosphate, dexamethasone and 1,25-dihydroxyvitamin D3 using alizarin red staining. In conclusion, bovine ASC are capable of multi-lineage differentiation. Poly-L-lysine or gelatin-A coating, the absence of FBS, and the presence of BSL and AsA favour optimal transdifferentiation into adipocytes. AsA supports transdifferentiation via a unique role in FABP4 induction, but this is not linearly related to the primarily BSL-driven lipid accumulation. Abbreviations: AcA: acetic acid; AsA: ascorbic acid; ASC: adipose-derived stem cells; BSL: bovine serum lipids; DAPI: 4´,6-diamidino-2-phenylindole; DLK: delta like non-canonical notch ligand; DMEM: Dulbecco’s modified Eagle’s medium; DPBS: Dulbecco’s phosphate-buffered saline; ENG: endoglin; FABP: fatty acid binding protein; FAS: fatty acid synthase; GLUT4: glucose transporter type 4; IBMX: 3-isobutyl-1-methylxanthine; LPL: lipoprotein lipase; MSC: mesenchymal stem cells; α-MEM: α minimum essential medium; NT5E: ecto-5ʹ-nucleotidase; PDGFRα: platelet derived growth factor receptor α; PPARγ: peroxisome proliferator activated receptor γ; RPS19: ribosomal protein S19; SEM: standard error of the mean; THY1: Thy-1 cell surface antigen; TRT: treatment; TRT-Con: treatment negative control; YWHAZ: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta

Effects of LncRNA SNHG15 on Osteogenic/Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Under Oxidative Stress by TGFβ/Smad Signaling Pathway

Oxidative stress can affect bone marrow mesenchymal stem cells (BMSCs). LncRNA SNHG15 involves in a variety of cellular physiological and pathological processes. However, LncRNA SNHG15’s effect on osteogenesis/adipogenic differentiation of BMSCs under oxidative stress remains unclear. BMSCs were divided into normal control group; oxidative stress group; SNHG15 group in which SNHG15 plasmid was transfected under oxidative stress condition followed by analysis of the expression of LncRNA SNHG15, RUNX2 and OPN by real-time quantitative PCR, reactive oxygen species (ROS) and superoxide dismutase (SOD) activity, FABP4 and PPARγ2 mRNA expression, alkaline phosphatase (ALP) activity and TGFβ/Smad signaling by Western blot. Under oxidative stress, the expression of SHHG15, RUNX2 and OPN mRNA was significantly decreased, ROS production was increased, SOD activity was reduced, along with increased expression of FABP4 and PPARγ2 protein, decreased ALP activity, as well as reduced expression of TGFβ1, Smad2, and Smad7 compared to control group (P < 0.05). Transfection of LncRNA SNHG15 plasmid significantly up-regulated the expression of SNHG15, RUNX2 and OPN mRNA, decreased ROS production, increased SOD activity, decreased expression of FABP4 and PPARγ2 mRNA, and increased ALP activity as well as increased expression of TGFβ1, Smad2, and Smad7 compared to control group (P < 0.05). The expression of SNHG15 is decreased in BMSCs under oxidative stress. Up-regulation of SNHG15 expression can improve the redox balance through TGFβ/Smad signaling pathway, promote osteogenic differentiation of BMSCs under oxidative stress, and inhibit its differentiation into fat.

Effect of stocker management program on beef cattle skeletal muscle growth characteristics, satellite cell activity, and paracrine signaling impact on preadipocyte differentiation.

