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Liver fatty acid binding protein is required for high rates of hepatic fatty acid oxidation but not for the action of PPAR in fasting mice¹

ERDAL EROL^*,2, LEENA S. KUMAR^*,2, GARY W. CLINE, GERALD I. SHULMAN, DANIEL P. KELLY and BERT BINAS^*,3

^* Department of Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, USA;
Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut, USA; and
Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, Missouri, USA

³ Correspondence: Department of Pathobiology, College of Veterinary Medicine, Texas A&M University, Raymond Stotzer Pkwy, College Station, TX 77843-4467, USA. E-mail: bbinas{at}cvm.tamu.edu

SPECIFIC AIMS

1. Liver fatty acid binding protein (L-FABP) is the main cytosolic binding site for long chain fatty acids (LCFA) in hepatocytes. Here we elucidated the significance of L-FABP for hepatic fatty acid oxidation in vivo.

2. In cell culture, L-FABP has been shown to increase activity and levels of peroxisome proliferator-activated receptor (PPAR), a transcription factor that boosts hepatic fatty acid oxidation and ketogenesis. We tested whether L-FABP is required for action of PPAR under fasting conditions.

PRINCIPAL FINDINGS

1. Long chain fatty acid oxidation and ketogenesis are reduced in L-FABP null liver
Blood ß-hydroxybutyrate levels were measured, as they are derived mainly from hepatic mitochondrial LCFA oxidation. Mice were subjected to starvation, high fat/low sugar (ketogenic) diet, standard diet, or high-fat/high-sugar (diabetogenic) diet. Figure 1 A shows that in null vs. wild-type mice, ß-hydroxybutyrate levels were significantly reduced under all ketogenic conditions whereas circulating free fatty acid levels were normal or slightly increased (Fig. 1B ). Fatty acid oxidation was assessed in suspensions of freshly isolated hepatocytes incubated with ¹⁴C-radiolabeled palmitic acid (1 mM). ß-Hydroxybutyrate production was reduced by 35.5% (P<0.05) and radiolabeled oxidation products (CO₂+acid soluble products) were reduced by 34.5% (P<0.003) in L-FABP null vs. wild-type cells. These results demonstrate that L-FABP-deficient mice exhibit a liver-intrinsic defect of long chain fatty acid oxidation and ketogenesis.

View larger version (24K): [in this window] [in a new window]   Figure 1. A liver-intrinsic defect impairs hepatic long chain, but not medium chain, fatty acid oxidation in L-FABP null mice. A, B) Serum levels of ß-hydroxybutyrate (A) and NEFA (B) in female (left panels) and male (right panels) mice subjected to starvation (overnight), standard diet, ketogenic diet (3 days), or high-fat diabetogenic diet (4 wk). Black columns, wild-type; white columns, L-FABP null. Each column pair represents mice matched for age, sex, and backcross generation; mice represented by different column pairs may slightly differ by age and backcross generation. n = 5–6 mice for each column. *P < 0.05; **P < 0.01, ***P < 0.001; comparisons between genotypes. C) Octanoate injection restores ketogenesis in L-FABP null but not PPAR null mice. L-FABP null mice (L-/-, empty circles; n=5) and wild-type littermates (L+/+, filled circles; n=5) as well as PPAR null mice (P-/-, empty triangles; n=4) and their wild-type littermates (P+/+, filled triangles; n=4) were injected with octanoic acid, and plasma ß-hydroxybutyrate levels were determined. All mice were females.

2. Capacities for long chain fatty acid oxidation and ketogenesis are normal in L-FABP null liver
We tested whether ketogenesis can be restored in vivo by injection of octanoic acid, a good but not physiologically important ketogenic substrate that is not a ligand of L-FABP and is poorly metabolized by PPAR null hepatocytes. When octanoic acid was intraperitoneally injected under fasting conditions, a massive increase of ß-hydroxybutyrate occurred in wild-type and L-FABP null mice, reaching identical, supraphysiological levels despite lower starting levels in the L-FABP null mice (Fig. 1C ). PPAR null mice that also showed low starting ß-hydroxybutyrate levels did not raise these levels significantly (Fig. 1C ). When palmitic acid (1 mM) oxidation was assessed in liver homogenates of wild-type and L-FABP null hepatocytes, no genotypic differences in the production of ß-hydroxybutyrate or labeled oxidation products (CO₂+acid soluble products) were seen. These results demonstrate that the capacity of L-FABP null hepatocytes for LCFA oxidation and ketogenesis is maintained.

