Impact of Dietary Phytol on Lipid Metabolism in SCP2/SCPx/L-FABP Null Mice

Abstract

In vitro studies suggest that liver fatty acid binding protein (L-FABP) and sterol carrier protein-2/sterol carrier protein-x (SCP2/SCPx) gene products facilitate uptake and metabolism and detoxification of dietary-derived phytol in mammals. However, concomitant upregulation of L-FABP in SCP2/SCPx null mice complicates interpretation of their physiological phenotype. Therefore, the impact of ablating both the L-FABP gene and SCP2/SCPx gene (L-FABP/SCP2/SCPx null or TKO) was examined in phytol-fed female wild-type (WT) and TKO mice. TKO increased hepatic total lipid accumulation, primarily phospholipid, by mechanisms involving increased hepatic levels of proteins in the phospholipid synthetic pathway. Concomitantly, TKO reduced expression of proteins in targeting fatty acids towards the triacylglycerol synthetic pathway. Increased hepatic lipid accumulation was not associated with any concomitant upregulation of membrane fatty acid transport/translocase proteins involved in fatty acid uptake (FATP2, FATP4, FATP5 or GOT) or cytosolic proteins involved in fatty acid intracellular targeting (ACBP). In addition, TKO exacerbated dietary phytol-induced whole body weight loss, especially lean tissue mass. Since individually ablating SCPx or SCP2/SCPx elicited concomitant upregulation of L-FABP, these findings with TKO mice help to resolve the contributions of SCP2/SCPx gene ablation on dietary phytol-induced whole body and hepatic lipid phenotype independent of concomitant upregulation of L-FABP.

Introduction

Much research has been directed towards exploring the effects of cytosolic lipid binding proteins on the metabolism of fatty acids, especially potentially toxic branched-chain fatty acids derived from dietary phytol. Phytol, a saturated sixteen-carbon chain-length fatty alcohol with four methyl branches, is released by ruminant bacterial cleavage of chlorophyll's side-chain for further conversion to branched-chain fatty acids [1]. While branched-chain fatty acids are often present at significant levels in meat and dairy products, serum levels are normally low, due to rapid hepatic uptake and metabolism [2-4]. However, in peroxisomal disorders of branched-chain fatty acid oxidation, serum and hepatic branched-chain fatty acid levels reach high toxic levels [2,3]. Branched-chain fatty acids such as phytanic acid have functional similarity to fibrates, hypolipidemic drugs used to treat cardiovascular disease, diabetes and metabolic syndrome [5,6]. Since branched-chain fatty acids are highly insoluble in aqueous, cytosolic lipid binding proteins have been proposed to act as ‘chaperones’ that facilitate their uptake, cytosolic transport, and targeting for degradation in oxidative organelles [7-11]. ‘Chaperones’ of particular interest are the protein products of the liver fatty acid binding protein (L-FABP) gene and of the sterol carrier protein-2/sterol carrier protein-x (SCP2/SCPx) gene.

While L-FABP has no enzymatic activity, it is quantitatively the most prevalent lipidic ligand ‘chaperone’ in liver cytosol [12-18]. L-FABP has high affinity for branched-chain lipids such as phytol-derived phytanic and pristanic acids [19,20], cholesterol [21], bile acids [22,23], and lipidic xenobiotics [24-26,26-31]. In vitro studies with cultured cells show that L-FABP enhances the uptake and peroxisomal oxidation of branched-chain fatty acids [7,9,10]. L-FABP has even been detected within peroxisomes, suggesting that it may not only chaperone bound branched-chain fatty acyl-CoA to peroxisomes but also to oxidative enzymes within peroxisomes [32].

SCP2 and SCPx, both encoded by the same SCP2/SCPx gene through alternate transcription sites, are also important contributors to branched-chain fatty acid metabolism [33]. Like L-FABP, SCP2 has no enzymatic activity, but binds branched-chain lipids such as phytol-derived phytanic and pristanic acids [19] and cholesterol [34]. SCP2 enhances branched-chain fatty acid cellular uptake and metabolism [8]. Hepatic SCP-2 concentration is about 6-8 fold less than that of L-FABP [13,35], with half in cytosol and the remainder primarily concentrated in peroxisomes [33,34,36]. Within the peroxisomal matrix, SCP2 directly interacts with fatty acid oxidative enzymes, suggesting a role in presenting bound branched-chain fatty acyl CoAs to these enzymes to facilitate their oxidation [37]. In contrast to SCP2 and L-FABP, SCPx is localized exclusively in peroxisomes [33,38,39]. SCPx functions as a ketothiolase enzyme with substrate specificity for both straight-chain and branched-chain fatty acids [38-42]. SCPx is the only known peroxisomal ketothiolase enzyme for β-oxidation of branched-chain fatty acids [41].

While gene ablation studies suggest physiological roles for both the L-FABP and SCP2/SCPx genes in branched-chain fatty acid uptake and metabolism, findings are complicated by concomitant upregulation of the non-ablated gene or genes. For example, individually ablating L-FABP [10,11], SCP2/SCPx [43-45], or SCPx [46] each impairs hepatic branched-chain fatty acid uptake and/or metabolism. However, individually ablating SCP2/SCPx or SCPx concomitantly elicits marked upregulation of L-FABP [45-47]. Therefore, the current study examined the impact of ablating both the L-FABP and SCP2/SCPx genes on whole body and hepatic lipid phenotype of female mice fed a defined phytol diet.

Experimental Procedures

Materials

The Bradford protein micro-assay (Cat # 500-0001, bovine gamma globulin) was obtained from Bio-Rad (Hercules, CA). Diagnostic kits from Wako Chemicals (Richmond, VA) were used to measure triacylglycerol (L-type Triglyceride M, TG), free cholesterol (free cholesterol, C), total cholesterol (cholesterol E, TC), phospholipid (phospholipid, PL) and non-esterified fatty acid (HR Series NEFA-HR, NEFA). Diagnostic kits from Stanbio Laboratory (Boerne, TX) were used to determine β-hydroxybutyrate (β-Hydroxybutyrate LiquiColor, β-OHB), high density lipoprotein cholesterol (Direct HDL-Cholesterol, HDL-C), aspartate aminotransferase (Stanbio Alkaline Phosphatase LiquiColor, AST), and alanine aminotransferase (Stanbio Alanine Aminotransferase LiquiColor, ALT). Diagnostic kits from Diazyme Labs (Poway, CA) were used to measure apolipoprotein B (apoB), and apolipoprotein A-I (apoA1).

