Liver Fatty Acid-binding Protein mRNA Research Articles

Oxidative stress-mediated developmental toxicity induced by isoniazide in zebrafish embryos and larvae.

Isoniazide (INH) is an important first-line drug that is used to treat tuberculosis. However, the effect of INH on fetal growth has not yet been elucidated, and the mechanism of INH-induced developmental toxicity is still unknown. In the present study, we employed zebrafish embryos and larvae to investigate the developmental toxicity of INH. The survival rates of the embryos and larvae as well as the hatching rates of embryos were significantly reduced. Morphological abnormalities, including spinal curvature, yolk retention, swimming bladder absence, tail bending and shorter body lengths were induced by INH. Histopathological analysis showed loose cell-to-cell contacts and large vacuoles in the larval hepatocytes. Thin intestinal walls, frayed gut villi and widespread cell lysis were observed in the intestines of the larvae in the higher concentration (8, 16 mm) exposure groups. In addition, exposure to high doses (≥ 6 mm) of INH significantly reduced the locomotor capacity of the zebrafish larvae. INH significantly increased the levels of reactive oxygen species and malondialdehyde and decreased the superoxide dismutase activity in zebrafish larvae, which suggested that oxidative stress was induced and that the antioxidant capacity was inhibited. Superoxide dismutase 1 and liver fatty acid-binding protein mRNA levels were significantly downregulated, while the GSTP2 and cytochrome P450 3A mRNA levels were significantly upregulated in the INH-exposed zebrafish larvae. The overall results indicated that INH caused a dose- and time-dependent increase in developmental toxicity and that oxidative stress played an important role in the developmental toxicity induced by INH in zebrafish larvae. Copyright © 2017 John Wiley & Sons, Ltd.

Effects of feed restriction and dietary fat type on mRNA expression of liver fatty acid-binding protein (L-FABP) in broilers

Background: Liver fatty acid-binding protein (L-FABP) is the main cytosolic binding site for long chain fatty acids in hepatocytes. FABPs enhance the uptake of fatty acids into the cell by increasing their concentration due to decreasing concentration of unbound fatty acids inside the cell. Objectives: The aim of this study was to evaluate the effects of dietary unsaturated to saturated fatty acid ratio and feed restriction on L-FABP mRNA expression. Methods: A total of 720, 10-day old male Ross 308 broiler chicks were fed diets with unsaturated to saturated fatty acid ratio (U/S) of 2, 3.5, 5, or 6.5 as ad libitum or skip-a-day feeding schedule (during 18–28 days of age). Relative expression of L-FABP mRNA in hepatocytes of broilers was determined using quantitative reverse transcription real-time polymerase chain reaction. Results: Our results show that feed restriction induced the expression of L-FABP gene in the liver of broilers. Moreover, L-FABP gene expression was increased by dietary U/S ratio of 6.5. There was no interaction between dietary U/S and feed restriction on the L-FABP gene expression. Conclusions: Results indicate that birds have a mechanism for regulation of fatty acid transfer under different nutritional conditions.

Differential expression of L-FABP and L-BABP between fat and lean chickens

Liver fatty acid-binding protein (L-FABP) and liver bile acid-binding protein (L-BABP), in the liver intra-cytoplasm of chicken, are members of the fatty acid-binding protein subfamily. This study was designed to analyze and compare L-FABP and L-BABP expression levels between fat and lean lines in chicken liver tissue, and to determine the relationship between their expression and lipid metabolism. Real-time polymerase chain reaction (PCR) and Western blotting were used to detect the mRNA and protein expression in liver tissue between the lean and fat lines. Real-time PCR showed that L-FABP mRNA expression in fat male chickens was higher than that in lean male chickens at 2, 3, 4, 5, 6, and 10 weeks of age (P < 0.05), and L-BABP mRNA expression in fat male chickens was higher than that in lean male chickens at 1, 2, 3, 4, 8, and 10 weeks of age (P < 0.05). Western blotting showed that the L-FABP protein expression in fat male chickens was higher than that in lean male chickens at 3, 5, 6, and 7 weeks of age (P < 0.05), and L-BABP protein expression in fat male chickens was higher than that in lean male chickens at 3, 4, 5, and 6 weeks of age (P < 0.05). These results suggested that chicken L-FABP and L-BABP affect abdominal fat deposition through differences in their expression level, and the possible mechanism is that a high expression level of L-FABP and L-BABP leads to a high lipogenesis rate and, ultimately, to lipid deposition.

