Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum.
ABSTRACT The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.
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ABSTRACT: The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal-FABP (IFABP; FABP2) and liver-FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally-coexpressed proteins, which both bind long chain FA, are functionally distinct. Here we directly compared IFABP-/- and LFABP-/- mice fed high-fat diets containing long-chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP-/- mice, while LFABP-/- mice had reduced monoacylglycerol (MG) incorporation in TG relative to PL, and reduced radiolabeled monoacylglycerol (MG) incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis: LFABP-/- mice fed high-fat diets became obese relative to WT, while IFABP-/- mice displayed an opposite, lean, phenotype. Fuel utilization followed adiposity, with LFABP-/- mice preferentially utilizing lipids, and IFABP-/- mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamine (AEA) were elevated in LFABP-/-, perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP-/- mice, result in divergent downstream effects at the systemic level.Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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ABSTRACT: BACKGROUND: The present study was designed to investigate the effect of albusin B on lipid metabolism and antioxidant defense in broiler chickens by a proteomic approach. The bacteriocin, albusin B of Ruminococcus albus 7, expressed by yeast was applied in this study. Three dietary treatments, consisting of the basal diet (control), basal diet + albusin B (2.5 g kg(-1) ), and basal diet + nosiheptide (2.5 mg kg(-1) ) were randomly fed to 90 broiler chickens from 1 to 35 days of age, respectively. After 35 days of supplementation, the growth performance, lipid metabolism and antioxidant proteins in the jejunum and liver, intestinal protein profile, and plasma lipid profile were analyzed. RESULTS: Broilers with albusin B supplementation had greater body weight than the control broilers. Compared with the control broilers, lower triglyceride and higher high-density lipoprotein concentration in the blood were observed in both broilers with albusin B and nosiheptide supplementation. In addition, albusin B suppressed the mRNA expression of fatty acid binding protein 2 and ATP binding cassette transporter G 5 in the jejunum. In the jejunal protein profiles, four antioxidant proteins were upregulated by albusin B and nosiheptide treatments. The jejunal antioxidant gene expression had a concordant pattern. Hepatic genes related to lipid metabolism, 3-hydroxy-3-methyl-glutaryl CoA reductase, and superoxide dismutase were upregulated by albusin B supplementation. CONCLUSION: Albusin B supplementation modulated lipid metabolism and activated systemic antioxidant defense, which might partially contribute to the performance of broiler chickens. Copyright © 2012 Society of Chemical Industry.Journal of the Science of Food and Agriculture 06/2012; · 1.76 Impact Factor
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ABSTRACT: Non-steroidal anti-inflammatory drugs (NSAIDs) play a significant role in the chemoprevention of cancer. We recently showed the chemopreventive response of a NSAID, 2-[(3-chloro-2-methylphenyl)amino]benzoic acid) known as tolfenamic acid (TA) in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in rats. Pre-clinical studies showed that TA inhibits Specificity protein (Sp) transcription factors and acts as an anti-cancer agent in several cancer models; however the pertinent mechanisms associated with its chemopreventive response in esophageal cancer are not known. Since the bioactivation of carcinogens through cytochrome P450 (CYP) is critical for the induction of cancer, we have studied the effect of TA on critical CYP isozymes in mouse liver samples. Athymic nude mice were treated with vehicle (corn oil) or TA (50 mg kg(-1), 3 times per week) for 4 weeks. Protein extracts (whole cell lysates and microsomal fractions) were prepared from liver tissue and the expression of various CYP isozymes was determined by Western blot analysis. Rat (Sprague-Dawley) livers were harvested and primary hepatocyte cultures were treated with vehicle (DMSO) or TA (50 μM) and cell viability was assessed at 2 and 5 days post-treatment. TA caused remarkable decrease in the expression of CYP2E1 in both liver lysates and sub-cellular fraction, while its response on other tested isozymes was marginal. TA did not affect the body weight of animals (mice) and viability of rat hepatocytes. These results demonstrate that TA modulates the expression of CYP2E1 which is associated with the bioactivation of carcinogens without causing apparent toxicity. These data suggest that TA-induced inhibition of CYP2E1 attenuates the bioactivation of carcinogens potentially leading to the chemoprevention of NMBA-induced esophageal tumorigenesis in rats.Integrative Biology 07/2012; 4(9):1122-9. · 4.32 Impact Factor