For hepatic mRNA quantitation, TaqMan® One-Step PCR Master Mix reagent kit and gene specific assays were purchased from Applied Biosystems (Foster City, CA) to determine hepatic expression of mouse glycerol-3-phosphate acyltransferase (GPAT) (Gpam; Mm00833328_m1), 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT) (Agpat2; Mm00458880_m1), Lipin (Lpin2; Mm00522390_m1), diacylglycerol acyltransferase (DGAT) (Dgat2; Mm00499536_m1), acetyl-coA carboxylase (ACC1) (Acaca; Mm01304285_m1), fatty acid synthase (FASN) (Fasn; Mm00662319_m1), 3-hydroxy-3-methylglutaryl-CoA (HMGCR) (Hmgcr; Mm01282492_m1), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS1) (Hmgcs1, Mm01282492_m1), sodium-taurocholate cotransporting polypeptide (NTCP) (Slc10; Mm01302718_m1), organic anion-transporting polypeptide 1 (OATP1) (Slco1; Mm01267414_m1), organic anion-transporting polypeptide 2, OATP2 (Slc22a7; Mm00460672_m1), ATP-binding cassette sub-family G member 5 (ABCG5) (Abcg5; Mm01226965_m1) and ATP-binding cassette sub-family G member 8 (ABCG8) (Abcg8; Mm00445977_m1).

For western blotting of hepatic proteins, the following antibodies were obtained: Rabbit and goat polyclonal antibody to mouse acyl-CoA-binding protein (ACBP) (SC-23474, apoA1 (SC-23606), brain fatty acid binding protein (B-FABP) (SC-30088), bile salt export pump (BSEP) (SC-17294), carnitine palmitoyltransferase 1a (Cpt1a) (SC-31128), carnitine palmitoyltransferase 2 (Cpt2) (SC-20671), cellular retinoic acid binding protein 1 (CRABP I) (SC-10062), fatty acid transport protein 4 (FATP4) (SC-5834), farnesoid x receptor (FxR), glyceraldehyde 3-phosphate dehydrogenase (GADPH) (SC-13063), intestinal fatty acid binding protein (I-FABP) (SC-16063), LDL-Receptor (LDL-R) (SC-11826), liver x receptor (LxR) (SC-1201), multidrug resistance protein (MDR) (SC-8313), retinoid x receptor α (RxRα) (SC-553), sterol regulatory element-binding protein 1 (SREBP1) (SC-367) and sterol regulatory element binding protein 2 (SREBP2) (SC-8151) were obtained from Santa Cruz Biotechnology (Dallas, TX). Rabbit and mouse polyclonal antibody to mouse acetyl-CoA acetyltransferase (ACAT2) (ab66259), apoB (ab31992), cytochrome c oxidase subunit IV (COX4) (ab16056), fatty acid transport protein 2 (FATP2) (ab83763) and fatty acid transport protein 5 (FATP5) (ab89008 were purchased from Abcam (Cambridge, MA). Sheep polyclonal antibody to bovine catalase (W90080C) was purchased from BioDesign (Dublin, OH). Rabbit polyclonal antibody to mouse glutamic oxaloacetic transaminase (GOT) (12248), LFABP (11294), p-thiolase (12464), SCP2 (12249), and SCPx (11306) were prepared as described [4,48]. Goat polyclonal antibody to mouse glutathione s-transferase (GST) (27457701V) was purchased from GE Life Sciences (Pittsburgh, PA). Bacterial polyclonal antibody to mouse 3alpha-hydroxysteroid reductase (3αHSD) (H9117-01) was purchased from US Biological (Peabody, MA). Rabbit polyclonal antibody to mouse PPARα (PA1-822A) was purchased from Pierce Antibody (Rockford, IL). Rabbit polyclonal antibody to mouse scavenger receptor class B member 1 (SR-B1) (NB400-104) was purchased from Novus Biological (Littleton, CO). Alkaline phosphatase-conjugated goat polyclonal antibody to rabbit IgG (product # A3687) as well as rabbit polyclonal antibody to goat IgG (product # A4187) was purchased from Sigma-Aldrich (St. Louis, MO). Alkaline phosphatase-conjugated rabbit polyclonal antibody to mouse IgG (product # ab6729-I) was obtained from Abcam (Cambridge, MA). Mouse monoclonal antibody to mouse GAPDH (MAB374) was purchased from Millipore (Billerica, MA). All reagents and solvents used were of the highest grade available.

Animals

Wild-type (WT) C57BL/6NCr mice were obtained from the National Cancer Institute (Frederick Cancer Research and Development Center, Frederick, MD). L-FABP/SCP-2/SCP-x null (TKO) mice were generated by our laboratory as previously described [49]. The term TKO is used since all three proteins encoded by the two genes (i.e. L-FABP as well as SCP-2 and SCP-x) were ablated. TKO mice were backcrossed >10 generations to the C57BL/6NCr background. Animals were housed in controlled conditions (T = 25 °C, H= 60-70% added humidity) under a 12:12 h light/dark cycle. All animal use protocols were approved by the Texas A&M Institutional Animal Care and Use Committee in compliance with the Guide for the Care and Use of Laboratory Animals. Animals were monitored for disease or injury daily as well as sentinel monitored quarterly, and were confirmed free of all known rodent pathogens.

Dietary phytol

One week before the study, all mice were moved from a standard pelleted rodent chow to a modified AIN-76A phytol-free, phytoestrogen-free pelleted control diet (5% calories from fat, diet no. D11243, Research Diets, New Brunswick, NJ). The control diet was used to minimize potential complications due to phytoestrogens [50,51] or phytol metabolites (e.g. phytanic acid), known to be the most potent naturally-occurring fatty acid ligand inducers of PPARα [20,52,53]. After one week, test mice were transferred to a modified AIN-76A pelleted rodent diet supplemented with 0.5% phytol (5% calories from fat, diet no. D01020601, Research Diets, New Brunswick, NJ), which is approximately ten times the total amount of free phytol and phytanic acid on average available in commercial laboratory rodent diets [6]. Two feeding groups contained 8 animals fed ad libitum: (a) WT mice fed the 0.5% phytol diet, and (b) TKO mice fed the 0.5% phytol diet. The study was conducted for seven days because TKO mice exhibited a low tolerance for the 0.5% phytol diet, which manifested as a 20% percent loss in body weight. Body weight and food intake were measured every other day.