Expression pattern of L-FABP gene in different tissues and its regulation of fat metabolism-related genes in duck

Liver fatty acid binding protein (L-FABP) is a member of intracellular lipid-binding proteins responsible for the transportation of fatty acids. The expression pattern of duck L-FABP mRNA was examined in this study by quantitative RT-PCR. The results showed that duck L-FABP gene was expressed in many tissues, including heart, lung, kidney, muscle, ovary, brain, intestine, stomach and adipocyte tissues, and highly expressed in liver. Several lipid metabolism-related genes were selected to detect the regulation of L-FABP in duck. The expression of L-FABP and lipoprotein lipase was promoted by oleic acid. The L-FABP knockdown decreased the expression levels of peroxisome proliferator-activated receptor α (PPARα), fatty acid synthase and lipoprotein lipase by 61.1, 42.3 and 53.7 %, respectively (P < 0.05), but had no influences on the mRNA levels of PPARγ and leptin receptor. L-FABP might function through the PPARα to regulate the fat metabolism-related gene expression and play important roles in lipid metabolism in duck hepatocytes.

Characterization of 4-HNE Modified L-FABP Reveals Alterations in Structural and Functional Dynamics

4-Hydroxynonenal (4-HNE) is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd1 = 0.395 µM and Kd2 = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a contributing factor in the pathogenesis of ALD.

Poultry fat decreased fatty acid transporter protein mRNA expression and affected fatty acid composition in chickens

BackgroundA study was undertaken to examine the effects of poultry fat (PF) compared with those of soybean oil (SBO) on intestinal development, fatty acid transporter protein (FATP) mRNA expression, and fatty acid composition in broiler chickens. A total of 144 day-old male commercial broilers were randomly allocated to 2 treatment groups (6 replicates of 12 chicks for each treatment) and fed isocaloric diets containing 3.0% PF or 2.7% SBO at 0 to 3 wk and 3.8% PF or 3.5% SBO at 4 to 6 wk, respectively.ResultsPF had no influence on intestinal morphology, weight, or DNA, RNA, or protein concentrations at 2, 4, and 6 wk of age. However, compared with SBO, PF significantly decreased FATP mRNA abundance at 4 wk (P = 0.009) and 6 wk of age (P < 0.001); decreased liver fatty acid-binding protein (L-FABP) mRNA abundance at 6 wk of age (P = 0.039); and decreased C18:2 (P = 0.015), C18:3 (P < 0.001), C20:2 (P = 0.018), Σ-polyunsaturated fatty acids (Σ-PUFA) (P = 0.020), and the proportion of PUFA (P < 0.001) in the intestinal mucosa and decreased C18:2 (P = 0.010), C18:3 (P < 0.001), C20:2 (P < 0.001), Σ-PUFA (P = 0.005), and the proportion of PUFA (P < 0.001) in breast muscle at 6 wk of age.ConclusionsPF decreases FATP and L-FABP mRNA expression and decreased the proportion of PUFA in the intestinal mucosa and breast muscle.

