Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.     

Decreased ANT content in Zucker fatty rats: relevance for altered hepatic mitochondrial bioenergetics in steatosis

Mitochondria from Zucker fatty (ZF) rats (a model for fatty liver disease) showed a delay in the repolarization after a phosphorylative cycle and a decrease on state 3 respiration, suggesting alterations at the phosphorylative system level. The ATPase activity showed no differences between control and ZF rats, implying alterations in other components of the phosphorylative system. A pronounced depletion in the content of the adenine nucleotide translocator (ANT) was observed by Western blotting, while no alterations were found in the mitochondrial voltage-dependent anion channel content. These data suggest that hepatic accumulation of fat impairs mitochondrial function, reflecting the loss of oxidative phosphorylation capacity caused by a decrease in the ANT content.     

A comparison of heart and liver fatty acid-binding proteins: interactions with fatty acids and possible functional differences studied with fluorescent fatty acid analogues

Summary Fatty acid-binding proteins (FABP) are distinct but related gene products which are found in many mammalian cell types. They are generally present in high abundance, and are found in those tissues where free fatty acid (ffa) flux is high. The function(s) of FABP is unknown. Also not known is whether all FABP function similarly in their respective cell types, or whether different FABP have unique functions. The purpose of these studies was to assess whether different members of the FABP family exhibit different structural and functional properties. Two fluorescent analogues of ffa were used to compare the liver (L-FABP) and heart (H-FABP) binding proteins. The propionic acid derivative of diphenylhexatriene (PADPH) was used to examine the physical properties of the ffa binding site on L- and H-FABP, as well as the relative distribution of ffa between FABP and membranes. An anthroyloxy-derivative of palmitic acid, 2AP, was used to monitor the transfer kinetics of ffa from liver or heart FABP to acceptor membranes, using a resonance energy transfer assay. The results demonstrate that the ffa binding sites of both FABP are hydrophobic in nature, although the L-FABP site is more nonpolar than the H-FABP site. Equilibration of PADPH between L-FABP and phosphatidylcholine (PC) bilayers resulted in a molar partition preference of > 20: 1, L-FABP : PC. Similar studies with H-FABP resulted in a PADPH partition preference of only 3:1, H-FABP : PC. Finally, the transfer of 2AP from H-FABP to acceptor membranes was found to be 50-fold faster than transfer from L-FABP. These studies demonstrate that important structural and functional differences exist between different members of the FABP family, and therefore imply that the roles of different FABP may be unique.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Liver FABP - H-FABP Heart FABP - SUV Small Unilamellar Vesicle - PADPH 3-[p-(6-Phenyl)-1,3,5-Hexatrienyl]-phenylpropionic acid - 2AP 2-(9-Anthroyloxy)Palmitic acid - Q Quantum yield - _F Fluorescence lifetime     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

Fatty acid metabolism in human breast cancer cells (MCF7) transfected with heart-type fatty acid binding protein

The human breast cancer cell line MCF7 does not express heart-type fatty acid binding protein (H-FABP), a marker protein for differentiated mammary gland. MCF7 cells transfected with the bovine H-FABP cDNA expressed the corresponding protein and were characterized by growth inhibition and lower tumorgenicity in nude mice [22]. By enzyme linked immunoassay we now determined the amount of bovine H-FABP in these cells as 638 ± 80 ng/mg protein and used the transfected cells to study the role of H-FABP in fatty acid metabolism. Compared to control cells the uptake of radioactively labelled palmitic acid and oleic acid into MCF7 cells after 30 or 60 min was increased by 67% in H-FABP expressing transfectants, demonstrating a stimulatory role for this FABP-type in fatty acid metabolism. However, preferential targeting of [¹⁴C]oleic acid into neutral or phospholipid classes was not observed by the criterion of high performance thin layer chromatography followed by autoradiography. A reason for the modest increase of fatty acid uptake in H-FABP transfected MCF7 cells may be the basal expression of epidermal-type FABP, which was detected for the first time in these cells. It appears that the small amount of E-FABP expressed in MCF7 cells fulfils the need of the cells for a cytosolic fatty acid carrier under culture conditions and that even high concentrations of another FABP do only slightly increase the uptake due to limitations of fatty acid transport through the plasma membrane or of metabolism.     

Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein.

