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Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Peroxisome proliferator-activated receptor-α (PPARα) is a major transcriptional regulator of lipid metabolism. It is activated by diverse chemicals such as fatty acids (FAs) and regulates the expression of numerous genes in organs displaying high FA catabolic rates, including the liver. The role of this nuclear receptor as a sensor of whole dietary fat intake has been inferred, mostly from high-fat diet studies. To delineate its function under low fat intake conditions (4.8% w/w), we studied the effects of five regimens with contrasted FA compositions on liver lipids and hepatic gene expression in wild-type and PPARα-deficient mice. Diets containing polyunsaturated FAs reduced hepatic fat stores in wild-type mice. Only sunflower, linseed, and fish oil diets lowered hepatic lipid stores in PPARα−/− mice, a model of progressive hepatic triglyceride accumulation. These beneficial effects were associated, in particular, with dietary regulation of Δ9-desaturase in both genotypes, and with a newly identified PPARα-dependent regulation of lipin. Furthermore, hepatic levels of 18-carbon essential FAs (C18:2ω6 and C18:3ω3) were elevated in PPARα−/− mice, possibly due to the observed reduction in expression of the Δ6-desaturase and of enoyl-coenzyme A isomerases. Effects of diet and genotype were also observed on the xenobiotic metabolism-related genes Cyp3a11 and CAR. Conclusion: Together, our results suggest that dietary FAs represent—even under low fat intake conditions—a beneficial strategy to reduce hepatic steatosis. Under such conditions, we established the role of PPARα as a dietary FA sensor and highlighted its importance in regulating hepatic FA content and composition. (HEPATOLOGY 2007;45:767–7777.)

The nuclear receptor (NR) superfamily in the mouse is composed of 49 transcriptional regulators of gene expression that respond to a wide variety of small lipophilic molecules. The NRs are implicated in numerous biological processes, including development, cell proliferation and differentiation, inflammation, energy, and xenobiotic metabolism. Among them, the peroxisome proliferator-activated receptors (PPARα, β/δ, and γ or NR1C1, C2 and C3) are important modulators of lipid metabolism. In organs displaying high fatty acid (FA) catabolic rates, PPARα regulates the expression of genes involved in most aspects of FA catabolism (FA uptake, activation, trafficking, α- and β-oxidation, ω-hydroxylation, ketogenesis), polyunsaturated FA (PUFA) biosynthesis (elongation and desaturation), and lipoprotein metabolism (apolipoprotein C-III, lipoprotein lipase). PPARα also plays important roles in inflammation, glucose and bile and amino acid metabolism, biotransformations, and hepatocarcinogenesis (reviewed by Mandard et al.1).

PPARα is activated by structurally diverse molecules known as peroxisome proliferators, which induce hepatic peroxisome proliferation and modulate the expression of several target genes in rodents.2 Among the peroxisome proliferators, fibrates are potent hypolipidemic drugs beneficially administered to humans. In vitro, several FAs bind to and activate PPARα at physiologically relevant concentrations.3 In vivo, high-fat (24% w/w)4 and fish oil (10% w/w)5 diets activate PPARα. Thus, activation of PPARα by dietary FAs may represent a promising strategy to counteract obesity-related complications, with the caveat that the benefits of activating this pathway are demonstrated under conditions of moderate fat intake.

It is well known that dietary PUFAs have effects on diverse biological processes such as fuel partitioning, insulin action, cardiovascular function, neuronal development and immune function. The ω3 and ω6 FAs are the major classes of dietary PUFAs and display differential effects on pathologies such as cancer6 and chronic inflammation.7 Additionally, dietary PUFAs activate—both directly and indirectly—other transcription factors such as liver X receptor, hepatocyte nuclear factor-4, and sterol regulatory element binding protein (reviewed by Jump et al.8), which mediate, to some extent, their effects on gene expression.

The current study evaluates the role of PPARα as a dietary FA hepatic sensor by exposing wild-type and PPARα-deficient mice to five selected FA mixes while maintaining 4.8% total dietary fat (usual fat content for mouse maintenance diets: 4%–6% w/w). We used 5 contrasted diets: an essential FA-deficient diet, a reference diet, an ω6 FA-rich diet, an ω3 FA-rich diet, and a fish oil–enriched diet, and studied their effects on hepatic lipids and gene expression.

