The Multifunctional Family of Mammalian Fatty Acid-Binding Proteins.

Fatty acid-binding proteins (FABPs) are small lipid-binding proteins abundantly expressed in tissues that are highly active in fatty acid (FA) metabolism. Ten mammalian FABPs have been identified, with tissue-specific expression patterns and highly conserved tertiary structures. FABPs were initially studied as intracellular FA transport proteins. Further investigation has demonstrated their participation in lipid metabolism, both directly and via regulation of gene expression, and in signaling within their cells of expression. There is also evidence that they may be secreted and have functional impact via the circulation. It has also been shown that the FABP ligand binding repertoire extends beyond long-chain FAs and that their functional properties also involve participation in systemic metabolism. This article reviews the present understanding of FABP functions and their apparent roles in disease, particularly metabolic and inflammation-related disorders and cancers.

The proximal intestinal Fatty Acid-Binding Proteins liver FABP (LFABP) and intestinal FABP (IFABP) differentially modulate whole body energy homeostasis but are not centrally involved in net dietary lipid absorption: Studies of the LFABP/IFABP double knockout mouse.

Proximal intestinal enterocytes expresses both intestinal-fatty acid binding protein (IFABP; FABP2) and liver-FABP (LFABP; FABP1). These FABPs are thought to be important in the net uptake of dietary lipid from the intestinal lumen, however their specific and potentially unique functions in the enterocyte remain incompletely understood. We previously showed markedly divergent phenotypes in LFABP-/- vs. IFABP-/- mice fed high-fat diets, with the former becoming obese and the latter remaining lean relative to wild-type (WT) mice, supporting different functional roles for each protein. Interestingly, neither mouse model displayed increased fecal lipid concentration, raising the question of whether the presence of one FABP was sufficient to compensate for absence of the other. Here, we generated an LFABP and IFABP double knockout mouse (DKO) to determine whether simultaneous ablation would lead to fat malabsorption, and to further interrogate the individual vs. overlapping functions of these proteins. Male WT, IFABP-/-, LFABP-/-, and DKO mice were fed a low-fat (10 % kcal) or high-fat (45 % kcal) diet for 12 weeks. The body weights and fat mass of the DKO mice integrated those of the LFABP-/- and IFABP-/- single knockouts, supporting the notion that IFABP and LFABP have distinct functions in intestinal lipid assimilation that result in downstream alterations in systemic energy metabolism. Remarkably, no differences in fecal fat concentrations were found in the DKO compared to WT, revealing that the FABPs are not required for net intestinal uptake of dietary lipid.

Structure-function relationships of cholesterol mobilization from the endo-lysosome compartment of NPC1-deficient human cells by ß-CD polyrotaxanes.

Niemann-Pick Type C is a rare metabolic disorder characterized by the cellular accumulation of cholesterol within endosomal and lysosomal compartments. 2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) containing polyrotaxanes represent an attractive approach for treating this disease due to their ability to circulate in the blood stream for longer periods of time as a prodrug form of HP-ß-CD. Once inside the cell, the macromolecular structure is thought to break down into the Pluronic precursor and the active cyclodextrin agent that promotes cholesterol mobilization from the aberrant accumulations within NPC-deficient cells. We now report that both cholesterol and decaarginine (R10) endcapped polyrotaxanes are able to remove cholesterol from NPC1 patient fibroblasts. R10 endcapped materials enter these cells and are localized within endosomes after 16 h. The cholesterol mobilization from endo-lysosomal compartments of NPC1 cells by the polyrotaxanes was directly related to their extent of endcapping and their threading efficiency. Incorporation of 4-sulfobutylether-ß-cyclodextrin (SBE-ß-CD) significantly improved cholesterol mobilization due to the improved solubility of the compounds. Additionally, in our efforts to scale-up the synthesis for preclinical studies, we prepared a library of polyrotaxanes using a solid phase synthesis method. These compounds also led to significant cholesterol mobilization from the cells, however, cytotoxicity studies showed that they were substantially more toxic than those prepared by the solvent-assisted method, thus limiting the therapeutic utility of agents prepared by this expedited method. Our findings demonstrate that complete endcapping of the polyrotaxanes and improved solubility are important design features for delivering high copy numbers of therapeutic ß-CD to promote enhanced sterol clearance in human NPC1-deficient cells.

Gut Microbiota and Phenotypic Changes Induced by Ablation of Liver- and Intestinal-Type Fatty Acid-Binding Proteins.

