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: Caspase-1 is known to activate the pro-inflammatory cytokines IL-1β and IL-18. Additionally, it can cleave other substrates including proteins involved in metabolism. Recently, we showed that caspase-1-deficiency in mice strongly reduces HFD-induced weight gain, at least partly caused by an increased energy production. Increased feces secretion by caspase-1-deficient mice suggests that lipid malabsorption possibly further reduces adipose tissue mass. In this study we therefore investigated whether caspase-1 plays a role in triglyceride-(TG)-rich-lipoprotein metabolism using caspase-1-deficient and wild-type mice. Caspase-1-deficiency reduced the postprandial TG-response to an oral lipid load, while TG-derived fatty acid (FA) uptake by peripheral tissues was not affected, demonstrated by unaltered kinetics of [3H]TG-labeled VLDL-like-emulsion-particles. An oral gavage of [3H]TG-containing olive oil revealed that caspase-1-deficiency reduced TG absorption and subsequent uptake of TG-derived FA in liver, muscle, and adipose tissue. Similarly, despite an elevated hepatic TG content, caspase-1-deficiency reduced hepatic VLDL-TG production. Intestinal and hepatic gene expression analysis revealed that caspase-1-deficiency did not affect FA oxidation or FA uptake, but rather reduced intracellular FA transport, thereby limiting lipid availability for the assembly and secretion of TG-rich lipoproteins. The current study reveals a novel function for caspase-1, or caspase-1-cleaved substrates, in controlling intestinal TG absorption and hepatic TG secretion.The Journal of Lipid Research 11/2012; · 4.39 Impact Factor
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ABSTRACT: Nascent very low-density lipoprotein (VLDL) exits the endoplasmic reticulum (ER) in a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Similar to protein transport vesicles (PTV), VTVs require coat complex II (COPII) proteins for their biogenesis from the ER membranes. Since the size of the VTV is large, we hypothesized that protein(s) in addition to COPII components might be required for VTV biogenesis. Our proteomic analysis supported by western blotting data show that a 26 kDa protein, cideB, is present in the VTV but not in other ER-derived vesicles such as PTV and PCTV. Western blotting and immuno-electron microscopy analyses suggest that cideB is concentrated in the VTV. Our co-immunoprecipitation data revealed that cideB specifically interacts with VLDL structural protein, apolipoproteinB100 (apoB100), but not with albumin, a PTV cargo protein. Confocal microscopic data indicate that cideB co-localizes with apoB100 in the ER. Additionally, cideB interacts with COPII components, Sar1 and Sec24. To investigate the role of cideB in VTV biogenesis, we performed an in vitro ER-budding assay. We show that the blocking of cideB inhibits VTV-budding indicating a direct requirement of cideB in VTV formation. To confirm our findings, we knocked down cideB in primary hepatocytes and isolated ER and cytosol to examine if they support VTV budding. Our data suggest that cideB knockdown significantly reduces VTV biogenesis. These findings suggest that cideB forms an intricate COPII-coat and regulates the VTV biogenesis.Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
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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