Even under low dietary fat intake, PUFA-containing diets reduced hepatic FA stores in a manner partially independent of PPARα. Dietary regulations of Lpin1 in wild-type and stearoyl–coenzyme A (CoA) desaturase-1 (SCD1) in both genotypes paralleled these effects on hepatic FA content. Moreover, hepatic FA profiles revealed that linolenic and linoleic acids accumulate in PPARα−/− livers, suggesting a role for this NR in the degradation and/or use of the essential precursors of ω3 and ω6 PUFAs. Consistent with this finding, we showed that in PPARα−/− mice, the hepatic expression of 3 enoyl-CoA isomerases as well as Δ6-desaturase were markedly reduced. In addition, we observed regulations of the xenobiotic metabolism-related genes Cyp3a11 and CAR. Overall, our study demonstrates that low-fat, PUFA-containing diets exert beneficial effects on hepatic fat stores. Moreover, it clarifies the role of PPARα in mediating dietary effects on hepatic gene expression, FA content, and composition.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals.

Chemicals for FA analyses were obtained from Prolabo (Paris, France) or Merck (Darmstadt, Germany). Standards for FA analyses, 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), and fenofibrate were obtained from Sigma (Lyon, France).

Animal Housing and Treatment.

PPARα-deficient mice with a C57BL/6J genetic background2, 9 were bred at INRA's transgenic rodent facility at 22 ± 2°C under 12-hour light/dark cycles. Age-matched male C57BL/6J mice were obtained from Charles River (Les Oncins, France) and were acclimated to local animal facility conditions for 2 weeks. In vivo studies were conducted under European Union guidelines for the use and care of laboratory animals and were approved by the institutional ethics committee.

Diet Study.

Ten to 11-week-old male mice were given experimental diets ad libitum for 8 weeks (pellets prepared by UPAE-INRA, Jouy-en-Josas, France, replaced weekly), with free access to water. Food consumption (groups of 6) and individual body weight were recorded weekly. All diets were isocaloric and contained 4.8% fat (w/w). Oils used for experimental diet preparation were corn and colza oils (50/50) for a reference diet (REF), hydrogenated coconut oil for a saturated FA diet (COC), sunflower oil for an ω6 FA-rich diet (SUN), linseed oil for an ω3-rich diet (LIN), and corn/colza/docosahexaenoic acid–enriched fish oils for the fish oil diet (FISH) (42.5/42.5/15). The fish oil (72% of C22:6n-3) was obtained from Polaris (Quimper, France). Other FAs were C22:5n-3 (17%), C22:5n-6 (2%), C22:4n-3 (2.4%), and C20:5n-3 (2%). The detailed composition of the diets is given in Table 1. FA composition was controlled via gas chromatography analysis of organic extracts from manufactured food pellets.

Table 1. Fatty Acid Composition of the Five Experimental Diets
 COCREFSUNLINFISH
  1. NOTE. Results are presented as a percentage of the total FAs present in the oil (except for the calculated ratios). All diets contained 4.8% fat, 45% starch, 22.2% sucrose, 1% cellulose, 21% casein, 4% salt mixture, 1% vitamin mixture (0.02% for vitamin E), and 1% agar.

  2. Abbreviations: ω3 or ω6, FAs of the ω3 or ω6 family, respectively; DHA, docosahexaenoic acid; MUFA, mono-unsaturated fatty acid; SFA, saturated fatty acid; UFA, unsaturated fatty acid.

SFA99.610.811.89.09.3
MUFA0.346.022.618.039.0
PUFA0.143.265.672.951.7
ω60.137.965.514.933.7
ω3 (DHA)0.0 (0.0)5.3 (0.0)0.1 (0.0)58.0 (0.0)18.0 (10.6)
UFA/SFA0.08.37.510.19.7
ω6/ω37.11091.70.31.9

Compound Administration.

Fenofibrate (100 mg/kg/day for 14 days) was administered via gavage and TCPOBOP (3 mg/kg/day for 3 days) was administered via intraperitoneal injection.

Tissue Sample Collection.