The objective of this study was to determine the effect of different stocker management programs on skeletal muscle development and growth characteristics, satellite cell (SC) activity in growing-finishing beef cattle as well as the effects of SC-conditioned media on preadipocyte gene expression and differentiation. Fall-weaned Angus steers (n = 76; 258 ± 28 kg) were randomly assigned to 1 of 4 stocker production systems: 1) grazing dormant native range (NR) supplemented with a 40% CP cottonseed meal-based supplement (1.02 kg · steer−1 · d−1) followed by long-season summer grazing (CON, 0.46 kg/d); 2) grazing dormant NR supplemented with a ground corn and soybean meal-based supplement fed at 1% of BW followed by short-season summer grazing (CORN, 0.61 kg/d); 3) grazing winter wheat pasture (WP) at high stocking density (3.21 steers/ha) to achieve a moderate rate of gain (LGWP, 0.83 kg/d); and 4) grazing winter WP at low stocking density (0.99 steers/ha) to achieve a high rate of gain (HGWP, 1.29 kg/d). At the end of the stocker (intermediate harvest, IH) and finishing (final harvest, FH) phases, 4 steers / treatment were harvested and longissimus muscles (LM) sampled for cryohistological immunofluorescence analysis and SC culture assays. At IH, WP steers had greater LM fiber cross-sectional area than NR steers; however, at FH, the opposite was observed (p < 0.0001). At IH, CORN steers had the lowest Myf-5+:Pax7+ SC density (p = 0.020), while LGWP steers had the most Pax7+ SC (p = 0.043). At FH, CON steers had the highest LM capillary density (p = 0.003) and their cultured SC differentiated more readily than all other treatments (p = 0.017). At FH, Pax7 mRNA was more abundant in 14 d-old SC cultures from HGWP cattle (p = 0.03). Preadipocytes exposed to culture media from proliferating SC cultures from WP cattle isolated at FH had more PPARγ (p = 0.037) and less FABP4 (p = 0.030) mRNA expression compared with NR cattle. These data suggest that different stocker management strategies can impact skeletal muscle growth, SC function, and potentially impact marbling development in growing-finishing beef cattle.

Temperature affects liver and muscle metabolism in photostimulated migratory redheaded buntings (Emberiza bruniceps).

We investigated the transcriptional effects of temperature on metabolism in the liver and flight muscle prior to the onset of spring migration in redheaded buntings. We measured changes in body mass, cellular lipid accumulation, and transcription of metabolic genes in the liver and flight muscle, during a week of exposure to 8-h short photoperiod (SP; 8L: 16D) and 13-h-long photoperiod (LP; 13L: 11D) at 22°C to ascertain the responsiveness to LP, and during 2.5weeks of LP at 22 and 38°C to examine the effect of temperature on LP-induced metabolic response. Particularly, we measured mRNA expression of genes involved in the fatty acids and triglycerides biosynthesis (srebf1, elovl6, scd, fads2, and dgat2), glucose and lipid metabolism (sirt1, foxo1, ppara, pparg, hdac3, and hmgcr) in the liver, and fatty acid transport (cd36 and fabp3) and metabolism (myod1) in the flight muscle. Birds gained weight and had elevated lipid accumulation at 22°C but not at 38°C in the liver and muscle tissues. We also found significant differences in gene expression patterns during 2.5weeks of LP exposure. For instance, as compared to week 1, there were decreased elovl6, dgat2, and srebf1 and increased foxo1 mRNA levels in the liver, and decreased cd36 and fabp3 mRNA levels in the muscle at 38°C after 2.5weeks of LP. It is suggested that a drastic change in temperature could alter the molecular metabolism in the liver and skeletal muscle with photoperiod-induced transition in the seasonal state in migratory songbirds.

Fibroblast growth factor homologous factor 1 stimulates Leydig cell regeneration from stem cells in male rats.

Fibroblast growth factor homologous factor 1 (FHF1) is an intracellular protein that does not bind to cell surface fibroblast growth factor receptor. Here, we report that FHF1 is abundantly present in Leydig cells with up‐regulation during its development. Adult male Sprague Dawley rats were intraperitoneally injected with 75 mg/kg ethane dimethane sulphonate (EDS) to ablate Leydig cells to initiate their regeneration. Then, rats daily received intratesticular injection of FHF1 (0, 10 and 100 ng/testis) from post‐EDS day 14 for 14 days. FHF1 increased serum testosterone levels without affecting the levels of luteinizing hormone and follicle‐stimulating hormone. FHF1 increased the cell number staining with HSD11B1, a biomarker for Leydig cells at the advanced stage, without affecting the cell number staining with CYP11A1, a biomarker for all Leydig cells. FHF1 did not affect PCNA‐labelling index in Leydig cells. FHF1 increased Leydig cell mRNA (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, Nr5a1 and Hsd11b1) and their protein levels in vivo. FHF1 increased preadipocyte biomarker Dlk1 mRNA level and decreased fully differentiated adipocyte biomarker (Fabp4 and Lpl) mRNA and their protein levels. In conclusion, FHF1 promotes Leydig cell regeneration from stem cells while inhibiting the differentiation of preadipocyte/stem cells into adipocytes in EDS‐treated testis.