Intestinal fatty acid-binding protein (IFABP; FABP2) and liver fatty acid-binding protein (LFABP; FABP1) are small intracellular lipid-binding proteins. Deficiency of either of these proteins in mice leads to differential changes in intestinal lipid transport and metabolism, and to markedly divergent changes in whole-body energy homeostasis. The gut microbiota has been reported to play a pivotal role in metabolic process in the host and can be affected by host genetic factors. Here, we examined the phenotypes of wild-type (WT), LFABP-/-, and IFABP-/- mice before and after high-fat diet (HFD) feeding and applied 16S rRNA gene V4 sequencing to explore guild-level changes in the gut microbiota and their associations with the phenotypes. The results show that, compared with WT and IFABP-/- mice, LFABP-/- mice gained more weight, had longer intestinal transit time, less fecal output, and more guilds containing bacteria associated with obesity, such as members in family Desulfovibrionaceae. By contrast, IFABP-/- mice gained the least weight, had the shortest intestinal transit time, the most fecal output, and the highest abundance of potentially beneficial guilds such as those including members from Akkermansia, Lactobacillus, and Bifidobacterium. Twelve out of the eighteen genotype-related bacterial guilds were associated with body weight. Interestingly, compared with WT mice, the levels of short-chain fatty acids in feces were significantly higher in LFABP-/- and IFABP-/- mice under both diets. Collectively, these studies show that the ablation of LFABP or IFABP induced marked changes in the gut microbiota, and these were associated with HFD-induced phenotypic changes in these mice.

Synthesis of Phosphatidyl Glycerol Containing Unsymmetric Acyl Chains Using H-Phosphonate Methodology.

Naturally occurring phospholipids, such as phosphatidyl glycerol (PG), are gaining interest due to the roles they play in disease mechanisms. To elucidate the metabolism of PG, an optically pure material is required, but this is unfortunately not commercially available. Our previous PG synthesis route utilized phosphoramidite methodology that addressed issues surrounding fatty acid substrate scope and glycerol backbone modifications prior to headgroup phosphorylation, but faltered in the reproducibility of the overall pathway due to purification challenges. Herein, we present a robust pathway to optically pure PG in fewer steps, utilizing H-phosphonates that features a chromatographically friendly and stable triethyl ammonium H-phosphonate salt. Our route is also amendable to the simultaneous installation of different acyl chains, either saturated or unsaturated, on the glycerol backbone.

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage.

Niemann-Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.

Impact of vitamin A transport and storage on intestinal retinoid homeostasis and functions.

Lecithin:retinol acyltransferase and retinol-binding protein enable vitamin A (VA) storage and transport, respectively, maintaining tissue homeostasis of retinoids (VA derivatives). The precarious VA status of the lecithin:retinol acyltransferase-deficient (Lrat-/-) retinol-binding protein-deficient (Rbp-/-) mice rapidly deteriorates upon dietary VA restriction, leading to signs of severe vitamin A deficiency (VAD). As retinoids impact gut morphology and functions, VAD is often linked to intestinal pathological conditions and microbial dysbiosis. Thus, we investigated the contribution of VA storage and transport to intestinal retinoid homeostasis and functionalities. We showed the occurrence of intestinal VAD in Lrat-/-Rbp-/- mice, demonstrating the critical role of both pathways in preserving gut retinoid homeostasis. Moreover, in the mutant colon, VAD resulted in a compromised intestinal barrier as manifested by reduced mucins and antimicrobial defense, leaky gut, increased inflammation and oxidative stress, and altered mucosal immunocytokine profiles. These perturbations were accompanied by fecal dysbiosis, revealing that the VA status (sufficient vs. deficient), rather than the amount of dietary VA per se, is likely a major initial discriminant of the intestinal microbiome. Our data also pointed to a specific fecal taxonomic profile and distinct microbial functionalities associated with VAD. Overall, our findings revealed the suitability of the Lrat-/-Rbp-/- mice as a model to study intestinal dysfunctions and dysbiosis promoted by changes in tissue retinoid homeostasis induced by the host VA status and/or intake.

Stereospecific synthesis of phosphatidylglycerol using a cyanoethyl phosphoramidite precursor.

Phosphatidylglycerols (PG) are a family of naturally occurring phospholipids that are responsible for critical operations within cells. PG are characterized by an (R) configuration in the diacyl glycerol backbone and an (S) configuration in the phosphoglycerol head group. Herein, we report a synthetic route to provide control over the PG stereocenters as well as control of the acyl chain identity.