The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539+/-8 A3 and 428 A2, respectively (1.4 A probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36-39% hydrophobic, 21-25% hydrophilic, and 37-43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity-with the exception of Asp74-are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.     

Localization of epidermal-type fatty acid binding protein in macrophages in advanced atretic follicles of adult mice

Summary The localization of epidermal-type fatty acid binding protein (E-FABP) in the mature mouse ovary was examined by immuno-light and electron microscopy. Numerous macrophages immunopositive for both anti-E-FABP and F4/80 antibodies, together with immunonegative cells, were found in advanced atretic follicles that had eccentric lumens containing deformed ova. While some E-FABP-immunopositive macrophages were spider in shape and appeared singly, others, especially close to the lumen, were round and voluminous and tended to be aggregated. The voluminous macrophages contained phagosomes of various sizes and they were regarded as those actively involved in the phagocytosis of apoptotic granulosa cells. E-FABP-immunopositive macrophages and their processes were often apposed to adjacent immunonegative cells, and some of them lined the lumen containing deformed ova. On the other hand, E-FABP-immunonegative cells in the atretic follicles were classified into two types: the one, a minority, was characterized by small mitochondria containing non-tubular cristae and presumably represented residual granulosa cells, while the other dominant type was characterized by large mitochondria containing tubular cristae and presumably represented theca cells originally surrounding the follicles to be atretic. The present detection of E-FABP-immunopositivity selectively in macrophages of the atretic follicles suggests possible involvement of E-FABP and/or its ligand fatty acids in the process of follicular atresia, and it makes more reliable the identification of the advanced atretic follicles with the antral spaces obliterated, which could provide further details on the histology of the follicular atresia than before.     

The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

Modeling fatty acid delivery from intestinal fatty acid binding protein to a membrane

Intestinal fatty acid binding protein (IFABP) interacts with biological membranes and delivers fatty acid (FA) into them via a collisional mechanism. However, the membrane-bound structure of the protein and the pathway of FA transfer are not precisely known. We used molecular dynamics (MD) simulations with an implicit membrane model to determine the optimal orientation of apo- and holo-IFABP (bound with palmitate) on an anionic membrane. In this orientation, the helical portal region, delimited by the alphaII helix and the betaC-betaD and betaE-betaF turns, is oriented toward the membrane whereas the putative beta-strand portal, delimited by the betaB-betaC, betaF-betaG, betaH-betaI turns and the N terminus, is exposed to solvent. Starting from the MD structure of holo-IFABP in the optimal orientation relative to the membrane, we examined the release of palmitate via both pathways. Although the domains can widen enough to allow the passage of palmitate, fatty acid release through the helical portal region incurs smaller conformational changes and a lower energetic cost.     

Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice with deletion of the liver fatty acid-binding protein gene

Liver fatty acid-binding protein (L-Fabp) is an abundant cytosolic lipid-binding protein with broad substrate specificity, expressed in mammalian enterocytes and hepatocytes. We have generated mice with a targeted deletion of the endogenous L-Fabp gene and have characterized their response to alterations in hepatic fatty acid flux following prolonged fasting. Chow-fed L-Fabp-/- mice were indistinguishable from wild-type littermates with regard to growth, serum and tissue lipid profiles, and fatty acid distribution within hepatic complex lipid species. In response to 48-h fasting, however, wild-type mice demonstrated a approximately 10-fold increase in hepatic triglyceride content while L-Fabp-/- mice demonstrated only a 2-fold increase. Hepatic VLDL secretion was decreased in L-Fabp-/- mice suggesting that the decreased accumulation of hepatic triglyceride was not the result of increased secretion. Fatty acid oxidation, as inferred from serum beta-hydroxybutyrate levels, was increased in response to fasting, although the increase in L-Fabp-/- mice was significantly reduced in comparison to wild-type controls, despite comparable induction of PPAR alpha target genes. Studies in primary hepatocytes revealed indistinguishable initial rates of oleate uptake, but longer intervals revealed reduced rates of uptake in fasted L-Fabp-/- mice. Oleate incorporation into cellular triglyceride and diacylglycerol was reduced in L-Fabp-/- mice although incorporation into phospholipid and cholesterol ester was no different than wild-type controls. These data point to an inducible defect in fatty acid utilization in fasted L-Fabp-/- mice that involves targeting of substrate for use in triglyceride metabolism.     