Arachidonic Acid Regulation of Intracellular Signaling Pathways and Target Gene Expression in Bovine Ovarian Granulosa Cells.

Simple SummaryArachidonic acid (AA) is one of the polyunsaturated fatty acids that presents in a very high proportion in the mammalian follicular fluid. However, the mechanism of its effects on bovine ovarian granulosa cells is not clear. In the present study, we found that arachidonic acid plays an important role in regulating cell proliferation, lipid accumulation and steroidogenesis of granulosa cells. In this sense, arachidonic acid can directly affect the functionality of granulosa cells and therefore follicular development and ovulation, which could provide useful information for future studies relating to increasing fecundity of bovine.In the present study, AA was used to challenge bovine ovarian granulosa cells in vitro and the related parameters of cellular and molecular biology were measured. The results indicated that lower doses of AA increased survival of bovine granulosa cells whereas higher doses of AA suppressed survival. While lower doses of AA induced accumulation of lipid droplet in granulosa cells, the higher dose of AA inhibited lipid accumulation, and AA increased abundance of FABP3, CD36 and SLC27A1 mRNA. Higher doses of AA decreased the secretion of E2 and increased the secretion of P4 accompanied by down-regulation of the mRNA abundance of CYP19A1, FSHR, HSD3B1 and STAR in granulosa cells. The signaling pathways employed by AA in the stimulation of genes expression included both ERK1/2 and Akt. Together, AA specifically affects physiological features, gene expression levels and steroid hormone secretion, and thus altering the functionality of granulosa cells of cattle.

Dietary Aroclor 1254-Induced Toxicity on Antioxidant Capacity, Immunity and Energy Metabolism in Chinese Mitten Crab Eriocheir sinensis: Amelioration by Vitamin A.

Effects of dietary Polychlorinated biphenyl (PCB) exposure and dietary vitamin A supplementation on Chinese mitten crab Eriocheir sinensis were studied with the aim to explain dietary PCB toxicity and toxic alleviation by vitamin A intake in crab. Four diets were used including three experimental diets containing 0, 80000 or 240000 IU/kg vitamin A with each experimental diet containing 10 mg PCB/kg diet, and a control diet (without vitamin A and PCB supplementation) in 56 days feeding trial. Crabs fed the PCB-only diet had significantly lower weight gain than those fed the control diet. No significant difference was observed in crab survival among all groups. Crabs fed the PCB-only diet had a significantly higher malondialdehyde content and antioxidase superoxide dismutase activity in the serum and hepatopancreas, and higher erythromycin N-demethylase and glutathione S-transferase activities in the hepatopancreas than those fed the control diet. However, supplementation of dietary vitamin A decreased the levels of all these parameters. The hepatopancreatic cytochrome P450 2 and 4 (CYP2, CYP4), fatty acid binding proteins 3 and 10 (FABP3, FABP10) and intracellular lipolytic enzyme (IL) Messenger Ribonucleic Acid (mRNA) levels in the PCB-only group were significantly higher than those in the control group, and dietary 240000 IU/kg vitamin A supplementation decreased hepatopancreatic CYP4, FABP3, FABP10 and IL enzyme mRNA level. The crabs fed 80000 IU/kg vitamin A supplementation diet had the highest level of retinoid X receptor mRNA in the hepatopancreas. The structure of the hepatopancreas was damaged and the deposit of lipid droplets decreased with dietary PCB exposure. Both levels of vitamin A supplementation alleviated the damage and increased lipid droplets in the hepatopancreas. Dietary PCB exposure significantly reduced total hemocyte count (THC), and phenoloxidase, acid phosphatase activities in the serum. Post-challenge survival of crab in the experimental PCB-only diet group was low compared with that in the control. Supplementation of 240000 IU/kg vitamin A significantly increased the THC and phenoloxidase activity in the serum and post-challenge survival compared with those in the PCB-only group. This study indicates that dietary vitamin A can improve the antioxidant capacity, immune response, detoxification enzymes activities, energy metabolism and hepatopancreas tissue structure of Chinese mitten crab fed PCB contaminated diets.