Wild bitter gourd extract up-regulates mRNA expression of PPARα, PPARγ and their target genes in C57BL/6J mice

Wild bitter gourd (Momordica charantia Linn. var. abbreviata ser.) was commonly used as a medicinal herb in Asia, Africa, and South America because of its anti-diabetic, antibacterial, anti-viral, and chemopreventive functions. C57BL/6J mice were orally administered with 250, 500 or 1000mg/kg BW of WBGE in 0.2mL/mouse of olive oil daily for 2 weeks. Compared to control (vehicle treated) mice, mice receiving WBGE showed significantly higher PPARα, ACO (acyl-CoA oxidase) and L-FABP (liver-fatty acid binding protein) mRNA expression, ACO activity and protein in the liver (P<0.05), as clofibrate-treated mice. WBGE treatment also resulted in significantly higher PPARγ and LPL (lipoprotein lipase) mRNA (P<0.05) in the epididymal adipose tissue. Liver triglyceride and non-esterified fatty acid concentration in WBGE treated mice were significantly lower than those of control mice (P<0.05). Plasma adiponectin level was significantly higher in mice receiving WBGE than in control mice (P<0.05), as the rosiglitazone treated mice. Results of this study demonstrated that WBGE also activates PPARα and PPARγ signaling pathway in vivo.

Regulation of liver fatty acid binding protein expression by clofibrate in hepatoma cells

Peroxisome proliferator-activated receptor (PPAR) agonists such as clofibrate are known to affect liver fatty acid binding protein (L-FABP) levels, which in turn influence hepatocellular oxidant status. The mechanism of clofibrate's modulation of L-FABP levels is not clear. In this study we used clofibrate (PPARα agonist), MK886 (PPARα antagonist), and GW9662 (PPARγ antagonist) in determining the regulating mechanism of L-FABP expression and its antioxidant activity in CRL-1548 hepatoma cells. Antioxidant activity was assessed by determining intracellular reactive oxygen species (ROS) using dichlorofluorescein (DCF) fluorescence. The effect of clofibrate on cytosolic activity of the intracellular antioxidant enzymes was also assessed. RT-PCR and mRNA stability assay showed that clofibrate treatment enhanced L-FABP mRNA stability, which resulted in increased L-FABP levels. A nuclear run-off assay and RT-PCR measurements of L-FABP mRNA revealed that clofibrate increased the L-FABP gene transcription rate. The increased L-FABP was associated with reduced cytosolic ROS. Levels of superoxide dismutase, glutathione peroxidase, and catalase were not affected by clofibrate treatment. L-FABP siRNA knockdown studies showed that a reduction in L-FABP expression was associated with increased DCF fluorescence. We conclude that clofibrate enhanced L-FABP gene transcription and mRNA stability, thus affecting L-FABP expression and ultimately cellular antioxidant activity.

The effect of glucagon-like peptide 2 injection on performance, small intestinal morphology, and nutrient transporter expression of stressed broiler chickens

An experiment was conducted to determine the effect of injecting glucagon-like peptide 2 (GLP-2) on the small intestinal weight, morphology, and nutrient transporter expression in pharmacologically stressed broiler chickens. A total of 144 seven-day-old birds were fed either a basal diet (CTRL) or a basal diet plus 30 mg of corticosterone (CORT)/kg of diet for a total of 14 d. Half of the birds from each group were injected daily with GLP-2 (6.7 nmol/kg of BW) or saline for 14 d. The average final BW, ADG, ADFI, and the ratio of feed intake to weight gain (F:G) was recorded over 21 d for the 4 groups of 36 birds, namely CTRL + saline, CTRL + GLP-2, CORT + saline, and CORT + GLP-2. In addition, the absolute and relative small intestinal weight, villus height (VH), and crypt depth (CD) of the duodenum and jejunum, as well as the abundance of sodium and glucose co-transporter 1 (SGLT-1), vitamin D-dependent calcium-binding protein-28,000 molecular weight (CaBP-D28k), and peptide transporter 1 (PepT-1) mRNA in the duodenum and of liver fatty acid-binding protein (L-FABP) mRNA in the jejunum. The total DNA, RNA, and protein content in small intestinal mucosa were also determined. The results showed that CORT administration significantly lowered average final BW, ADG, ADFI, absolute small intestinal weight, VH, and CD of duodenum and jejunum (P < 0.05) while increasing the relative small intestinal weight, F:G, relative abundance of SGLT-1, CaBP-D28k, PepT-1, and L-FABP mRNA (P < 0.05). Glucagon-like peptide 2 injection increased the average final BW, ADG, VH, and CD in duodenum and jejunum and relative abundance of SGLT-1, CaBP-28k, PepT1, and PepT1 mRNA of broiler chickens, respectively (P < 0.05), and decreased F:G (P < 0.05). In chickens fed basal diet plus CORT, injecting GLP-2 decreased F:G (P < 0.05); increased VH and CD of duodenum and CD of jejunum; and increased relative abundance of SGLT-1, CaBP-D28k, PepT-1, and L-FABP mRNA, RNA, and total protein content in small intestine compared with the injection of saline (P < 0.05). In birds fed the basal diet, GLP-2 injection decreased F:G (P < 0.05) and increased final BW, ADG, small bowel weight, CD of jejunum, and relative abundance of CaBP-D28k and PepT-1 mRNA compared with injecting saline (P < 0.05). In conclusion, GLP-2 injection reversed the negative effect of stress on the weight and morphology and the absorptive function of small bowel of broiler chickens. Glucagon-like peptide 2 injection also had a positive effect on the growth performance of healthy broiler chickens.

