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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. Expected final online publication date for the Annual Review of Nutrition, Volume 43 is August 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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.

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., Instituto de Investigaciones Bioquímicas de La Plata

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.

Niemann Pick Type C (NPC) Disease is a rare lysosomal storage disorder in which one of the genes that codes for either the NPC-1 or NPC-2 pro-tein is mutated, causing cell lysosomes to accumu-late cholesterol and lipids. Previous studies discov-ered that a unique late endosomal/lysosomal phos-pholipid, lysobisphosphatidic acid (LPBA), is in-volved in cholesterol clearance from late endo-somes. It has also been shown that exogenous treat-ment of the NPC-1 deficient cells with LBPA’s precur-sor, phosphatidylglycerol (PG), leads to LBPA enrich-ment and subsequent endolysosomal cholesterol clearance. Autophagy is a mechanism of cellular clearance in the endolysomal system and we are in-terested to see if it is a partial route in cholesterol clearance during PG treatment of NPC-1 deficient cells. To do so, we silenced the gene that codes for an essential protein in the autophagy pathway, mak-ing the cells autophagy deficient. We then treated the cells with PG, measured the amount of choles-terol clearance in those cells, and compared it to cells with normal autophagy. We found significantly less cholesterol clearance by PG in cells with defec-tive autophagy, confirming that autophagy is in-volved as a partial route in cholesterol clearance dur-ing PG treatment, but not enough of a difference to conclude that it is a major underlying mechanism.

Retinol-binding protein 2 (RBP2) binds 2-monoacylglycerols affecting their intestinal levels and enteroendocrine responses., 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.

8 pages, 4 figures, 1 table, 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, This work was supported by the Spanish Ministerio de Economía y Competitividad (AGL2013-49079-C2-1-R) and the National Institutes of Health (R01-DK38389). The Consejo Superior de Investigaciones Científicas and the University of Santiago de Compostela are gratefully acknowledged for the doctoral fellowship to Gabriel Dasilva. Xunta de Galicia and Axencia Galega de Innovación are also thankfully recognized for the financial support to Gabriel Dasilva

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., Instituto de Investigaciones Bioquímicas de La Plata

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.

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.

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., eLife digest Cholesterol is a type of fat that is essential for many processes in the body, such as repairing damaged cells and producing certain hormones. Normally, cholesterol enters cells from the bloodstream and is then moved to the parts of the cell that need it via a process known as ‘trafficking’. When cholesterol trafficking goes wrong, abnormally large amounts of cholesterol and other fats accumulate within the cell. Over time, these fatty deposits become toxic to cells and eventually damage the affected tissues. Niemann-Pick type C disease (NPC) is a severe genetic disorder affecting cholesterol trafficking. It is characterized by cholesterol build-up in multiple tissues, including the brain, which ultimately causes degeneration and death of nerve cells. Two proteins, NPC1 and NPC2, are involved in NPC disease. Both proteins normally help move cholesterol out of important trafficking compartments (known as the endosomal and lysosomal compartments) to other areas of the cell where it is needed. Patients with the disease can have mutations in either the gene for NPC1 or the gene for NPC2. This means that cells from NPC1 patients do not make enough functional NPC1 protein (but contain working NPC2), and vice versa. Previous studies had shown that giving cells with NPC1 mutations large amounts of the small molecule lysobisphosphatidic acid (LBPA for short) could compensate for the loss of NPC1, and stop the toxic build-up of cholesterol. McCauliff, Langan, Li et al. therefore wanted to explore exactly how LBPA was doing this. They had shown that LBPA dramatically increased the ability of purified NPC2 protein to transport cholesterol, and wondered if the effect of LBPA in the cells without NPC1 depended on NPC2. They predicted that boosting LBPA levels would not work in cells lacking NPC2. Biochemical experiments using purified protein showed that LBPA and NPC2 did indeed interact directly with each other. Systematically changing different building blocks of NPC2 revealed that a single region of the protein is sensitive to LBPA, and when this region was altered, LBPA could no longer interact with NPC2. Since LBPA is naturally produced by cells, they then stimulated cells grown in the laboratory to generate more LBPA using its precursor phosphatidylglycerol. They used cells from patients with mutations in either NPC1 or NPC2 and demonstrated that LBPA’s ability to reverse the accumulation of cholesterol was dependent on its interaction with NPC2. Thus, increasing LBPA levels in cells from patients with NPC1 mutations was beneficial, but had no effect on cells from patients with NPC2 mutations. These results shed new light not only on how cells transport cholesterol, but also on potential methods to combat disorders of cellular cholesterol trafficking. In the future, LBPA could be developed as a genetically tailored, patient-specific therapy for diseases like NPC.