Two fatty acid-binding proteins expressed in the intestine interact differently with endocannabinoids.

Two different members of the fatty acid-binding protein (FABP) family are found in enterocyte cells of the gastrointestinal system, namely liver-type and intestinal fatty acid-binding proteins (LFABP and IFABP, also called FABP1 and FABP2, respectively). Striking phenotypic differences have been observed in knockout mice for either protein, for example, high fat-fed IFABP-null mice remained lean, whereas LFABP-null mice were obese, correlating with differences in food intake. This finding prompted us to investigate the role each protein plays in directing the specificity of binding to ligands involved in appetite regulation, such as fatty acid ethanolamides and related endocannabinoids. We determined the binding affinities for nine structurally related ligands using a fluorescence competition assay, revealing tighter binding to IFABP than LFABP for all ligands tested. We found that the head group of the ligand had more impact on binding affinity than the alkyl chain, with the strongest binding observed for the carboxyl group, followed by the amide, and then the glycerol ester. These trends were confirmed using two-dimensional 1 H-15 N nuclear magnetic resonance (NMR) to monitor chemical shift perturbation of the protein backbone resonances upon titration with ligand. Interestingly, the NMR data revealed that different residues of IFABP were involved in the coordination of endocannabinoids than those implicated for fatty acids, whereas the same residues of LFABP were involved for both classes of ligand. In addition, we identified residues that are uniquely affected by binding of all types of ligand to IFABP, suggesting a rationale for its tighter binding affinity compared with LFABP.

Retinol-binding protein 2 (RBP2) binds monoacylglycerols and modulates gut endocrine signaling and body weight.

Expressed in the small intestine, retinol-binding protein 2 (RBP2) facilitates dietary retinoid absorption. Rbp2-deficient (Rbp2-/- ) mice fed a chow diet exhibit by 6-7 months-of-age higher body weights, impaired glucose metabolism, and greater hepatic triglyceride levels compared to controls. These phenotypes are also observed when young Rbp2-/- mice are fed a high fat diet. Retinoids do not account for the phenotypes. Rather, RBP2 is a previously unidentified monoacylglycerol (MAG)-binding protein, interacting with the endocannabinoid 2-arachidonoylglycerol (2-AG) and other MAGs with affinities comparable to retinol. X-ray crystallographic studies show that MAGs bind in the retinol binding pocket. When challenged with an oil gavage, Rbp2-/- mice show elevated mucosal levels of 2-MAGs. This is accompanied by significantly elevated blood levels of the gut hormone GIP (glucose-dependent insulinotropic polypeptide). Thus, RBP2, in addition to facilitating dietary retinoid absorption, modulates MAG metabolism and likely signaling, playing a heretofore unknown role in systemic energy balance.

Mechanisms underlying reduced weight gain in intestinal fatty acid-binding protein (IFABP) null mice.

Intestinal-fatty acid binding protein (IFABP; FABP2) is a 15-kDa intracellular protein abundantly present in the cytosol of the small intestinal (SI) enterocyte. High-fat (HF) feeding of IFABP-/- mice resulted in reduced weight gain and fat mass relative to wild-type (WT) mice. Here, we examined intestinal properties that may underlie the observed lean phenotype of high fat-fed IFABP-/- mice. No alterations in fecal lipid content were found, suggesting that the IFABP-/- mice are not malabsorbing dietary fat. However, the total excreted fecal mass, normalized to food intake, was increased for the IFABP-/- mice relative to WT mice. Moreover, intestinal transit time was more rapid in the IFABP-/- mice. IFABP-/- mice displayed a shortened average villus length, a thinner muscularis layer, reduced goblet cell density, and reduced Paneth cell abundance. The number of proliferating cells in the crypts of IFABP-/- mice did not differ from that of WT mice, suggesting that the blunt villi phenotype is not due to alterations in proliferation. IFABP-/- mice were observed to have altered expression of genes and proteins related to intestinal structure, while immunohistochemical analyses revealed increased staining for markers of inflammation. Taken together, these studies indicate that the ablation of IFABP, coupled with high-fat feeding, leads to changes in gut motility and morphology, which likely contribute to the relatively leaner phenotype occurring at the whole-body level. Thus, IFABP is likely involved in dietary lipid sensing and signaling, influencing intestinal motility, intestinal structure, and nutrient absorption, thereby impacting systemic energy metabolism.NEW & NOTEWORTHY Intestinal fatty acid binding protein (IFABP) is thought to be essential for the efficient uptake and trafficking of dietary fatty acids. In this study, we demonstrate that high-fat-fed IFABP-/- mice have an increased fecal output and are likely malabsorbing other nutrients in addition to lipid. Furthermore, we observe that the ablation of IFABP leads to marked alterations in intestinal morphology and secretory cell abundance.