Tissue Specific Expression of Lipid Metabolism Related Molecules in Digestive Organs of Miniature Pigs

The miniature pig is a very suitable donor species in xenotransplantation of human organs. Lipid metabolism is an important process that involves the creation and degradation of lipids, which is associated with the function of the gastro-intestinal tract. However, the distribution of lipid metabolism related molecules in the gastro-intestinal tract in the miniature pig is unclear. The present study examined the expression of farnesoid X-receptor (FXR), liver X- receptor (LXR), retinoid X-receptor (RXR), liver fatty acid binding protein (L-FABP), fatty acid synthase (FAS) mRNA in the digestive organs of miniature pigs. FXR and LXR mRNA were not expressed in the stomach but were expressed at high and low density in the small and large intestines, respectively. RXR mRNA was expressed in stomach with moderate density, small intestine with high density and in the large intestine with low density. L-FABP and FAS mRNA were expressed in the stomach and large intestine with low density and in the small intestine with high density. L-FABP mRNA was expressed in the liver and kidney with high density, and in pancreas with low density. FAS mRNA was expressed in the liver with high density, and in pancreas and kidney with low density.

Renal L-type fatty acid-binding protein mediates the bezafibrate reduction of cisplatin-induced acute kidney injury

Fibrates, the PPAR alpha ligand-like compounds increase the expression of proximal tubule liver fatty acid binding protein (L-FABP) and significantly decrease cisplatin-induced acute kidney injury. To study whether the bezafibrate-mediated upregulation of renal L-FABP was involved in this cytoprotective effect we treated transgenic mice of PPAR agonists inducible human L-FABP expression with cisplatin in the presence or absence of bezafibrate. Blood urea nitrogen was unchanged in the first day but increased 3 days after cisplatin. While urinary L-FABP increased over 100-fold 1 day after cisplatin treatment in the transgenic mice it was significantly reduced when these transgenic mice were pretreated with bezafibrate. Cisplatin-induced renal necrosis and apoptosis were significantly reduced in bezafibrate pretreated transgenic mice and this correlated with decreased accumulation of lipid and lipid peroxidation products. Immunohistochemical analysis of kidney tissue of bezafibrate-cisplatin-treated transgenic mice showed preservation of cytoplasmic L-FABP in the proximal tubule, but this was reduced in transgenic mice treated only with cisplatin. L-FABP mRNA and protein levels were significantly increased in bezafibrate-cisplatin-treated transgenic mice when compared to mice not fibrate treated. Our study shows that the bezafibrate-mediated upregulation of proximal tubule L-FABP plays a pivotal role in the reduction of cisplatin-induced acute kidney injury.

