A protein of molecular weight approximately 12,000 which binds long-chain fatty acids and certain other lipids has been identified in cytosol of intestinal mucosa, liver, myocardium, adipose tissue, and kidney. Binding is noncovalent and is greater for unsaturated than for saturated and medium-chain fatty acids. This protein appears to be identical with the smaller of two previously described cytoplasmic anion-binding proteins. Binding of long-chain fatty acids by this protein is greater than that of other anions tested, including sulfobromophthalein, and does not depend on negative charge alone. The presence of this binding protein may explain previously observed differences in intestinal absorption among fatty acids, and the protein may participate in the utilization of long-chain fatty acids by many mammalian tissues.
A B S T R A C T A soluble fatty acid-binding protein (FABP), mol wt 12,000 is present in intestinal mucosa and other tissues that utilize fatty acids, including liver, myocardium, adipose, and kidney. This protein binds long chain fatty acids both in vivo and in vitro.FABP was isolated from rat intestine by gel filtration and isoelectric focusing. It showed a reaction of complete -immunochemical identity with proteins in the 12,000 mol wt fatty acid-binding fractions of liver, myocardium, and adipose tissue supernates. (The presence of immunochemically nonidentical 12,000 mol wt FABP in these tissues is not excluded.) By quantitative radial immunodiffusion, supernatant FABP concentration in mucosa from proximal and middle thirds of jejuno-ileum significantly exceeded that in distal third, duodenum, and liver, expressed as micrograms per milligram soluble protein, micrograms per gram DNA, and micrograms per gram tissue. FABP concentration in villi was approximately three times greater than in crypts. Small quantities of FABP were present in washed nuclei-cell membrane, mitochondrial and microsomal fractions. However, the amount of FABP solubilized per milligram membrane protein was similar for all particulate fractions, and total membrane-associated FABP was only about 16% of supernatant FABP. Intestinal FABP concentration was significantly greater in animals maintained on high fat diets than on low fat; saturated and unsaturated fat diets did not differ greatly in this regard.The preponderance of FABP in villi from proximal and middle intestine, its ability to bind fatty acids in vivo as well as in vitro, and its response to changes in dietary fat intake support the concept that this protein participates in cellular fatty acid transport during fat
Kinetic analysis of the uptake of carbon-14-labeled oleate in a single-pass perfusion of rat liver and saturable and specific binding of iodine-125-labeled albumin to hepatocytes in suspension suggest the existence of a receptor for albumin on the liver cell surface. The putative receptor appears to mediate uptake of albumin-bound fatty acids by the cell and may account for the efficient hepatic extraction of many other substances tightly bound to albumin.
Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14-15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABPpm). These proteins have not been fully characterized to date, but strong evidence suggest that they function in the transport of long-chain fatty acids across the plasma membrane.
A B S T R A C T The transport of endogenous lipids in the lipoproteins of mesenteric lymph was studied in fasting rats with mesenteric lymph fistulas. The lymph was found to contain, in addition to chylomicrons (Sf > 400), a significant amount of another, more dense, triglyceride-rich fraction, the very low density lipoproteins (VLDL), which showed a peak Sf of 102. The VLDL differed from chylomicrons not only in flotation, but also in per cent lipid composition and electrophoretic mobility in agarose gel. The VLDL fraction was found to contain 47% of the triglyceride and 54% of the cholesterol of fasting lymph and, in the fasting state, was the major lipoprotein species present.When cholestyramine resin was administered intraduodenally, or bile flow was acutely diverted from the intestine, it was demonstrated that the lipids in lymph VLDL, like those in chylomicrons, were derived from the intestine and bile. These data indicate that the VLDL in intestinal lymph are not derived from the plasma but are of intestinal origin.Because certain properties of lymph VLDL were similar to those reported for plasma VLDL (per cent lipid composition, flotation coefficient, and continuing entry into plasma in the fasting state), additional comparisons between these fractions were made. Although lymph VLDL moved to the as region in agarose gel, when they were mixed with VLDL-free serum immediately before electrophoresis they showed the a, mobility of rat serum VLDL. Furthermore, immunoelectrophoretic comparison of partially delipidated lymph and serum VLDL revealed that these fractions shared in