Intracellular cholesterol trafficking is dependent upon NPC2 interaction with lysobisphosphatidic acid.

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein.

Fatty acid-binding proteins: functional understanding and diagnostic implications.

PURPOSE OF REVIEW: Fatty acid-binding proteins (FABPs) are a family of small, abundant proteins with highly tissue-specific expression patterns whose different functions remain incompletely understood. The purpose of this review is to summarize recent findings regarding FABP functions and mechanisms of action, including their potential utilization as serum markers of tissue-specific metabolic diseases. RECENT FINDINGS: FABPs are important not only in their tissues of origin but also appear to influence the metabolism and function of tissues distal to their sites of expression. This may be secondary to metabolic changes in their primary tissues, and/or a result of FABP secretion from these tissues leading to effects on distal sites. Their levels in the circulation are increasingly explored as potential biomarkers for tissue-specific disease prognosis and progression. SUMMARY: The nine fatty acid-binding members of the FABP family have unique tissue-specific functions and important secondary effects on tissues in which they are not expressed. For many of the FABPs, circulating levels may be indicative of disease processes related to their primary tissues, and may influence physiological function in distal tissues.

Muscle metabolic reprogramming underlies the resistance of liver fatty acid-binding protein (LFABP)-null mice to high-fat feeding-induced decline in exercise capacity.

Liver fatty acid-binding protein (LFABP) binds long-chain fatty acids with high affinity and is abundantly expressed in the liver and small intestine. Although LFABP is thought to function in intracellular lipid trafficking, studies of LFABP-null (LFABP-/-) mice have also indicated a role in regulating systemic energy homeostasis. We and others have reported that LFABP-/- mice become more obese than wildtype (WT) mice upon high-fat feeding. Here, we show that despite increased body weight and fat mass, LFABP-/- mice are protected from a high-fat feeding-induced decline in exercise capacity, displaying an approximate doubling of running distance compared with WT mice. To understand this surprising exercise phenotype, we focused on metabolic alterations in the skeletal muscle due to LFABP ablation. Compared with WT mice, resting skeletal muscle of LFABP-/- mice had higher glycogen and intramuscular triglyceride levels as well as an increased fatty acid oxidation rate and greater mitochondrial enzyme activities, suggesting higher substrate availability and substrate utilization capacity. Dynamic changes in the respiratory exchange ratio during exercise indicated that LFABP-/- mice use more carbohydrate in the beginning of an exercise period and then switch to using lipids preferentially in the later stage. Consistently, LFABP-/- mice exhibited a greater decrease in muscle glycogen stores during exercise and elevated circulating free fatty acid levels postexercise. We conclude that, because LFABP is not expressed in muscle, its ablation appears to promote interorgan signaling that alters muscle substrate levels and metabolism, thereby contributing to the prevention of high-fat feeding-induced skeletal muscle impairment.

Relative levels of dietary EPA and DHA impact gastric oxidation and essential fatty acid uptake.

Previous research showed that increasing the proportion of docosahexaenoic acid (DHA) in marine lipid supplements significantly reduces associated health benefits compared with balanced eicosapentaenoic acid (EPA):DHA supplementation Dasilva et al., 2015 [1]. It was therefore hypothesized that the EPA and DHA molecules might have differential resistance to oxidation during gastric digestion and that the oxidation level achieved could be inversely correlated with intestinal absorption and, hence, with the resultant health benefits. Accordingly, we tested this proposed mechanism of action by investigating the degree of oxidation in the stomach, and the levels of bioaccessible lipids, of varying molar proportions of DHA and EPA (2:1, 1:1 and 1:2) using the dynamic gastrointestinal tract model TIM-1. In addition, small intestine enterocyte absorption and metabolism were simulated by Caco-2 cell monolayers that were incubated with these same varying proportions of DHA and EPA, and comparing oxidized and nonoxidized polyunsaturated fatty acids (PUFAs). The results show an inverse correlation between lipid oxidation products in the stomach and the levels of bioaccessible lipids. The balanced 1:1 EPA:DHA diet resulted in lower oxidation of PUFAs during stomach digestion relative to the other ratios tested. Finally, cell-based studies showed significantly lower assimilation of oxidized EPA and DHA substrates compared to nonoxidized PUFAs, as well as significant differences between the net uptake of EPA and DHA. Overall, the present work suggests that the correct design of diets and/or supplements containing marine lipids can strongly influence the stability and bioaccessibility of PUFAs during gastrointestinal digestion and subsequent absorption. This could modulate their health benefits related with inflammation, oxidative stress and metabolic disorders.