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 by the LE/LY phospholipid lysobisphosphatidic acid (LBPA) and in these studies sought to determine their functional relationship in normal LE/LY cholesterol egress. Here we demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Using its precursor phosphatidylglycerol (PG), we show that PG-induced LBPA enrichment results in clearance of accumulated cholesterol from NPC1-deficient cells but is ineffective in cells lacking functional NPC2. Together these studies reveal a heretofore unknown aspect of intracellular cholesterol trafficking, in which NPC2 and LBPA function together in an obligate step of sterol egress from the LE/LY compartment, which appears to be independent of NPC1.

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.

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.

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.

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2(-/-) fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.

Monoacylglycerol lipase (MGL) is a ubiquitously expressed enzyme that catalyzes the hydrolysis of monoacylglycerols (MGs) to yield FFAs and glycerol. MGL contributes to energy homeostasis through the mobilization of fat stores and also via the degradation of the endocannabinoid 2-arachidonoyl glycerol. To further examine the role of MG metabolism in energy homeostasis, MGL(-/-) mice were fed either a 10% (kilocalories) low-fat diet (LFD) or a 45% (kilocalories) high-fat diet (HFD) for 12 weeks. Profound increases of MG species in the MGL(-/-) mice compared with WT control mice were found. Weight gain over the 12 weeks was blunted in both diet groups. MGL(-/-) mice were leaner than WT mice at both baseline and after 12 weeks of LFD feeding. Circulating lipids were decreased in HFD-fed MGL(-/-) mice, as were the levels of several plasma peptides involved in glucose homeostasis and energy balance. Interestingly, MGL(-/-) mice had markedly reduced intestinal TG secretion following an oral fat challenge, suggesting delayed lipid absorption. Overall, the results indicate that global MGL deletion leads to systemic changes that produce a leaner phenotype and an improved serum metabolic profile.

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., Highlights • An efficient purification scheme has been developed for adipose fatty acid-binding protein (AFABP). • AFABP monomers have been separated from a minor dimer form. • The AFABP dimer is linked by a disulfide bond between N-terminal cysteine residues. • Exclusion of oxygen blocks formation of AFABP dimers.

Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22-C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in the liver but not in the adipose tissue or skeletal muscle of the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [VH]-palmitate was also abrogated in the hepa- tocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in the CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22-C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids.

Five polyrotaxanes were synthesized by threading 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) onto a variety of α,ω-ditriethylenediamino-N-carbamoyl-poly-(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronic) triblock copolymers using a two-pot strategy under heterogeneous, nonaqueous conditions. The threaded HP-β-CD units were retained on the pseudopolyrotaxane precursors by end-capping the branched diamine termini with sodium 2,4,6-trinitrobenzene sulfonate. Inclusion of the Pluronic copolymers within the HP-β-CD cavities was more favorable in nonpolar solvents, such as diethyl ether and n-hexane, both of which gave better coverage ratios than polar solvents. (1)H NMR and MALDI-TOF were used to estimate the average molecular weights of the purified polyrotaxane products. A globular morphology of aggregated polyrotaxanes was observed by tapping-mode AFM imaging of dried samples. Treatment of Niemann-Pick C (NPC) type 2-deficient fibroblasts with the polyrotaxane derivatives produced substantial reductions in sterol accumulation, as seen by diminished filipin staining in these cells, suggesting that Pluronic-based polyrotaxanes may be promising vehicles for delivery of HP-β-CD to cells with abnormal cholesterol accumulation.