Whole body phenotype analysis and animal euthanasia

PIXImus images were taken of representative mice in the beginning (day 0) as well as all mice at the end of the study (day 8) as previously described [54]. Animals were anesthetized with a ketamine/xylazine mixture (0.01 mL/g body weight; 10 mg ketamine/mL and 1 mg xylazine/mL in 0.9% saline solution). Dual-energy X-ray absorptiometry (DEXA) using a Lunar PIXImus densitometer (Lunar Corp., Madison, WI) was performed as previously described [4] after calibration using a phantom mouse with known bone mineral density and fat tissue mass as described [4,55]. To obtain an in vivo measurement of whole body fat tissue mass (FTM) and bone-free lean tissue mass (LTM), the entire mouse, minus the head region, was exposed to sequential beams of high- and low-energy X-rays and an image was taken of the X-rays impacting a luminescent panel. Bone mass was differentiated from soft tissue mass by measuring the ratios of attenuation at different energies. The soft tissue mass was then separated into FTM and LTM as previously described [4]. At the end of the study, blood was collected via cardiac puncture followed by cervical dislocation as the secondary form of euthanasia according to the AVMA Guidelines for the Euthanasia of Animals. Blood was processed to serum and stored at −80 °C for subsequent lipid and protein analysis, and final DEXA images were taken.

Liver collection and analysis

After euthanasia, livers were harvested, weighed, snap frozen with dry ice, and stored at −80°C for lipid analysis, western blotting and reverse transcriptase-PCR analysis. Liver samples (∼0.1 gram) were extensively minced, 0.5 mL PBS (pH 7.4) added, and homogenized using a motor-driven pestle (Tekmar Co, Cincinnati, OH) operating at 2000 rpm. Liver homogenate protein concentration was evaluated using the Bradford protein micro-assay (Bio-rad, Hercules, CA) in accordance with the manufacturer's instructions using Costar 96-well assay plates (Corning, Corning, NY) and the BioTek Synergy 2 micro-plate reader (BioTek Instruments, Winooski, VT). Liver homogenate lipids were measured using two different techniques. First, liver homogenate (5mg protein), as well as appropriate lipid standards (C, CE, TG, NEFA, and PL) were solvent-extracted and analyzed by thin layer chromatography (TLC) as described [17]. Second, liver homogenate lipids were quantified using Wako diagnostic kits in accordance with the manufacturer's instructions. To support the use of 96-well plates and the BioTek Synergy 2 micro-plate reader in measuring the liver lipids, sample/reagent volumes were adjusted accordingly. There was no significant difference in quatitative lipid measurements when comparing the two lipid analysis procedures described above. To determine cholesteryl ester concentration (CE), free (non-esterified) cholesterol concentration was subtracted from the total concentration. No significant differences were recognized between the solvent/extraction technique/TLC procedure and the diagnostic kit procedure.

Liver Histopathology and Serum Markers of Liver Toxicity

At the time of liver collection, liver slices were taken near the porta hepatis, fixed for 24 h in 10% neutral buffered formalin, put into individual cassettes with 70% alcohol, processed and embedded in paraffin, sectioned (4-6 microns), and stained with hematoxylin and eosin for histological evaluation [4,6]. Serum was obtained after overnight blood coagulation at 4°C, followed by centrifugation at 14,000 rpm for 20 min at 4°C and removal of the serum fraction. Stanbio diagnostic kits (Boerne, TX) were used to determine serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and β-beta-hydroxybutyrate (β-OHB).

Serum lipid and apolipoprotein analysis

Serum lipids [free cholesterol (FC), total cholesterol (TC), HDL-cholesterol (HDL-C)], apoA1 and apoB levels were determined using commercially available diagnostic kits in accordance with the manufacturer's instructions (see above). The assays were modified to support the use of 96-well plates and micro-plate reader as described above. Serum cholesteryl ester (CE) concentrations were calculated by subtraction of serum FC from serum TC. Serum non-HDL-C was calculated by subtracting serum HDL-C from serum TC.

Real-time qRT-PCR

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed on total mRNA isolated from livers and purified using an RNeasy minikit (Quiagen, Valencia, CA) in accordance with the manufacturer's protocol. mRNA concentrations and quality were measured using the ND-1000 method (Nanodrop Technologies, Inc., Wilmington, DE) where a 260/280 ratio of 1.9-2.1 was accepted as mRNA of good quality. For qRT-PCR, expression patterns were assessed using TaqMan One Step PCR Master Mix Reagent Kit, gene-specific TaqMan PCR probes, and primers. mRNA expression was quantitated with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) and using the following thermal cycling conditions: 48 degrees C for 30 minutes, 95 degrees C for 10 minutes before the first cycle, 95 degrees C for 15 seconds and 60 degrees C for 1 minute, repeated 40 times. Before amplification, total mRNA was reverse transcribed in the first step of the thermal cycler protocol (48 degrees C for 10 minutes) using TaqMan one step chemistry. For specific probes and primers, Assay-on-Demand products for mouse glycerol-3-phosphate acyltransferase-1 (Gpam; Mm00833328_m1), acylglycerolphosphate acyltransferase-2 (Agpat2; Mm00458880_m1), Lipin-2 (Lpin2; Mm00522390_m1), diacylglycerol acyltransferase-2 (Dgat; Mm00499536_m1), ATP-binding cassette subfamily G member 5 (Abcg5; Mm01226965), ATP-binding cassette subfamily G member 8 (Abcg8; Mm00445977_m1), acetyl CoA carboxylase-1 (Acc1; Mm01304285_m1), acetyl CoA carboxylase-2, cholesteryl ester hydrolase/hormone-sensitive lipase (Lipe/Ceh/Hsl; Mm00495359_m1), fatty acid synthase (Fasn; Mm00662319_m1), HMC-CoA synthase (Hmgcs1, Mm01304569_ml), HMG-CoA reductase (Hmgcr, Mm01282492_ml), Na+-taurocholate cotransporting polypeptide (Ntcp/Slc10a1; Mm01302718) and organic anion transporting polypeptide 1 (Oatp1a1/Slco1a1; Mm01267414_m1), organic anion transporting polypeptide 2 (Oatp2/Slco1c1; Mm00460672_m1) were obtained from Applied Biosystems. Experiments were analyzed with ABI Prism 7000 SDS software (Applied Biosystems) to resolve the threshold cycle (C_T) from each well. Cycle number and primer concentrations were optimized to ensure that the reactions were analyzed in the linear phase of amplification. To analyze qRT-PCR data, mRNA expression was normalized to 18S RNA (a housekeeping gene) as described [56] and made relative to the control mouse group (male WT mice on control diet) set to one for final calculations.