Effects of soy-lecithin on lipid metabolism and hepatic expression of lipogenic genes in broiler chickens

The purpose of this experiment was to determine the effects of soy-lecithin on the lipid metabolism and expression of lipogenic genes in the liver of broiler chickens. Arbor Acres (AA) chicks growing from 1 to 42 days of age were randomly divided into 4 groups and fed corn–soybean basal diets containing 0% soy-lecithin (control), 0.5% soy-lecithin (SL1), 1% soy-lecithin (SL2), 2% soy-lecithin (SL3), respectively. At the end of experiment, samples of serum were taken for analyses of metabolites/hormones and liver tissue was collected to quantify expression of selected genes. The abdominal fat was removed and samples of thigh muscle and breast muscle also were collected. The hepatic expression of the genes encoding malic enzyme (ME), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), sterol regulatory element binding protein-1 (SREBP-1), stearoyl-CoA (Δ9) desaturase 1 (SCD1) and liver fatty acid binding protein (L-FABP) were determined with reverse transcription-polymerase chain reaction (RT-PCR) using SYBR green as a flourophore monitored in a real time mode. The study showed that the percentage of abdominal fat and liver fat were not significantly affected by soy-lecithin ( P > 0.05). SL2 group had the highest percentage of thigh muscle fat compared with other groups ( P < 0.05), but soy-lecithin had no significant effect on the percentage of breast muscle fat and the width of inter-muscular fat ( P > 0.05). Serum concentration of total serum cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were reduced by soy-lecithin, whereas high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) were improved ( P < 0.05). The thyroid-stimulating hormone (TSH) and insulin (INS) were elevated in SL3 group ( P < 0.05). Furthermore, abundance of ME, FAS, ACC, SREBP-1, L-FABP and SCD1 mRNA were greater ( P < 0.05) in SL3 group. The results indicate that soy-lecithin alters the serum hormone levels and affects hepatic gene expression and thereby regulates fat metabolism of broilers.

Hepatocyte Retinoid X Receptor α-Dependent Regulation of Lipid Homeostasis and Inflammatory Cytokine Expression Contributes to Alcohol-Induced Liver Injury

Hepatocyte retinoid X receptor alpha (RXRalpha)-deficient mice are more sensitive to ethanol toxicity than wild-type mice. Because RXRalpha-mediated pathways are implicated in lipid homeostasis and the inflammatory response, we hypothesized that a compromise in lipid metabolism and associated production of proinflammatory mediators are responsible for the hepatotoxicity observed in ethanol-treated hepatocyte RXRalpha-deficient mice. Wild-type and hepatocyte RXRalpha-deficient mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. After ethanol treatment, serum ALT levels increased significantly (4-fold) in hepatocyte RXRalpha-deficient mice, but not in the wild-type mice. Hepatic liver fatty acid binding protein (L-FABP) mRNA and protein levels were reduced due to RXRalpha deficiency. Ethanol induced L-FABP mRNA and protein in wild-type mice and provided protection against nonesterified fatty acid toxicity; however, this effect was absent in the mutant mice. Accordingly, hepatic nonesterified fatty acid level was increased in ethanol-fed mutant mice. Ethanol increased nuclear factor (NF)-kappaB binding activity in hepatocyte RXRalpha-deficient mice, but not in wild-type mice. In agreement, hepatic mRNA levels of proinflammatory cytokines and chemokines were increased to a greater extent in the mutant than in wild-type mice. Furthermore, signal transducer and activator of transcription factor (STAT) 3 and associated Bcl-xL induction was observed in ethanol-fed wild-type mice but not in ethanol-fed hepatocyte RXRalpha-deficient mice. Taken together, after ethanol treatment, hepatocyte RXRalpha deficiency results in lack of L-FABP induction, increased hepatic free fatty acids, NF-kappaB activation, and proinflammatory cytokines production and a lack of STAT3 activation, which in part may contribute to alcohol-induced liver damage.