Impact of HIV/simian immunodeficiency virus infection and viral proteins on adipose tissue fibrosis and adipogenesis

HIV-infected patients receiving antiretroviral treatment (ART) often present adipose tissue accumulation and/or redistribution. adipose tissue has been shown to be an HIV/SIV reservoir and viral proteins as Tat or Nef can be released by infected immune cells and exert a bystander effect on adipocytes or precursors. Our aim was to demonstrate that SIV/HIV infection per se could alter adipose tissue structure and/or function. Morphological and functional alterations of subcutaneous (SCAT) and visceral adipose tissue (VAT) were studied in SIV-infected macaques and HIV-infected ART-controlled patients. To analyze the effect of Tat or Nef, we used human adipose stem cells (ASCs) issued from healthy donors, and analyzed adipogenesis and extracellular matrix component production using two dimensional (2D) and three-dimensional (3D) culture models. Adipocyte size and index of fibrosis were determined on Sirius red-stained adipose tissue samples. Proliferating and adipocyte 2D-differentiating or 3D-differentiating ASCs were treated chronically with Tat or Nef. mRNA, protein expression and secretion were examined by RT-PCR, western-blot and ELISA. SCAT and VAT from SIV-infected macaques displayed small adipocytes, decreased adipogenesis and severe fibrosis with collagen deposition. SCAT and VAT from HIV-infected ART-controlled patients presented similar alterations. In vitro, Tat and/or Nef induced a profibrotic phenotype in undifferentiated ASCs and altered adipogenesis and collagen production in adipocyte-differentiating ASCs. We demonstrate here a specific role for HIV/SIV infection per se on adipose tissue fibrosis and adipogenesis, probably through the release of viral proteins, which could be involved in adipose tissue dysfunction contributing to cardiometabolic alterations of HIV-infected individuals.

Linoleic Acid Increases Prostaglandin E2 Release and Reduces Mitochondrial Respiration and Cell Viability in Human Trophoblast-Like Cells.

The omega 6 fatty acid (FA) linoleic acid (LA) is required for embryonic development; however, omega 6 FAs can alter cellular metabolism via inflammation or modulation of mitochondrial function. Fetal LA is obtained from the maternal diet, and FAs are transported to the fetus via placental FA transporters (FATPs) and binding proteins (FABPs), but specific proteins responsible for LA transport in placental trophoblasts are unknown. Dietary LA consumption is increasing, but the effect of elevated LA on trophoblast function is not clear. Swan71 trophoblasts were exposed to physiological and supraphysiological concentrations of LA for 24 hours. Quantification of mRNA was determined using real time PCR, and protein concentration was determined by Western blot analysis. Cell viability, citrate synthase activity and mitochondrial respiration were determined. Exposure to 300 and 500 μM LA increased FATP1 and FATP4 mRNA expression. 500 μM LA increased FATP1 and FATP4 protein expression. Exposure to 500 μM increased FABP5 mRNA expression, while exposure to 100 to 500 μM LA decreased FABP3 mRNA expression. 300 and 500 μM LA decreased FABP3 protein expression. Cell viability was decreased by exposure to LA (100 to 1000 μM). Citrate synthase activity and routine mitochondrial respiration were significantly decreased by exposure to 300 and 500 μM LA, and maximal respiration and spare respiratory capacity were decreased by exposure to 100 to 500 μM LA. 300 and 500 μM LA increased reactive oxygen species generation in human trophoblasts. Moreover, exposure to 300 and 500 μM LA decreased IL-6 secretion. Exposure to 500 μM LA increased IL-8, NF-κB and PPAR-γ mRNA expression, but decreased NF-κB protein expression. 300 μM LA decreased IL-8 protein expression. Further, exposure to 100 to 500 μM LA increased prostaglandin E2 and leukotriene B₄ release. Exposure to LA decreases cell viability, alters mRNA expression of FA transport related proteins, mitochondrial respiration and function, and inflammatory responses in trophoblasts. These findings may have implications on placental function when women consume high levels of LA.

Inhibition of FAO in AML co-cultured with BM adipocytes: mechanisms of survival and chemosensitization to cytarabine