3. Hepatic expression of PPAR target genes is normal in fasting L-FABP null mice, but HMG CoA synthase mRNA is reduced in fed L-FABP null mice
The expression of key genes of lipid oxidation was compared between L-FABP null, PPAR null, and wild-type mice. Under fasting conditions, PPAR null vs. wild-type mice showed significantly reduced levels of mRNAs encoding CYP4A3, MCAD, ACO, mitochondrial HMG CoA synthase, and L-FABP whereas mRNAs encoding L-CPT1 and LCAD showed little or no reduction (Fig. 2 A, C), in agreement with the literature. In contrast, the above mRNAs (except L-FABP) as well as PPAR mRNA were not changed in L-FABP null vs. wild-type livers (Fig. 2A, C ). When Northern blot experiments were repeated under feeding (standard diet), a similar tendency to reduced gene expression was seen in PPAR null vs. wild-type mice (Fig. 2B, D ). In fed L-FABP null vs. wild-type mice, a significant reduction was seen for mitochondrial HMG CoA synthase mRNA and a similar tendency for L-CPT1 that did not reach statistical significance. Other mRNA levels measured were not affected by an absence of L-FABP.

View larger version (45K): [in this window] [in a new window]   Figure 2. Expression of key genes of lipid oxidation in female L-FABP null and PPAR null livers. Shown are representative Northern blots prepared from A) mice starved overnight (14–16 h); B) mice fed standard chow. L, L-FABP; P, PPAR; +/+, wild-type; -/-, null. Separate matched wild-type controls were used for L and P mice. For each genotype, 2 samples from 2 different mice were applied to neighboring lanes on the gel. A, B) Two blots hybridized multiple times. Identical results were obtained with a second pair of Northern blots (not shown) prepared with RNA samples from additional mice, resulting in 4 analyzed mice for each of the 4 groups of mice. C) Quantification of expression levels in starved mice; D) quantification of expression levels in fed mice (except for CYP4A3 because of low levels). Signals of all bands of all blots were normalized to the respective GAPDH signals; resulting numbers from null mice were divided by those from wild-type control mice. White columns, (L-/-)/(L+/+) ratio; black columns, (P-/-)/(P+/+) ratio. *P<0.05; **P<0.01, ***P<0.001; comparison between null and wild-type. , Ratio equals zero by definition.

CONCLUSIONS AND SIGNIFICANCE

We have addressed two related questions: 1) is L-FABP important for hepatic LCFA oxidation in vivo; 2) under conditions favoring hepatic LCFA oxidation, is L-FABP required for the action of its postulated target, the transcription factor PPAR, a master on-switch of fatty acid oxidation? The results answer the first question to the affirmative but argue strongly against the second possibility.

We showed here that in both in vivo and hepatocyte incubations, L-FABP is a limiting factor in the produc-tion of ß-hydroxybutyrate, the final product of (mainly) hepatic fatty acid oxidation. Males showed a weaker phenotype than females, in line with the known gender difference in L-FABP levels. Since ketogenesis from octanoate was normal in vivo, the defect must be upstream of medium chain acyl CoA dehydrogenase (MCAD). That no genotypic difference in palmitic acid oxidation was seen after liver homogenization and addition of the LCFA in albumin-bound form argues that the defect was located upstream of long chain acyl CoA synthase and hence at the level of substrate availability, as opposed to enzymatic capacity for oxidation. mRNA levels of MCAD and mitochondrial HMG CoA synthase remained normal. Since these RNAs are known to depend on PPAR, this finding provides further support against a role of L-FABP in the action of PPAR under fasting conditions, a conclusion strengthened by the normal expression of additional PPAR target genes. These results contrast with the literature demonstrating FABP-dependent activation of PPARs in cultured cell lines. However, the low degree of differentiation of these cell lines may not support high LCFA fluxes; we therefore suggest that FABPs might be more important for cognate transcription factors under conditions of low lipid metabolism. In this context, our observation that mitochondrial HMG CoA synthase mRNA levels were reduced in L-FABP null versus wild type liver under standard diet (i.e., at lower circulating fatty acid levels) may be of interest. We suggest that the relative importance of L-FABP in substrate provision and gene expression may vary with physiological condition (Fig. 3 ), a concept that may be useful for future investigation of various members of the FABP family.

View larger version (18K): [in this window] [in a new window]   Figure 3. Hypothetical role of L-FABP in fatty acid availability. Intracellular availability (columns) of LCFA and their CoA esters may have to be distinguished from their total cellular levels. Availability may be determined by L-FABP (black) and factors (white) that may include other proteins as well as total cellular LCFA/LCFA-CoA levels. In fasted L-FABP null mice, enough LCFA/LCFA-CoA is presented by these other factors to the transcription machinery but availability of LCFA/LCFA-CoA is not high enough to saturate mitochondrial lipid oxidation. In fed mice, fatty acid availability is too low for maximum activation of some transcription factors and can be increased by L-FABP.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0330fje

² These authors contributed equally to this work

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