The pathogenesis of ethanol versus methionine and choline deficient diet-induced liver injury

The differences and similarities of the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) were examined. Mice (six/group) received one of four Lieber-Decarli liquid diets for 6 weeks: (1) paired-fed control diet; (2) control diet with ethanol (ethanol); (3) paired-fed methionine/choline deficient (MCD) diet; and (4) MCD plus ethanol (combination). Hepatotoxicity, histology, and gene expression changes were examined. Both MCD and ethanol induced macrovesicular steatosis. However, the combination diet produced massive steatosis with minor necrosis and inflammation. MCD and combination diets, but not ethanol, induced serum ALT levels by 1.6- and 10-fold, respectively. MCD diet, but not ethanol, also induced serum alkaline phosphatase levels suggesting bile duct injury. Ethanol increased liver fatty acid binding protein (L-FABP) mRNA and protein levels. In contrast, the combination diet decreased L-FABP mRNA and protein levels and increased hepatic free fatty acid and lipid peroxide levels. Ethanol, but not MCD, reduced hepatic S-adenosylmethionine (SAM) and GSH levels. Hepatic TNFα protein levels were increased in all treatment groups, however, IL-6, a hepatoprotective cytokine which promotes liver regeneration was increased in ethanol-fed mice (2-fold), but decreased in the combination diet-treated mice. In addition, the combination diet reduced phosphorylated STAT3 and Bcl-2 levels. While MCD diet might cause bile duct injury and cholestasis, ethanol preferentially interferes with the SAM-GSH oxidative stress pathway. The exacerbated liver injury induced by the combination diet might be explained by reduced L-FABP, increased free fatty acids, oxidative stress, and decreased IL-6 protein levels. The combination diet is an efficient model of steatohepatitis.

Decreased expression of peroxisome proliferator‐activated receptor α and liver fatty acid binding protein after partial hepatectomy of rats and mice

Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.

Antioxidative function of L-FABP in L-FABP stably transfected Chang liver cells

Liver fatty acid binding protein (L-FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L-FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L-FABP mRNA level. Full-length L-FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA-FABP). Chang cells were stably transfected with pc-DNA-FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 micromol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L-FABP was documented by regular reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and Western blot. The pcDNA-FABP-transfected cells expressed full-length L-FABP mRNA, which was absent from vector-transfected control cells. Western blot showed expression of 14-kd L-FABP protein in pcDNA-FABP-transfected cells, but not in vector-transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L-FABP expression. Lower LDH release was observed in the higher L-FABP-expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L-FABP gene. The L-FABP-expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L-FABP has a significant role in oxidative stress.

Statin Induction of Liver Fatty Acid-Binding Protein (L-FABP) Gene Expression Is Peroxisome Proliferator-activated Receptor-α-dependent

Statins are drugs widely used in humans to treat hypercholesterolemia. Statins act by inhibiting cholesterol synthesis resulting in the activation of the transcription factor sterol-responsive element-binding protein-2 that controls the expression of genes involved in cholesterol homeostasis. Statin therapy also decreases plasma triglyceride and non-esterified fatty acid levels, but the mechanism behind this effect remains more elusive. Liver fatty acid-binding protein (L-FABP) plays a role in the influx of long-chain fatty acids into hepatocytes. Here we show that L-FABP is a target for statins. In rat hepatocytes, simvastatin treatment induced L-FABP mRNA levels in a dose-dependent manner. Moreover, L-FABP promoter activity was induced by statin treatment. Progressive 5'-deletion analysis revealed that the peroxisome proliferator-activated receptor (PPAR)-responsive element located at position -67/-55 was responsible for the statin-mediated transactivation of the rat L-FABP promoter. Moreover, treatment with simvastatin and the PPARalpha agonist Wy14,649 resulted in a synergistic induction of L-FABP expression (mRNA and protein) in rat Fao hepatoma cells. This effect was also observed in vivo in wild-type mice but not in PPARalpha-null animals demonstrating the direct implication of PPARalpha in L-FABP regulation by statin treatment. Statin treatment resulted in a rise in PPARalpha mRNA levels both in vitro and in vivo and activated the mouse PPARalpha promoter in a reporter assay. Altogether, these data demonstrate that L-FABP expression is up-regulated by statins through a mechanism involving PPARalpha. Moreover, PPARalpha might be a statin target gene. These effects might contribute to the triglyceride/non-esterified fatty acid-lowering properties of statins.