Western Blotting

Western blot analysis was performed as in [57] to determine protein levels of hepatic proteins involved in cholesterol synthesis and metabolism (ACAT2, SREBP1, SREBP2), cholesterol uptake (SR-B1, LDL-R), biliary bile acid transport (OATP1, OATP2, NTCP, BSEP, MRP2), detoxification of peroxidized lipids (GST), cholesterol oxidation to bile acid (3αHSD, LXR1), fatty acid transport (FATP5, FATP2, FATP4, GOT, LFABP, IFABP, BFABP, ACBP, SCP2, CRABP I), fatty acid synthesis and oxidation (CPT1, CPT2, catalase, p-thiolase, 58 SCPX, 43 SCPX), and cholesterol transport to bile (PPARα, RXRα). Because each protein of interest and the housekeeping proteins COX4 and GADPH were easily determined by size on the tricene gels, membrane blots were cut into two. This was so that each western blot was probed with antisera against the protein of choice and against COX4 or GADPH to ensure uniform protein loading. Protein bands were visualized with alkaline-phosphatase conjugated goat anti-rabbit or mouse IgG and Sigma Fast 5-bromo-4-chloro-3-indolyn phosphate/nitroblue tetrazolium tablets (Sigma Chemical Co, St. Louis, MO). To quantitate protein levels in the blots images were obtained using single-chip CCD video camera and a computer workstation (IS-500 system, Alpha Innotech, San Leandro, CA). Image proteins (mean 8-bit grayscale density) were quantitated by densitometric analysis using NIH Image (available by anonymous FTP). Expression of each protein was normalized to the mean expression of COX4 or GAPDH. Linear standard curves were produced from Western blots where pure protein (L-FABP, SCP2, ACBP) was available for quantitative analysis. Band intensities on western blots were analyzed as described above and then plotted against the protein amount to produce a standard curve within the linear range of each protein. Changes in protein expression between liver homogenate samples were measured by comparing the sample with the standard curve on each blot. Proteins with no source of pure protein were expressed as relative fold differences between samples as described [46].

Statistics

Each feeding group contained 8 animals. All values were expressed as averages ± standard error of the mean (SEM) with n and p values indicated in figure legends. Statistical analysis was performed using t-test (GraphPad Prism, San Diego, CA, San Jose, CA). Values with p < 0.05 were considered statistically significant.

Results

Body Weight, Liver Weight, Liver Histopathology and Serum AST and ALT

Dietary phytol significantly decreased weight gain in WT mice, an effect exacerbated by TKO (Fig 1A). The phytol-induced weight loss was attributable to both reduced food consumption and reduced energy intake. Phytol decreased food consumption by 16±3% in WT and nearly twice as much by 29±2% in TKO mice (not shown). This suggested that TKO exacerbated the effect of dietary phytol on weight loss by decreasing food intake.

Figure 1. Body Weight, Liver Weight and Serum AST and ALT.

Percent change in body weight (A), ratio of body weight per gram of liver weight (B), liver weight (C) and serum levels of ALT (D) and AST (E) were determined for wild-type (WT) and L-FABP/SCP2/SCPx gene ablated (TKO) female mice fed a 0.5% phytol diet as described in Experimental Procedures. Means +/- SE; n= 8 animals/group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

Dietary phytol was associated with mild hepatocellular necrosis in both WT and TKO (not shown) with no difference between the phytol fed mouse groups in liver weight/body weight ratio or liver weight (Fig 1B,C). These observations were supported by measurement of serum values of ALT and AST, other indicators of hepatocyte damage. Although dietary phytol increase ALT and AST values similarly in both WT and TKO mice, all values were within the normal range of mouse serum ALT and AST values (less than 150 and 330 units/l, respectively) shown for blood chemistry and hematology in 8 inbred strains of mice, MPD: Eumorphia. Mouse Phenome Database web site, The Jackson Laboratory, Bar Harbor, Maine USA. http://phenome.jax.org [Cited 29 Oct, 2014] (Fig 1D,E). Taken together, these measures indicated that the major changes in whole body and liver lipid phenotype elicited by TKO in phytol fed mice detailed in the following sections were not due to marked hepatotoxicity.

Body Composition: Fat Tissue Mass and Lean Tissue Mass

PIXImus dual-energy x-ray absorptiometry (DEXA) scans were done to determine if any decline in weight gain, was due to selective loss of fat or protein. Representative PIXImus images of mice from the beginning (Fig 2A,B) and end (Fig. 2C,D) of the dietary study suggested gross differences in body composition. Phytol-fed WT mice appeared visually smaller at the end of the study (Fig 2A,C). This difference appeared even more marked in phytol-fed TKO mice (Fig 2B,C). DEXA analysis of multiple mice to resolve FTM (Fig 2E) and LTM (Fig 2F) showed that dietary phytol increased FTM while concomitantly decreasing LTM (Fig 2E,F). In contrast, TKO decreased both FTM and LTM (Fig. 2E,F).

Figure 2. Body Composition of Fat Tissue Mass and Lean Tissue Mass.