Intestinal fatty acid binding protein mRNA is one of the most abundant mRNA species in the rat small intestinal epithelium. RNA We were interested in defining the structure of this protein for several reasons. (i) Its precise function in lipid metabolism is not known. Partially purified preparations of this polypeptide have been shown to bind long-chain fatty acids, having higher affinity for unsaturated compared to saturated forms (2). However, it is unclear whether this protein actually participates in cellular fatty acid transport across the intestinal mucosa (1-3) or simply represents an intracellular FABP. (ii) The rat small intestine contains another, immunologically distinct FABP termed "liver" FABP because of its initial identification in rat hepatocytes (4, 5). The structural and functional relationships between these two proteins have not been determined. The mRNAs encoding these two FABPs are the most abundant mRNA species that accumulate in the small intestinal mucosa, comprising 3.3% (liver FABP) and 2% (intestinal FABP) of translatable RNA (5). We have recently determined the complete sequence of liver FABP mRNA from an analysis of cloned cDNA (6). Liver FABP is identical to Z protein (7) and is probably the same as sterol carrier protein (8), amino azo dye-binding protein A (9), and band C (10). (iii) These small peptides could serve as a useful model system for detailing primary and secondary structural characteristics required for lipid-prQtein interactions.In this report we describe the primary structure of this protein obtained from analysis of cloned cDNA and compare the structures of both liver and intestinal FABPs. Possible functional roles played by these peptides in intestinal lipid metabolism are discussed. MATERIALS AND METHODSMale Sprague-Dawley rats (250 g) were maintained on a standard rat chow diet and fasted overnight before being sacrificed. 3H-and 35S-labeled amino acids were purchased from Amersham. Monospecific antiserum against intestinal FABP was identical to that described previously (2). This antibody does not form precipitin lines against rat liver cytosol or purified rat liver FABP on Ouchterlony plates but reacts well with intestinal FABP and intestinal cytosol (data not shown).
A B S T R A C T The role of nonchylomicron very low density lipoproteins (VLDL, in the transport of triglyceride and cholesterol was studied during lipid absorption. Various long chain fatty acids were infused intraduodenally in the form of mixed fatty acid-monoolein-taurocholate micelles; control animals received saline or taurocholate.As compared with controls, all fatty acids (palmitic, oleic, linoleic) resulted in significant increases in chylomicron (Sf > 400) triglyceride. In addition, palmitic acid resulted in a twofold increase in VLDL triglyceride, whereas with the absorption of oleic or linoleic acid VLDL triglyceride did not change significantly. Differences in triglyceride fatty acid composition between chylomicrons and VLDL were observed during lipid absorption.Although the absolute amount of endogenous choles- These studies demonstrate that dietary long chain fatty acids differ significantly in their effects upon the transport of triglyceride and cholesterol by lipoproteins of rat intestinal lymph. These findings, together with the observed differences in rates of removal of chylomicrons and VLDL from plasma, suggest that variations in lipoprotein production at the intestinal level may be reflected in differences in the subsequent metabolism of absorbed dietary and endogenous lipids. INTRODUCTIONIn previous studies (1) we have shown that in the fasting state very low density lipoproteins (VLDL) play a major role in the transport of endogenous lipids in intestinal lymph. This class of triglyceride-rich lipoproteins can be operationally distinguished from the less dense chylomicrons by its flotation rate (VLDL, Sf 20-400; chylomicrons, Sf > 400). Although it is possible that VLDL and the more often studied chylomicrons represent two ends of a single spectrum of particle size and density, these two fractions differ also in other respects, including electrophoretic mobility in agarose gel and per cent lipid and protein composition (1, 2). Accordingly, intestinal lymph VLDL bear a greater similarity to plasma VLDL than to chylomicrons. These observations have suggested that the metabolic role of in-
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