Adipocytes are the prevalent stromal cell type in adult bone marrow (BM), and leukemia cells continuously adapt to deficiency of nutrients acquiring chemoresistant profiles in the BM microenvironment. We have previously shown that fatty acid metabolism is a key energy pathway for survival of acute myeloid leukemia (AML) cells in the adipocyte-abundant BM microenvironment. The novel fatty acid β-oxidation (FAO) inhibitor avocatin B, an odd-numbered carbon lipid derived from the avocado fruit, induced apoptosis and growth inhibition in mono-cultured AML cells. In AML cells co-cultured with BM adipocytes, FAO inhibition with avocatin B caused adaptive stimulation of free fatty acid (FFA) uptake through upregulation of FABP4 mRNA, enhanced glucose uptake and switch to glycolysis. These changes reflect the compensatory response to a shortage of FFA supply to the mitochondria, and facilitate the protection of AML cells from avocatin B–induced apoptosis in the presence of BM adipocytes. However, the combination treatment of avocatin B and conventional anti-AML therapeutic agent cytarabine (AraC) increased reactive oxygen species and demonstrated highly synergistic effects on AML cells under BM adipocyte co-culture condition. These findings highlight the potential for combination regimens of AraC and FAO inhibitors that target bone marrow-resident chemoresistant AML cells.

Beneficial Effects of Korean Red Ginseng in the Progression of Non-Alcoholic Steatohepatitis via FABP4 Modulation.

Korean red ginseng (KRG) is a traditional herbal medicine used to prevent several geriatric diseases due to its therapeutic effects on metabolic disorder, including type 2 diabetes and fatty liver disease. In this study, we investigated the effects of KRG on the progression of nonalcoholic steatohepatitis (NASH) in mice. NASH was induced by feeding a methionine- and choline-deficient high-fat or high-fat/high-sucrose diet for 6 or 13 weeks, respectively. Each diet group was also orally administered saline (group G0) or KRG extract (100, 200, or 400 mg/kg/day; groups G1, G2, and G4, respectively). KRG showed anti-inflammatory and antifibrogenic effects in the diet-induced NASH models. Furthermore, the expression levels of lipid metabolism-related genes were markedly decreased with KRG treatment in both diet-induced NASH groups. We next confirmed the expression levels of FABP4 in the liver and its ability to regulate inflammation and/or oxidative stress. We observed decreased levels of FABP4 mRNA and protein in the KRG-treated groups indicating that KRG affects the pathogenesis of NASH-related inflammatory responses by modulating FABP4 expression. Results of in vitro experiments showed similar patterns in cells treated with KRG, indicating that KRG treatment regulates the expression of FABP4 and subsequently reduces NASH related inflammation. Our findings suggest a novel role of KRG in NASH-related inflammatory responses via modulation of FABP4 expression in the liver. KRG may be a safe alternative therapy to prevent NASH progression.

Altered fatty acid‐binding protein 4 (FABP4) expression and function in human and animal models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality. Risk factors for developing HCC include viral hepatitis, alcohol and obesity. Fatty acid-binding proteins (FABPs) bind long-chain free fatty acids (FFAs) and are expressed in a tissue-specific pattern; FABP1 being the predominant hepatic form, and FABP4 the predominant adipocyte form. The aims of this study were to investigate the expression and function of FABPs1-9 in human and animal models of obesity-related HCC. FABP1-9 expression was determined in a mouse model of obesity-promoted HCC. Based on these data, expression and function of FABP4 was determined in human HCC cells (HepG2 and HuH7) in vitro. Serum from patients with different underlying hepatic pathologies was analysed for circulating FABP4 levels. Livers from obese mice, independent of tumour status, exhibited increased FABP4 mRNA and protein expression concomitant with elevated serum FABP4. In vitro, FABP4 expression was induced in human HCC cells by FFA treatment, and led to FABP4 release into culture medium. Treatment of HCC cells with exogenous FABP4 significantly increased proliferation and migration of human HCC cells. Patient serum analysis demonstrated significantly increased FABP4 in those with underlying liver disease, particularly non-alcoholic fatty liver disease (NAFLD) and HCC. These data suggest FABP4, an FABP not normally expressed in the liver, can be synthesized and secreted by hepatocytes and HCC cells, and that FABP4 may play a role in regulating tumour progression in the underlying setting of obesity.

Association of genetic variants and expression levels of porcine FABP4 and FABP5 genes.