Whole body phenotype of WT and TKO mice fed a 0.5% phytol diet was determined by Lunar PIXImus dual-energy x-ray absorptiometry (DEXA). DEXA resolved whole body fat tissue mass (FTM) and lean tissue mass (LTM). A and B are representative images of control diet fed WT and TKO mice at the end of the study, respectively. C and D are representative images of phytol diet fed WT and TKO mice at the end of the study, respectively. The change in FTM (E) and LTM (F) was determined for 8 mice per group. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

Hepatic Lipid and Glyceride Accumulation

Total hepatic lipid was markedly higher in phytol-fed TKO mice than phytol-fed WT mice, both when expressed on the basis of nmol/mg liver protein (Fig 3A) or when expressed on the basis of nmol/mg liver wet weight (Supplementary Fig. 3A). This change was attributed primarily to a decrease in neutral lipid, expressed both on the basis of nmol/mg liver protein (Fig 3B) or when expressed on the basis of nmol/mg liver wet weight (Supplementary Fig. 3B). It was also due in part to the nearly 3-fold increase in phospholipid expressed both on the basis of nmol/mg liver protein (Fig 3C) or when expressed on the basis of nmol/mg liver wet weight (Supplementary Fig. 3C). In contrast, triacylglycerol actually decreased about 25% when expressed both on the basis of nmol/mg liver protein (Fig 3D) or when expressed on the basis of nmol/mg liver wet weight (Supplementary Fig. 3D). Hepatic phospholipid accumulation was associated with a more than two-fold increase in expression of the key enzymes in the synthesis of phosphatidic acid (precursor of both phospholipids and triacylglycerols), i.e. Gpam (Fig 3E) and Agpat (Fig 3F) and despite no change in SREBP1 (Fig 4A), increased expression of Acc1, the rate limiting enzyme in de novo fatty acid synthesis (Fig 4B). Though Dgat is a key enzyme in the synthesis of triacylglycerol and was significantly higher in TKO mice than WT mice (Fig 3H), this increase was not reflected in hepatic triacylglycerol levels. On the contrary, triacylglycerol levels were significantly less in TKO mice. This decrease correlated with decreased expression of Fasn, a key enzyme in fatty acid synthesis (Fig 4C), and increased expression of Lipe, a key enzyme in triacyglycerol hydrolysis (4G). However it is equally plausible that phytol toxicity caused decreases in triacyglycerol and Fasn expression independently. Taken together, these findings suggested that the TKO induced hepatic lipid accumulation by favoring fatty acyl targeting towards phospholipids at the expense of triacylglycerol in phytol-fed TKO phytol-fed mice.

Figure 3. Hepatic Lipid Levels and Expression of Key Proteins in Glyceride Synthesis.

WT and TKO mice were fed a 0.5% phytol diet as described in Experimental Procedures. Levels of total hepatic lipid (A), hepatic neutral lipid (B), hepatic phospholipid (C) and hepatic triacylglycerol (D) were measured and qRT-PCR was performed to measure the expression of Gpam (E), Agpat2 (F), Lipin2 (G) and Dgat2 (H) as in Experimental Procedures. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

Figure 4. Hepatic Expression of SREBPs and Target Genes in de novo Fatty Acid and Cholesterol Synthesis.

Livers from WT and TKO mice fed a 0.5% diet phytol diet were examined by western blotting as in Experimental Procedures to measure relative hepatic protein levels of SREBP1 (A) and SREBP2 (D). The housekeeping gene GADPH or COX4 was used as a loading control to normalize protein expression for SREBP1 and SREBP2, respectively. Inset in Panels A and D show representative western blots of relative protein expression in each mouse group. qRT-PCR was performed to determine relative transcription of SREBP1 target genes Acc1 (C) and Fasn (D), transcription of SREBP2 target genes Hmgcs1 (E) and Hmgcr (F) and Lipe (G) also as in Experimental Procedures. 18S rRNA was used to normalize mRNA expression levels. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

Hepatic Fatty Acid Oxidation

Hepatic glyceride accumulation in phytol-fed TKO mice may arise not only from increased de novo lipogenesis, but also from increased uptake and/or decreased oxidation of fatty acids.

While loss of L-FABP and SCP-2, both facilitators of fatty acid uptake, was expected to increase serum non-esterified fatty acid (NEFA) level, on the contrary TKO did not significantly changes serum NEFA (Fig 5A). This lack of increase in serum NEFA was attributable in part to concomitant downregulation of hepatic membrane fatty acid transporter FATP5 (Fig 5B) (FATP2 and FATP4, Fig 5C,D, remained unchanged) and downregulation of GOT (Fig 5E).

Figure 5. Fatty Acid Uptake and Expression of Membrane Fatty Acid Transport Proteins.

WT and TKO mice were fed a 0.5% phytol diet as described in Experimental Procedures. Serum NEFA was measured (A) and western blotting was performed to measure the expression of FATP5 (B), FATP2 (C), FATP4 (D), and GOT (E) as in Experimental Procedures. The housekeeping gene COX4 was used as a loading control to normalize FATP5, FATP2 and GOT expression. GADPH was used as a loading control to normalize FATP4 expression. Insets in Panels B-E show representative western blots of relative protein expression in each mouse group. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

TKO did not decrease serum (Fig 6A) or hepatic (Fig 6B) levels of β-hydroxybutyrate (β-OHB), a measure of hepatic fatty acid oxidation. This was reflected in no change in the levels of hepatic expression of key enzymes in mitochondrial (CPT1A, CPT2) fatty acid β-oxidation (Fig. 6C,D) or peroxisomal fatty acid β-oxidation (Fig 6 E,F). As expected, the other major enzymes in peroxisomal fatty acid β-oxidation, i.e. SCPx (58kDa) and SCPx (43kDa), were not detectable in these TKO mice (Fig 6G,H). In addition, there was no significant change in the expression of nuclear receptors regulating transcription of fatty acid β-oxidative enzymes (Fig 6I,J).

Figure 6. Fatty Acid Oxidation.

WT and TKO mice were fed a 0.5% phytol diet as described in Experimental Procedures. Serum (A) and liver (B) concentrations of β-hydroxybutyrate (β-OHB) were determined and western blotting was performed to measure the hepatic expression of CPT1A (C), CPT2 (D), catalase (E), p-thiolase (F), SCPx (G and H), PPARα (I) and RxRα (J) as described in Experimental Procedures. The housekeeping gene COX4 was used as a loading control to normalize CPT1A, CPT2 and p-thiolase expression. GADPH was used as a loading control to normalize catalase, SCPx, PPARα and RxRα expression. Inset in Panels C-J show representative western blots of relative protein expression in each mouse group. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

Thus, the TKO-induced increase in hepatic glycerides was not associated with either increased expression of membrane transport proteins involved in fatty acid uptake or a decrease in fatty acid β-oxidation. Instead, the TKO-induced increase in hepatic glyceride was associated with increased fatty acid synthesis, represented by an increase in key enzymes, particularly an increase in Acc1, the rate limiting enzyme in fatty acid synthesis (Fig 4B).