In this chapter we describe the use of stopped flow fluorescence spectroscopy to analyze the kinetic mechanisms of protein mediated cholesterol transfer to, from, and between model membranes. These assays allow for the detection of protein-membrane interactions that may occur during cholesterol transfer by simply modifying donor or acceptor concentrations, membrane composition, or buffer properties, and analyzing resultant transfer rates.

The intracellular fatty acid-binding proteins (FABPs) are abundantly expressed in almost all tissues. They exhibit high affinity binding of a single long-chain fatty acid, with the exception of liver FABP, which binds two fatty acids or other hydrophobic molecules. FABPs have highly similar tertiary structures consisting of a 10-stranded antiparallel β-barrel and an N-terminal helix-turn-helix motif. Research emerging in the last decade has suggested that FABPs have tissue-specific functions that reflect tissue-specific aspects of lipid and fatty acid metabolism. Proposed roles for FABPs include assimilation of dietary lipids in the intestine, targeting of liver lipids to catabolic and anabolic pathways, regulation of lipid storage and lipid-mediated gene expression in adipose tissue and macrophages, fatty acid targeting to β-oxidation pathways in muscle, and maintenance of phospholipid membranes in neural tissues. The regulation of these diverse processes is accompanied by the expression of different and sometimes multiple FABPs in these tissues and may be driven by protein-protein and protein-membrane interactions.

The metabolic fates of radiolabeled sn-2-monoacylglycerol (MG) and oleate (FA) in rat and mouse intestine, added in vivo to the apical (AP) surface in bile salt micelles, or to the basolateral (BL) surface via albumin-bound solution, were examined. Mucosal lipid products were quantified, and the results demonstrate a dramatic difference in the esterification patterns for both MG and FA, depending upon their site of entry into the enterocyte. For both lipids, the ratio of triacylglycerol to phospholipid (TG:PL) formed was approximately 10-fold higher for delivery at the AP relative to the BL surface. Further, a 3-fold higher level of FA oxidation was found for BL compared with AP substrate delivery. Incorporation of FA into individual PL species was also significantly different, with >2-fold greater incorporation into phosphatidylethanolamine (PE) and a 3-fold decrease in the phosphatidylcholine:PE ratio for AP- compared with BL-added lipid. Overnight fasting increased the TG:PL incorporation ratio for both AP and BL lipid addition, suggesting that metabolic compartmentation is a physiologically regulated phenomenon. These results support the existence of separate pools of TG and glycerolipid intermediates in the intestinal epithelial cell, and underscore the importance of substrate trafficking in the regulation of enterocyte lipid metabolism.

Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the αI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the αI-helix of LFABP (α(I)LβIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from α(I)LβIFABP compared to IFABP. The results indicate that the αI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the αII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the αI helix of IFABP in its physical interaction with membranes. Fil: Franchini, Gisela Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Storch, J.. Rutgers University; Estados Unidos Fil: Córsico, Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina

Intestinal monoacylglycerol (MG) metabolism is well known to involve its anabolic reesterification to triacylglycerol (TG). We recently provided evidence for enterocyte MG hydrolysis and demonstrated expression of the monoacylglycerol lipase (MGL) gene in human intestinal Caco-2 cells and rodent small intestinal mucosa. Despite the large quantities of MG derived from dietary TG, the regulation of MG metabolism in the intestine has not been previously explored. In the present studies, we examined the mRNA expression, protein expression, and activities of the two known MG-metabolizing enzymes, MGL and MGAT2, in C57BL/6 mouse small intestine, as well as liver and adipose tissues, during development and under nutritional modifications. Results demonstrate that MG metabolism undergoes tissue-specific changes during development. Marked induction of small intestinal MGAT2 protein expression and activity were found during suckling. Moreover, while substantial levels of MGL protein and activity were detected in adult intestine, its regulation during ontogeny was complex, suggesting post-transcriptional regulation of expression. In addition, during the suckling period MG hydrolytic activity is likely to derive from carboxyl ester lipase rather than MGL. In contrast to intestinal MGL, liver MGL mRNA, protein and activity all increased 5-10-fold during development, suggesting that transcriptional regulation is the primary mechanism for hepatic MGL expression. Three weeks of high fat feeding (40% kcal) significantly induced MGL expression and activity in small intestine relative to low fat feeding (10% kcal), but little change was observed upon starvation, suggesting a role for MGL in dietary lipid assimilation following a high fat intake.