Enhanced expression of cytosolic fatty acid binding protein and fatty acid uptake during liver regeneration in rats

Cytoplasmic liver fatty acid binding protein (L-FABP) has been suggested to be associated with cellular mitotic activity but the changes in L-FABP mRNA and protein levels during liver regeneration following partial hepatectomy (PHx) are not clear. In the present study, we determined the time course of L-FABP mRNA expression and L-FABP levels following 70% PHx using Northern and Western blot, respectively. To elucidate one of the roles for L-FABP in PHx, [3H]-palmitic acid clearance in hepatocytes isolated from 24 h post-PHx and control animals was assessed. L-FABP mRNA increased at 30 min, peaked at approximately 1 h (163 +/- 17%; mean +/- SE, n = 5), and returned to control levels 6 h post-PHx. L-FABP level also increased at 1 h but peaked at 24-h (219 +/- 41%; mean +/- SE, n = 5). Hepatocyte [3H]-palmitic acid clearance increased by 29% at 24-h post-PHx, suggesting an increased intracellular transport (or binding) function by L-FABP. Pre-treatment with dexamethasone statistically reduced L-FABP levels (29%) and suppressed the regenerative process (mitotic activity). L-FABP mRNA increased sharply in response to PHx but the increase was short lived, while L-FABP level increased at a later stage. Both L-FABP content and fatty acid uptake increased significantly during liver regeneration induced by PHx in rats. It is likely that L-FABP is one of the factors responsible for hepatic regeneration.

Effects of fatty acids and growth hormone on liver fatty acid binding protein and PPARalpha in rat liver.

The aim of this study was to investigate the interaction between long-chain fatty acids (LCFA) and growth hormone (GH) in the regulation of liver fatty acid binding protein (LFABP) and peroxisome proliferator-activated receptor-alpha (PPARalpha). Cultured rat hepatocytes were given oleic acid (OA; 500 microM) and GH (100 ng/ml) for 3 days. LFABP mRNA increased 3.6-fold by GH and 5.7-fold by OA, and combined incubation with GH and OA increased LFABP mRNA 17.6-fold. PPARalpha mRNA was decreased 50% by GH, but OA had no effect. Hypophysectomized (Hx) female rats were treated with L-thyroxine, cortisol, GH, and dietary fat for 7 days. PPARalpha mRNA levels were three- to fourfold higher in Hx than in normal female rats. GH decreased PPARalpha mRNA 50% in Hx rats. Dietary triglycerides (10% corn oil) increased LFABP mRNA and cytosolic LFABP about twofold but had no effect on PPARalpha mRNA in Hx rats. GH and dietary triglycerides had an additive effect on LFABP expression. Dietary triglycerides increased mitochondrial hydroxymethylglutaryl-CoA synthase mRNA only in the presence of GH. The diet increased serum triglycerides in Hx rats, and GH treatment prevented this increase. Addition of cholesterol to the diet did not influence LFABP levels but mitigated increased hepatic triglyceride content. In summary, these studies show that GH regulates LFABP expression independently of PPARalpha. Moreover, GH has different effects on PPARalpha-responsive genes and does not counteract the effect of LCFA on the expression of these gene products.

Differential involvement of peroxisome-proliferator-activated receptors α and δ in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (α, γ and δ) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARα-null mice, we have found that PPARα in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARδ/α agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARα-null mice. These findings demonstrate that PPARδ can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARδ target gene in the small intestine. We propose that PPARδ contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.