Hepatic Cholesterol Accumulation

Despite there being no change in SREBP2, which plays a major role in cholesterol homeostasis (Fig 4D), the finding that TKO increased total neutral lipid, despite decreasing triacylglycerol, suggested upregulation of the other major neutral lipid species, cholesterol and/or cholesteryl ester perhaps as a result of the upregulated expression of target genes in cholesterol synthesis (Hmgcs1, Hmgcr) (Fig 4E,F). However, there was no effect on hepatic total cholesterol, free cholesterol or cholesteryl ester levels (Fig 7A-C) nor was there a change in the expression of the cholesteryl ester synthetic enzyme (acyl CoA cholesterol acyltransferase, ACAT 2) (Fig 7D). In contrast, there was marked upregulation of hepatic expression of the Lipe gene, which codes for the degradative enzyme cholesteryl ester hydrolase (CEH) (Fig 4G). Thus, upregulation of Lipe did not actively decrease hepatic cholesterol levels in phytol-fed TKO.

Figure 7. Expression of Proteins in the Cholesteryl Ester Synthesis/Hydrolysis Cycle.

WT and TKO mice were fed a 0.5% phytol diet as described in Experimental Procedures. Levels of total hepatic cholesterol (A), hepatic free cholesterol (B), and hepatic cholesteryl ester (C) and were assayed; western blots were performed to measure the expression of ACAT2 as described in Experimental Procedures. The housekeeping gene COX4 was used as a loading control to normalize protein expression. Inset in Panel D shows representative western blotting of relative protein expression in each mouse group. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

Serum Levels of Cholesterol and Lipoproteins in Serum Cholesterol Transport

In phytol-fed mice, TKO significantly increased both serum cholesterol and cholesterol ester, regardless whether data were expressed on the basis of nmol lipid/ml serum (Fig 8A,B) or nmol lipid/mg serum protein (Supplementary Fig. 2A,B). With no changes in HDL-C, lipoprotein rich in apoA1 or apoA1/HDL ratio (Fig 8 C,D,E), this increase is attributed to increased lipoprotein rich in apoB as well as increased non-HDL-C (Fig. 8F, G) in the serum. However, these increases in serum cholesterol did not correlate with any change in hepatic accumulation of cholesterol (Fig. 7A-C). These findings suggest that TKO, in combination with phytol, has a “protective” effect against hepatic cholesterol accumulation.

Figure 8. Serum Lipid and Lipoprotein Levels.

WT and TKO mice were fed a 0.5% phytol diet as described in Experimental Procedures. Serum levels of free cholesterol (A), cholesteryl ester (B), HDL cholesterol (C), apoA1 (D), non-HDL-C (F) and apoB (G) were measured and the apoA1/HDL-C (E) and apoB/non-HDL-C (H) ratios were calculated as described in Experimental Procedures. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

Hepatic Expression of Proteins Involved in Cholesterol Basolateral Uptake/Efflux and Canalicular Secretion

TKO and dietary phytol differentially impacted hepatic expression of proteins involved in hepatic cholesterol uptake and loss.

TKO did not alter hepatic levels of scavenger receptor B1 (SR-B1) or low density lipoprotein receptor (LDL-R), the key receptors in bidirectional uptake/efflux of high density lipoprotein (HDL) cholesterol and low density lipoprotein (LDL) cholesterol (Fig 9C, D), or apoA1, involved in hepatic cholesterol secretion to nascent-HDL (Fig 9A) at the basolateral membrane. In contrast, TKO markedly decreased hepatic accumulation of apoB, the key apoprotein in LDL (Fig 9B). Concomitantly, TKO did not affect expression of ATP binding cassette proteins localized in the canalicular membrane for transporting hepatic cholesterol into bile, i.e. Abcg5, Abcg8 (Fig 9E,F). Low level of apoB in the liver was reflected by high levels of apoB in the serum in TKO mice.

Figure 9. Expression of Hepatic Proteins Involved in Cholesterol Uptake, Efflux and Biliary Secretion.

WT and TKO mice were fed a 0.5% phytol diet as described in Experimental Procedures. Levels of hepatic apoA1 (A), apoB (B), SR-B1 (C) and LDL-R (D were determined by western blotting as in Experimental Procedures. The housekeeping gene COX4 was used as a loading control to normalize protein expression. Inset in Panels A-D show representative western blots of relative protein expression in each mouse group. qRT-PCR was performed to determine relative transcription of Abcg5 (E) and Abcg8 (F) as in Experimental Procedures. 18S rRNA was used to normalize mRNA expression levels. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO.

Bile Acid and Transport

Since bile acid level in large part drives biliary cholesterol secretion [58-60], hepatic expression of proteins involved in bile acid reuptake from serum (Oatp1, Oatp2, Ntcp), bile acid cytosolic transport (L-FABP, SCP2, 3αHSD, GST), and biliary secretion of bile acid (BSEP) and phospholipid (MDR) at the canalicular membrane was examined.

With regards to basolateral proteins in bile acid uptake from serum, TKO significantly decreased hepatic expression of Oatp1 (Fig 10A), but increased that of Oatp2 (Fig 10B) and Ntcp (Fig 10A,C). TKO decreased expression of the nuclear regulatory protein FxR, but did not affect the nuclear regulatory protein LxR (Fig 10H,I). These proteins are responsible for regulating transcription of target genes in bile acid metabolism and uptake.

Figure 10. Hepatic Expression of Proteins for Bile Acid Transport.

WT and TKO mice were fed a 0.5% phytol diet as described in Experimental Procedures. qRT-PCR was performed to measure the expression of Oatp1 (A), Oatp2 (B) and Ntcp (C). 18S rRNA was used to normalize mRNA expression levels. Western blotting was performed to measure the expression of 3αHSD (D), GST (E), LxR (F), MDR (G), BSEP (H), and FxR (I). The housekeeping gene COX4 was used as a loading control to normalize protein expression. Inset in Panels D-I show representative western blots of relative protein expression in each mouse group. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

Regarding cytosolic bile acid binding/transport proteins, TKO abolished expression of the major murine cytosolic bile acid binding/transport protein L-FABP (Fig 11A), and this null effect was not compensated for by upregulation of the other known cytosolic bile acid binding/transport proteins (3αHSD, GST, Fig 10D-E).

Figure 11. Lipid Binding and Transport Proteins.