Selected organs were dissected, rinsed, weighed, snap-frozen in liquid nitrogen, and stored at −80°C until analysis.

FA Composition.

Hepatic FA composition was assayed as described previously.10 Triglycerides (TGs) and phospholipids were separated via thin-layer chromatography before FA methyl esters analysis via gas chromatography (Agilent Technologies 6890N, Bios Analytique, Toulouse, France).

Enzyme Activities.

FA-desaturase activities were assayed on postmitochondrial fractions using 14C-labeled FAs.10

Northern Blot, Macroarray, and Quantitative PCR Analysis.

Total RNA was extracted with TRIzol reagent (Invitrogen, France). Northern blot analysis was performed as described.10, 11 Production of INRARRAY, which comprises the selection, cloning, amplification and spotting of complementary DNAs onto nylon membranes, has been described previously.11 Briefly, INRARRAY 01.2 consists of 120 complementary DNA fragments comprising class II NRs, target genes for each NR, housekeeping genes, and external controls (Lucidea Universal Scorecard, Amersham Biosciences, Orsay, France). Protocols for RNA radiolabeling, INRARRAY hybridization, and image analysis have been described.11 Forty hepatic RNA samples (4 mice/group) were screened with INRARRAY. For real-time quantitative PCR (Q-PCR), total RNA samples (2 μg) were reverse-transcribed with SuperScript II (Invitrogen). All assays were performed on an ABI Prism 7000 (Applied Biosystems, Courtaboeuf, France) using standard PCR conditions. Primers and Taqman probes for TATA-box binding protein (Mm00446973_m1), Lpin1 (Mm00550511_m1), CARβ (Mm00437986_m1), PECI (Mm00478725_m1), ECI (Mm00494452_m1), FADS1 (Mm00507605_m1), and FADS2 (Mm00517221_m1) were purchased from Applied Biosystems Assays-on-Demand. Primers for SYBR Green assays were as follows: SCD1-F, 5′-CCGGAGACCCCTTAGATCGA-3′; SCD1-R, 5′-TAGCCTGTAAAAGATTTCTGCAAACC-3′; PMDCI-F, 5′-GGAAAGATGTTCACTTCAGGTATTGAC-3′; PMDCI-R, 5′-CGGGCCGCATCATCTC-3′. A pool of all complementary DNA samples was used to generate calibration curves. All Q-PCR data were normalized by TATA-box binding protein levels.

Statistical Analysis.

Data analyses were performed using SPlus 2000 (Insightful, Toulouse, France) and R (http://www.r-project.org/) software. Data are expressed as the mean ± SD (n = 4–6 per group). Differential effects were analyzed via ANOVA with the appropriate factors and interactions. When an effect was significant, a post hoc 2-tailed Student t test with a pooled variance estimate was used to compare the groups. For the macroarray data, the Benjamini-Hochberg procedure12 was used to control the false discovery rate at 5% (multitest package from www.bioconductor.org). Only the genes displaying a minimum of 1 significant modulation of at least 1.5-fold amplitude are presented.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Food Consumption, Body, and Organ Weights.

No significant effect of genotype or diet was observed on body weight gain or food consumption. PPARα−/− mice displayed significantly higher epididymal WAT (white adipose tissue) weight compared with controls (3.1 ± 1.0% and 2.3 ± 0.6% of body weight, respectively; n = 30 per genotype), without significant diet effect. Only the LIN diet significantly reduced liver weight in PPARα−/− mice (Table 2), which consistently displayed heavier livers than their wild-type counterparts.

Table 2. Genotype and Diet Effects on Liver Weight and Hepatic FA Content in the Total Lipid, Triglyceride, and Phospholipid Fractions
PPARαDietLiver Weight (% Body Weight)Total FA Content (μg/mg of Liver)TG–FA Content (μg/mg of Liver)PL-FA Content (μg/mg of Liver)
  • a

    Significant genotype effect (same diet; n = 6 per group).

  • b, c, b,c

    Significant difference versus the COC or REF diet, respectively (same genotype; n = 6 per group).