Rat liver fatty acid-binding protein (LFABP) is distinctive among intracellular lipid-binding proteins (iLBPs): more than one molecule of long-chain fatty acid and a variety of diverse ligands can be bound within its large cavity, and in vitro lipid transfer to model membranes follows a mechanism that is diffusion-controlled rather than mediated by protein-membrane collisions. Because the apoprotein has proven resistant to crystallization, nuclear magnetic resonance spectroscopy offers a unique route to functionally informative comparisons of molecular structure and dynamics for LFABP in free (apo) and liganded (holo) forms. We report herein the solution-state structures determined for apo-LFABP at pH 6.0 and for holoprotein liganded to two oleates at pH 7.0, as well as the structure of the complex including locations of the ligands. 1H, 13C, and 15N resonance assignments revealed very similar types and locations of secondary structural elements for apo- and holo-LFABP as judged from chemical shift indices. The solution-state tertiary structures of the proteins were derived with the CNS/ARIA computational protocol, using distance and angular restraints based on 1H-1H nuclear Overhauser effects (NOEs), hydrogen-bonding networks, 3J(HNHA) coupling constants, intermolecular NOEs, and residual dipolar (NH) couplings. The holo-LFABP solution-state conformation is in substantial agreement with a previously reported X-ray structure [Thompson, J., Winter, N., Terwey, D., Bratt, J., and Banaszak, L. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates, J. Biol. Chem. 272, 7140-7150], including the typical beta-barrel capped by a helix-turn-helix portal. In the solution state, the internally bound oleate has the expected U-shaped conformation and is tethered electrostatically, but the extended portal ligand can adopt a range of conformations based on the computationally refined structures, in contrast to the single conformation observed in the crystal structure. The apo-LFABP also has a well-defined beta-barrel structural motif typical of other members of the iLBP protein family, but the portal region that is thought to facilitate ligand entry and exit exhibits conformational variability and an unusual "open cap" orientation with respect to the barrel. These structural results allow us to propose a model in which ligand binding to LFABP occurs through conformational fluctuations that adjust the helix-turn-helix motif to open or close the top of the beta-barrel, and solvent accessibility to the protein cavity favors diffusion-controlled ligand transport.

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.

Objective Niemann–Pick type C (NPC) disease is a fatal, neurodegenerative, lysosomal storage disorder characterized by intracellular accumulation of unesterified cholesterol (UC) and other lipids. While its mechanism of action remains unresolved, administration of 2‐hydroxypropyl‐β‐cyclodextrin (HPβCD) has provided the greatest disease amelioration in animal models but is ototoxic. We evaluated other cyclodextrins (CDs) for treatment outcome and chemical interaction with disease‐relevant substrates that could pertain to mechanism. Methods NPC disease mice treated for 2 weeks with nine different CDs were evaluated for UC, and GM2 and GM3 ganglioside accumulation using immunohisto/cytochemical and biochemical assays. Auditory brainstem responses were determined in wild‐type mice administered CDs. CD complexation with UC, gangliosides, and other lipids was quantified. Results Four HPβCDs varying in degrees of substitution, including one currently in clinical trial, showed equivalent storage reduction, while other CDs showed significant differences in relative ototoxicity and efficacy, with reductions similar for the brain and liver. Importantly, HPγCD and two sulfobutylether‐CDs showed efficacy with reduced ototoxicity. Complexation studies showed: incomplete correlation between CD efficacy and UC solubilization; an inverse correlation for ganglioside complexation; substantial interaction with several relevant lipids; and association between undesirable increases of UC storage in Kupffer cells and UC solubilization. Interpretation CDs other than HPβCD identified here may provide disease amelioration without ototoxicity and merit long‐term treatment studies. While direct interactions of CD‐UC are thought central to the mechanism of correction, the data show that this does not strictly correlate with complexation ability and suggest interactions with other NPC disease‐relevant substrates should be considered.