WT and TKO mice were fed a 0.5% phytol diet as described in Experimental Procedures. Quantitative western blotting was performed to measure the expression of L-FABP (A), SCP2 (B) and ACBP (C) as described in Experimental Procedures. Additional western blotting was performed to measure the relative expression of I-FABP (D), B-FABP (E), A-FABP (F) and CRABP-1 (G). The housekeeping gene GADPH was used as a loading control to normalize protein expression. Inset in Panels D-G show representative western blots of relative protein expression in each mouse group. Means +/- SE; n= 8 animals per group; ^#p < 0.05 between phytol-fed WT versus phytol-fed TKO mice.

With regards to canalicular membrane proteins involved in hepatic bile acid export into bile, TKO did not affect expression of the bile salt export protein BSEP (Fig 10H) or the canalicular membrane protein for phospholipid transport into bile (MDR, Fig 10G).

Other Fatty Acid Binding and Transport Proteins

The possibility of potential compensatory upregulation of other fatty acid and cholesterol binding proteins occurred in response to loss of L-FABP and SCP2/SCPx (TKO) was examined. As expected, TKO alone resulted in complete loss of L-FABP (Fig 11A) as well as all SCP2/SCPx gene products, SCP2 (Fig 11B), SCPx (58kDa) (Fig 6G), and SCPx (43kDa) (Fig 6H). These ablations resulted in compensatory upregulation of other known FABP family members including I-FABP (Fig 11D) and A-FABP (Fig 11F) with no compensatory upregulation of B-FABP (Fig 11E) or CRABP1 (Fig 11G).

Discussion

Since accumulation of branched-chain fatty acids derived from dietary phytol is toxic [2,3], most research has focused on identification of the individual peroxisomal enzymes in their metabolism and the pathological consequences of genetic mutations therein [61-65]. In contrast, much less is known about how these poorly soluble branched-chain lipids traffic through the hepatocyte cytosol for metabolism. In vitro ligand binding [7,9,10,19] and transfected cell [7-10] studies suggest L-FABP and SCP2/SCPx gene products act as their potential ‘chaperones’. Further, a highly prevalent human L-FABP SNP (26-38% allele frequency) [66-72] resulting in L-FABP T94A substitution alters uptake/metabolism of another branched-chain lipid, cholesterol [29,67,71,73]. Human subjects expressing the L-FABP T94A variant exhibit marked triglyceride accumulation in liver [29,71], elevated triglyceride and LDL cholesterol in serum [67,74,75], and increased incidence of cardiovascular disease [67,69,74]. Although less prevalent, a mutation in the human SCP-x gene completely abolishes SCP-x activity, markedly elevates serum levels of branched chain fatty acids (phytanic acid, pristanic acid), and induces neurodegeneration [76,77]. While gene-targeted mice ablated in L-FABP [10,11,78-84], SCPx [46], or SCP2/SCPx [5,44,45,47,85] have proven useful in resolving respective physiological roles, concomitant upregulation of the non-ablated chaperone, especially L-FABP in SCP2/SCPx null or SCPx null mice, complicates interpretation of phenotype [45-47]. The current study with mice ablated in both SCP2/SCPx and L-FABP genes (SCP2/SCPx/L-FABP triple gene ablation or TKO) provided several important insights:

First, ablating both L-FABP and SCP2/SCPx significantly affected whole body phenotype in mice fed a high phytol diet as compared to ablating each individually. In earlier findings, feeding dietary phytol alone decreased body weight of female wild-type (WT) mice [6], an effect not further exacerbated upon ablating L-FABP [11]. In contrast, singly ablating SCPx or SCP2-SCPx markedly exacerbated phytol diet-induced weight loss several-fold [45,46]. Likewise, ablating L-FABP in SCP2/SCP-x null mice (i.e. TKO) nearly doubled the dietary phytol-induced weight loss, an effect attributed to decreased food consumption.. These data indicated that concomitant upregulation of L-FABP in SCP2/SCPx null or SCPx null mice [45-47,57] did not diminished and/or obscure the impact of SCP2/SCPx ablation on dietary phytol-induced weight loss. This suggested that SCP2/SCPx may play a greater role than L-FABP in exacerbating phytol-induced weight loss.

Second, the TKO data presented herein suggested that SCP2/SCPx gene products, much more than the L-FABP, contributed to phytol oxidation. In response to dietary phytol, peroxisomal oxidation products of phytanic acid and pristanic acid accumulate concomitant with upregulation of L-FABP, SCP2, SCP-x and peroxisomal oxidative enzymes in WT mice [6]. L-FABP gene ablation did not further exacerbate this dietary phytol-induced increase likely due to concomitant upregulation of SCP-x, a key peroxisomal enzyme in phytol oxidation [11]. In contrast, singly ablating SCPx or SCP2-SCPx markedly decreased dietary phytol-induced accumulation of phytol metabolites despite concomitant upregulation of L-FABP [45-47,57] and some fatty acid oxidative enzymes [45,46]. Ablating L-FABP in SCP2/SCP-x null mice (i.e. TKO) did not further exacerbate dietary-phytol induced oxidation and upregulation of fatty acid oxidative enzymes. These findings suggested that SCP2/SCPx gene products may play a greater role in phytol oxidation than L-FABP. Consistent with this possibility, nearly half of SCP2 is peroxisomal, while all of SCPx is peroxisomal and SCPx is the only known peroxisomal ketoacyl-CoA thiolase involved in branched chain fatty acid oxidation [33]. Despite these findings, however, L-FABP may serve a lesser role in phytol metabolism, i.e. in re-uptake of excess phytol metabolites from serum [10]. L-FABP enhances fatty acid uptake [86-88], accounts for 80-90% of hepatic cytosol binding/transport capacity for fatty acids and fatty acyl-CoAs [17,18], and is the most prevalent cytosolic transporter for targeting these ligands to mitochondria and peroxisomes for oxidation [85,89,90]. Loss of L-FABP alone decreases hepatocyte uptake of phytanic acid [10]. In contrast, ablation of SCP2/SCPx significantly decreases serum NEFA, attributable in large part to marked upregulation of L-FABP [45,57]. TKO offset the effect of L-FABP upregulation in SCP2/SCPx ablated mice since serum NEFA levels were unaltered. Taken together, these findings suggested that in the context of high dietary phytol, SCP2/SCPx has a greater role in oxidation while L-FABP may have a role in reuptake.