The FABP4 and FABP5 genes, coding for fatty acid transport proteins, have long been studied as positional candidate genes for SSC4 QTL affecting fat deposition and composition traits in pigs. Polymorphisms in these genes, FABP4:g.2634_2635insC and FABP5:g.3000T>G, have previously been associated with fatness traits in an Iberian by Landrace cross (IBMAP). The aim of the present work was to evaluate the functional implication of these genetic variants. For this purpose, FABP4 and FABP5 mRNA expression levels in 114 BC1_LD animals (25% Iberian×75% Landrace) were analyzed using real-time quantitative PCR in backfat and muscle. FABP4 gene expression in backfat, but not in muscle, was associated with FABP4:g.2634_2635insC. In contrast, FABP5:g.3000T>G was not associated with gene expression levels. An expression-based genome-wide association study highlighted the FABP4:g.2634_2635insC polymorphism as the polymorphism most associated with FABP4 gene expression in backfat. Furthermore, other genomic regions associated in trans with the mRNA expression of FABP4 in backfat and FABP5 in muscle were also identified. Finally, two putative transcription binding sites for PPARG and NR4A2 may be affected by the FABP4:g.2634_2635insC polymorphism, modifying FABP4 gene expression. Our results reinforce FABP4 as a candidate gene for fatness traits on SSC4.

Abstract 023: Inducible Deletion of Adipocyte Prorenin Receptor Reverses Obesity Related Hypertension

Obesity contributes to approximatively 2.5 million deaths every year and is associated with life threatening conditions including hypertension. Recently, we found that constitutive deletion of adipocyte (pro)renin-receptor (PRR) prevented high-fat diet-induced obesity through a drastic decrease in fat mass. However, adipocyte PRR deficient mice were characterized by a fatty liver and by an elevated systolic blood pressure (SBP), classic features of models of lipodystrophy. The purpose of this study was to investigate whether the temporally-controlled deletion of adipocyte PRR in obese mice reverses obesity related hypertension. After 18 weeks of high fat diet, inducible adipocyte-PRR deficient ( PRR ERT ) and control ( PRR fl/Y ) male mice (n=7-11 mice/ group) were injected intraperitoneally with tamoxifen (TMX) for 5 consecutive days. Body weight, body composition and blood pressure, measured by radiotelemetry in a subgroup of mice (n=2-4 mice/ group), were recorded before and after TMX injection. The inducible deletion of adipocyte PRR in PRR ERT mice decreased significantly body weights ( PRR fl/fl , 46.6 ± 1.3 g; PRR ERT , 42.1 ± 1.4 g, P&lt;0.05) and fat mass ( PRR fl/fl , 15.8 ± 1.0 g; PRR ERT , 8.1 ± 0.7 g, P&lt;0.05) compared to control mice. PPARγ, FABP4 and FAS mRNA levels were significantly decreased by 68% (6.8 out 10), 80% (8 out 10) and 68% (6.8 out 10) respectively in white adipose tissues of PRR ERT mice suggesting that PRR positively regulated adipogenesis and lipid metabolism in adipose tissue. In addition, the inducible deletion of adipocyte PRR in PRR ERT mice decreased significantly SBP compared to control mice ( PRR fl/fl , -4.3 ± 3.2 g; PRR ERT , -10.2 ± 2.4 g, P&lt;0.05). Interestingly, adipocyte angiotensinogen mRNA abundance was significantly decreased in adipose tissue of PRR ERT mice fed a standard diet suggesting that the decrease in blood pressure might be mediated by a local renin angiotensin system (RAS). The measurement of local (liver, kidney, adipose tissue and brain) and systemic RAS in HF-fed mice is under investigation. Taken together, our results highlight a new signaling pathway in which PRR regulates adipogenesis, lipid metabolism and blood pressure. PRR could represent a new potential therapeutic target for obesity and hypertension.

The effect of mechanical stress on the proliferation, adipogenic differentiation and gene expression of human adipose-derived stem cells.

To allow for a better implementation of external volume expansion to clinical applications for soft tissue regeneration, it is necessary to comprehensively understand the underlying mechanisms. As human adipose-derived stem cells (hASCs) play a crucial role in soft tissue enlargement, we investigated the impact of cyclic stretch on gene expression, proliferation rate and adipogenic differentiation of these cells. After cyclic stretching, RNA was extracted and subjected to DNA microarray analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Also, the expression of FABP4 mRNA was analysed by RT-qPCR to test whether mechanical stretch affected adipogenic differentiation of hASCs. The proliferation rate was assessed using the alamarBlue assay and Ki-67 staining. A cell cycle analysis was performed with flow cytometry and Western blot. We found that cyclic stretch significantly induced the expression of CYP1B1 mRNA. Furthermore, the adipogenic differentiation of hASCs was impaired, as was the proliferation. This was partly due to a decrease in extracellular signal-regulated kinase (ERK) 1/2 and histone H3 phosphorylation, suggesting a growth arrest in the G2 /M phase of the cell cycle. Enrichment analyses demonstrated that stretch-regulated genes were over-represented in pathways and biological processes involved in extracellular matrix organization, vascular remodelling and responses to cell stress. Taken together, mechanical stress impaired both proliferation and adipogenic differentiation, but led to a tissue-remodelling phenotype of hASCs. These data suggest that extracellular matrix remodelling and neoangiogenesis may play a more important role in external volume expansion than proliferation and adipogenesis of hASCs. Copyright © 2017 John Wiley & Sons, Ltd.