Interaction of rat liver microsomes containing saturated or unsaturated fatty acids with fatty acid binding protein: Peroxidation effect

In the studies described here rat liver microsomes containing labeled palmitic, stearic, oleic or linoleic acids were incubated with fatty acid binding protein (FABP) and the rate of removal of¹⁴C-labeled fatty acids from the membrane by the soluble protein was measured using a model system. More unsaturated than saturated fatty acids were removed from native liver microsomes incubated with similar amounts of FABP. Thein vitro peroxidation of microsomal membranes mediated by ascorbate-Fe⁺⁺, modified its fatty acid composition with a considerable decrease of the peroxidizability index. These changes in the microsomes facilitated the removal of oleic and linoeic acids by FABP, but the removal of palmitic and stearic acids was not modified. This effect is proposed to result from a perturbation of membrane structure following peroxidation with release of free fatty acids from susceptible domains.Abbreviations BSA bovine serum albumin - FABP fatty acid binding protein     

Hypermethylation of Fads2 and altered hepatic fatty acid and phospholipid metabolism in mice with hyperhomocysteinemia

Alterations in lipid metabolism may play a role in the vascular pathology associated with hyperhomocysteinemia (HHcy). Homocysteine is linked to lipid metabolism through the methionine cycle and the synthesis of phosphatidylcholine (PC) by phosphatidylethanolamine (PE) methyltransferase, which is responsible for the synthesis of 20-40% of liver PC. The goal of the present study was to determine if the reduced methylation capacity in HHcy is associated with alterations in liver phospholipid and fatty acid metabolism. Mice heterozygous for disruption of cystathionine beta-synthase (Cbs+/-) fed a diet to induce HHcy (HH diet) had higher (p<0.001) plasma total homocysteine (30.8+/-4.4 microM, mean+/-S.E.) than C57BL/6 mice (Cbs+/+) fed the HH diet (7.0+/-1.1 microM) or Cbs+/+ mice fed a control diet (2.3+/-0.3 microM). Mild and moderate HHcy was accompanied by lower adenosylmethionine/adenosylhomocysteine ratios (p<0.05), higher PE (p<0.05) and PE/PC ratios (p<0.01), lower PE methyltransferase activity (p<0.001), and higher linoleic acid (p<0.05) and lower arachidonic acid (p<0.05) in PE. Mice with moderate HHcy also had higher linoleic acid and alpha-linolenic acid (p<0.05) and lower arachidonic acid and docosahexaenoic acid (p<0.05) in liver PC. The first step in the desaturation and elongation of linoleic acid and linolenic acid to arachidonic acid and docosahexaenoic acid, respectively, is catalyzed by Delta6-desaturase (encoded by Fads2). We found hypermethylation of the Fads2 promoter (p<0.01), lower Fads2 mRNA (p<0.05), and lower Delta6-desaturase activity (p<0.001) in liver from mice with HHcy. These findings suggest that methylation silencing of liver Fads2 expression and changes in liver fatty acids may contribute to the pathology of HHcy.     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

Impact of Fabp1/Scp-2/Scp-x gene ablation (TKO) on hepatic phytol metabolism in mice

Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.     

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

Time-dependent changes in lipid metabolism in mice with methionine choline deficiency-induced fatty liver disease

Methionine and choline-deficient diet (MCD)-induced fatty liver is one of the best-studied animal models of fatty liver disease. The present study was performed to clarify the relative contributions of individual lipid metabolic pathways to the pathogenesis of MCD-induced fatty liver. Hepatic lipogenesis mediated by the sterol regulatory element-binding protein (SREBP-1c) was increased at 1 week, but not at 6 weeks, of MCD feeding. On the other hand, ¹⁴C-palmitate oxidation did not change at 1 week, but significantly decreased at 6 weeks. This decrease was associated with increased expression of fatty acid translocase, a key enzyme involved in fatty acid uptake. Expression of endoplasmic reticulum stress markers was increased in mice given MCD for both 1 and 6 weeks. These findings suggest the presence of time-dependent differences in lipid metabolism in MCD-induced fatty liver disease: SREBP-1c-mediated lipogenesis is important in the early stages of fatty liver disease, whereas increased fatty acid uptake and decreased fatty acid oxidation become more important in the later stages.     

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3α-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7α-hydroxylase, sterol 27α-hydroxylase, Na⁺/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.