Third, TKO markedly increased hepatic lipid accumulation, primarily due to increased phospholipid, in phytol-fed female mice. Earlier it was shown that dietary phytol alone increased hepatic lipid accumulation, primarily by increasing phospholipid in female wild-type (WT) mice [6], an effect exacerbated upon L-FABP ablation [11]. L-FABP is known to stimulate the rate limiting enzyme in phospholipid synthesis (GPAT) in vitro [91-93]. L-FABP overexpression in transfected cells increases fatty acid uptake and incorporation into triglyceride, albeit quantitatively not as much as into phospholipid [86]. In contrast, singly ablating SCPx or SCP2-SCPx decreased hepatic total lipid, primarily by decreasing triglyceride [45,46] concomitant with upregulation of L-FABP [45-47,57]. Ablating L-FABP in SCP2/SCPx mice (i.e. TKO) fed phytol mice reflected a combined phenotype where phospholipid was increased nearly 5-fold at the expense of triglyceride. The increased hepatic phospholipid was associated with increased transcription of the rate limiting gene in de novo fatty acid synthesis (Acc1) and enzymes in phospholipid synthesis (Gpam, Agpat). Taken together, these findings suggested that in the context of phytol-diet, L-FABP and SCP2/SCPx play somewhat different roles in hepatic lipid accumulation, differing in preferentially impacting phospholipid vs triglyceride.

Fourth, phytol diet TKO increased cholesterol in the serum, but not liver. Dietary phytol alone did not alter hepatic cholesterol accumulation in female wild-type (WT) mice [11]. However, L-FABP gene ablation significantly increased only hepatic free cholesterol, but not cholesteryl ester or total cholesterol, in phytol-fed females [11]. Likewise, singly ablating SCPx did not significantly alter hepatic total, free, or esterified cholesterol in female mice [46] while ablating SCP2/SCPx increased hepatic total and esterified cholesterol level [57]—effects likely attributed to concomitant greater upregulation of L-FABP [45-47,57]. L-FABP overexpression in cultured cells enhances cholesterol uptake and intracellular targeting for esterification [94,95]. Concomitant L-FABP upregulation in SCP2 overexpression mice also induces hepatic cholesterol accumulation [96-98]. Taken together, these data suggested that L-FABP upregulation may counteract the effect of ablating SCP2/SCPx or SCPx on hepatic cholesterol.

In summary, the results presented herein with TKO mice shed significant new insights into the roles of L-FABP and SCP2/SCPx genes in whole body weight gain and hepatic lipid metabolism in response to dietary phytol. TKO exacerbated dietary phytol-induced whole body weight loss, especially lean tissue mass (LTM). TKO also increased hepatic total lipid accumulation, primarily phospholipid, in response to upregulation of hepatic levels of proteins in the phospholipid synthesis. Increased hepatic lipid accumulation was not attributable to any concomitant upregulation of membrane fatty acid transport/translocase proteins involved in fatty acid uptake (FATP2, FATP4, FATP5 or GOT) or cytosolic proteins involved in fatty acid intracellular targeting (ACBP). Since individually ablating SCPx or SCP2/SCPx elicited concomitant upregulation of L-FABP, these findings with TKO mice significantly contribute to our understanding of the respective roles of these genes in dietary phytol metabolism.

Highlights.

• FABP1/SCP2/SCPx ablation (TKO) impacts liver lipids in phytol-fed female mice.

• TKO increased liver phospholipid and enzymes of phospholipid synthetic pathway.

• TKO reduced liver triacylglycerol and enzymes of triacylglycerol synthetic pathway.

• TKO exacerbated dietary phytol-induced whole body weight loss, especially lean tissue mass.

• TKO helps resolve contributions of FABP1 and SCP2/SCPx genes in female mice.

Abbreviations

3αHSD

3alpha-hydroxysteroid reductase

ABCG5 or G8

ATP-binding cassette sub-family G member 5 or 8

ACAT2

acetyl-CoA acetyltransferase

ACBP

acyl-CoA-binding protein

ACC1

Acetyl-CoA carboxylase

AFABP

adipocyte fatty acid binding protein

AGPAT

1-acylglycerol-3-phosphate-O-acyltransferase

ALT

alanine aminotransferase

apoA1

apolipoprotein A-I

apoB

apolipoprotein B

AST

aspartate aminotransferase

B-FABP

brain fatty acid binding protein

β-OHB

β-hydroxybutyrate

BSEP

bile salt export pump

C

free cholesterol

CE

cholesteryl ester

COX4

cytochrome c oxidase subunit IV

CPT1a or 2

carnitine palmitoyltransferase 1a or 2

CRABP I

cellular retinoic acid binding protein 1

DEXA

dual-energy X-ray absorptiometry

DGAT

diacylglycerol acyltransferase

FASN

fatty acid synthase

FATP2, 4 and 5

fatty acid transport protein 2, 4 and 5

FTM

fat tissue mass

FxR

farnesoid x receptor

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GOT

glutamic oxaloacetic transaminase

GPAT/Gpam

glycerol-3-phosphate acyltransferase

GST

glutathione s-transferase

HDL-C

high density lipoprotein cholesterol

HMGCR

3-hydroxy-3-methylglutaryl-CoA reductase

HMGCS1, HMGCR

3-hydroxy-3-methylglutaryl-CoA synthase

I-FABP

intestinal fatty acid binding protein

LDL-R

low density lipoprotein receptor

L-FABP

liver fatty acid binding protein

LTM

lean tissue mass

LxR

liver x receptor

MDR

multidrug resistance protein

NEFA

non-esterified fatty acid

non-HDL-C

non-HDL cholesterol

NTCP

sodium-taurocholate cotransporting polypeptide

OATP1 or 2

organic anion-transporting polypeptide 1 or 2

PL

phospholipid

PPARα

peroxisome proliferator activated receptor alpha

qRT-PCR

quantitative real-time polymerase chain reaction

RxRα

retinoid x receptor α

SCP2

sterol carrier protein 2

SCPx

sterol carrier protein x

SEM

standard error of the mean

SR-B1

scavenger receptor class B member 1

SREBP1 and 2

sterol regulatory element-binding protein 1 and 2

TC

total cholesterol

TG

triglyceride/triacylglycerol

TKO

L-FABP/SCP2/SCPx null

TLC

thin layer chromatography

WT

wild-type