Fatty Acids Have Different Adipogenic Differentiation Potentials in Stromal Vascular Cells Isolated from Abdominal Fat in Laying Hens.

This study was conducted to examine the effects of fatty acids (FA) with/without chicken serum (CS) on the expression of adipogenic transcripts and adipogenesis in chicken stromal vascular cells (SVC). In experiment 1, SVC were grown in DMEM containing 10% FBS (Control) and treated with 300µM oleic acid (OLA)+FBS, linoleic acid (LNA)+FBS, palmitic acid (PAM)+FBS, or stearic acid (STA)+FBS for 48h. In experiment 2, cells were grown in DMEM containing 5% CS and treated with 300µM OLA (CS+OLA), PAM (CS+PAM), STA (CS+STA) or 200µM LNA (CS+LNA) for 48h. Adipogenesis was determined using Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. The proportion of OLA, PAM, or STA was increased (P<0.05) in SVC grown in either FBS or CS with OLA, PAM or STA. Adipogenesis was induced in FBS+OLA, FBS+LNA, FBS+PAM, FBS+STA, CS+OLA, CS+LNA, CS+PAM, or CS+SAT compared to FBS. GPDH activity was significantly higher in FBS+OLA and FBS+LNA than one in FBS. Compared to FBS, the expression of FABP4 mRNA increased (P<0.05) in FBS+OLA, FBS+LNA, or FBS+PAM, whereas that of C/EBPα, C/EBPβ, and ATGL increased (P<0.05) in FBS+OLA or FBS+LNA cells. Expression of FABP4 and C/EBPβ mRNA was higher in CS, CS+OLA, CS+LNA, CS+PAM, or CS+SAT compared with (FBS, whereas the expression of ATGL and C/EBPα was higher in CS, CS+OLA, or CS+LNA than FBS cells. In conclusion, these results showed that FA have different potentials to induce adipogenesis, LNA is the most potent among the tested FA, and these potentials can be improved in the presence of CS.

Influence of Bovine Serum Lipids and Fetal Bovine Serum on the Expression of Cell Surface Markers in Cultured Bovine Preadipocytes

To establish the influence of fetal bovine serum (FBS) and bovine serum lipids (BSL) on cell differentiation marker expression, bovine adipose-derived stem cells from subcutaneous tissue were incubated for 14 days in 4 types of differentiation media containing 10% FBS and 10 µL/mL BSL (TRT-1), no FBS and 10 µL/mL of BSL (TRT-2), 10% FBS and no BSL (TRT-3), or no supplements (TRT-4). Cells were subjected to Nile red staining, immunocytochemistry (CD73, CD90, CD105, DLK1, FabP4), and quantitative real-time PCR (CD73, CD90, CD105, FabP4). The number of cells presenting FabP4 and the percentage of mature adipocytes with large lipid droplets were increased in TRT-2, accompanied by a robust increase in FabP4 mRNA abundance and a decrease in DLK1-positive cells. In preadipocytes, CD73 was present around the nucleus and translocated towards cell membranes during differentiation. Although the percentage of CD73-positive cells was not different among treatments, its mRNA abundance, immunocytochemical staining intensity, and translocation towards cell membranes were decreased when the medium contained no FBS (TRT-2 and TRT-4). All cells showed a diffuse distribution of CD90 and CD105 and remained positive for these markers irrespective of the treatment. However, the CD90 and CD105 mRNA abundance was decreased in TRT-2 and TRT-4; i.e., in media containing no FBS. The presence of FBS increased the absolute number of cell nuclei as assessed by DAPI fluorescence. Our results suggest that bovine subcutaneous preadipocytes display typical stem cell markers. The differentiation into mature adipocytes is promoted by BSL, whereas FBS endorses cell proliferation.