Protocols and pitfalls in obtaining fatty acid-binding proteins for biophysical studies of ligand-protein and protein-protein interactions.

Adipocyte fatty acid-binding protein (AFABP: FABP4) is a member of the intracellular lipid-binding protein family that is thought to target long-chain fatty acids to nuclear receptors such as peroxisome proliferator-activated receptor gamma (PPARγ), which in turn plays roles in insulin resistance and obesity. A molecular understanding of AFABP function requires robust isolation of the protein in liganded and free forms as well as characterization of its oligomerization state(s) under physiological conditions. We report development of a protocol to optimize the production of members of this protein family in pure form, including removal of their bound lipids by mixing with hydrophobically functionalized hydroxypropyl dextran beads and validation by two-dimensional NMR spectroscopy. The formation of self-associated or covalently bonded protein dimers was evaluated critically using gel filtration chromatography, revealing conditions that promote or prevent formation of disulfide-linked homodimers. The resulting scheme provides a solid foundation for future investigations of AFABP interactions with key ligand and protein partners involved in lipid metabolism.

FABP1 knockdown in human enterocytes impairs proliferation and alters lipid metabolism.

Fatty Acid-Binding Proteins (FABPs) are abundant intracellular proteins that bind long chain fatty acids (FA) and have been related with inmunometabolic diseases. Intestinal epithelial cells express two isoforms of FABPs: liver FABP (LFABP or FABP1) and intestinal FABP (IFABP or FABP2). They are thought to be associated with intracellular dietary lipid transport and trafficking towards diverse cell fates. But still their specific functions are not well understood. To study FABP1's functions, we generated an FABP1 knockdown model in Caco-2 cell line by stable antisense cDNA transfection (FABP1as). In these cells FABP1 expression was reduced up to 87%. No compensatory increase in FABP2 was observed, strengthening the idea of differential functions of both isoforms. In differentiated FABP1as cells, apical administration of oleate showed a decrease in its initial uptake rate and in long term incorporation compared with control cells. FABP1 depletion also reduced basolateral oleate secretion. The secreted oleate distribution showed an increase in FA/triacylglyceride ratio compared to control cells, probably due to FABP1's role in chylomicron assembly. Interestingly, FABP1as cells exhibited a dramatic decrease in proliferation rate. A reduction in oleate uptake as well as a decrease in its incorporation into the phospholipid fraction was observed in proliferating cells. Overall, our studies indicate that FABP1 is essential for proper lipid metabolism in differentiated enterocytes, particularly concerning fatty acids uptake and its basolateral secretion. Moreover, we show that FABP1 is required for enterocyte proliferation, suggesting that it may contribute to intestinal homeostasis.

Bacterial communities in the small intestine respond differently to those in the caecum and colon in mice fed low- and high-fat diets.

Bacterial communities in the mouse caecum and faeces are known to be altered by changes in dietary fat. The microbiota of the mouse small intestine, by contrast, has not been extensively profiled and it is unclear whether small intestinal bacterial communities shift with dietary fat levels. We compared the microbiota in the small intestine, caecum and colon in mice fed a low-fat (LF) or high-fat (HF) diet using 16S rRNA gene sequencing. The relative abundance of major phyla in the small intestine, Bacteriodetes, Firmicutes and Proteobacteria, was similar to that in the caecum and colon; the relative abundance of Verrucomicrobia was significantly reduced in the small intestine compared to the large intestine. Several genera were uniquely detected in the small intestine and included the aerotolerant anaerobe, Lactobacillus spp. The most abundant genera in the small intestine were accounted for by anaerobic bacteria and were identical to those identified in the large intestine. An HF diet was associated with significant weight gain and adiposity and with changes in the bacterial communities throughout the intestine, with changes in the small intestine differing from those in the caecum and colon. Prominent Gram-negative bacteria including genera of the phylum Bacteroidetes and a genus of Proteobacteria significantly changed in the large intestine. The mechanistic links between these changes and the development of obesity, perhaps involving metabolic endotoxemia, remain to be determined.