+/+COC4.8 ± 0.260.5 ± 21.416.4 ± 3.913.3 ± 2.3
 REF4.1 ± 0.4b38.3 ± 4.0b9.9 ± 2.2b13.9 ± 1.0
 SUN4.3 ± 0.2b37.6 ± 5.2b8.7 ± 2.5b14.2 ± 1.9
 LIN4.5 ± 0.3c31.5 ± 6.0b6.2 ± 2.2b,c13.0 ± 0.7
 FISH4.6 ± 0.3c42.3 ± 6.7b7.8 ± 2.0b15.0 ± 5.8
−/−COC5.3 ± 0.4a219.5 ± 82.3a96.1 ± 65.4a12.6 ± 2.4
 REF5.3 ± 0.7a212.4 ± 44.6a73.1 ± 26.5a12.8 ± 2.9
 SUN5.0 ± 0.4a128.5 ± 54.0ab,c46.4 ± 32.5ab13.8 ± 2.3
 LIN4.5 ± 0.4b,c96.9 ± 42.0ab,c42.9 ± 24.2ab15.8 ± 1.3
 FISH5.6 ± 0.6a76.3 ± 29.2ab,c26.8 ± 12.8ab16.1 ± 2.8

Effect of Diet and Genotype on Hepatic FA Content and Composition.

Hepatic total FA content was significantly higher in PPARα−/− versus wild-type mice for all diets (Table 2). In wild-type mice fed PUFA-containing diets (REF, SUN, LIN, and FISH), hepatic total FA content was significantly reduced compared with the COC group. Conversely, in PPARα−/− mice, the REF diet no longer reduced hepatic FA, whereas the SUN, LIN, and FISH diets did efficiently reduce hepatic FA stores. Similar results were obtained for the hepatic TG–FA content, whereas no significant effects were observed on hepatic phospholipid–FA content (Table 2). Histological examination of neutral lipid-staining in frozen liver sections (Fig. 1) confirmed these quantitative data (Table 2). In wild-type mice (Fig. 1A–C), increased TG accumulation was also observed in the COC-fed group. As previously reported,9 PPARα-deficiency favored macrovacuolar, centrilobular steatosis. However, the level of hepatic neutral lipid accumulation was reduced in FISH-fed PPARα−/− mice (Fig. 1F) compared with mice fed the COC (Fig. 1D) and REF (Fig. 1E) diets.

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Figure 1. Liver neutral lipids in mice fed the COC, REF, and FISH diets. Representative Oil Red O–stained frozen sections of liver from (A-C) wild-type and (D-F) PPARα−/− mice fed the (A, D) COC, (B, E) REF, or (C, F) FISH diet. (Original magnification ×200.) Neutral lipids appear in red.

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Twenty-one hepatic FAs were assayed via gas chromatography in the total lipid, TG, and phospholipid fractions (Table 3 and supplementary material). Livers from both genotypes displayed specific accumulations of FA families that were present in the diets. Mice fed the COC diet preferentially accumulated saturated FA (except C18:0) and especially mono-unsaturated FAs in their livers. Mice fed the SUN diet had livers enriched in ω6 FAs, whereas those fed the LIN and FISH diets preferentially accumulated ω3 FAs. Regarding FA composition, the REF diet corresponded to a balanced profile compared with the other diets (Table 3 and supplementary material). Generally, PPARα−/− livers when compared with wild-type livers accumulated 16-carbon and 18-carbon monounsaturated FAs and, notably, displayed a robust increase in linoleic (C18:2ω6) and α-linolenic (C18:3ω3) acid proportions. Conversely, most saturated FAs and 20- to 22-carbon PUFAs were reduced in PPARα−/− livers. Hepatic TG and phospholipid analyses yielded comparable conclusions (see Supplementary material).

Table 3. Hepatic FA Composition of Liver Total Lipids
  1. NOTE. Data are expressed as the mean percentage of FA ± SD (n = 6 per group).

  2. Abbreviations: HUFA, highly unsaturated FA (at least 3 double bonds); LHUFA, long-chain highly unsaturated FA (HUFA with more than 20 carbons); ND, not detected in any sample of the group; SFA, saturated fatty acid.

  3. aSignificant difference versus wild-type mice fed the same diet (Student t test; P < .05).

  4. b,c,d,e,fSignificant difference versus the same genotype fed the COC, REF, SUN, LIN, or FISH diet, respectively (Tukey test; P < .05).

  5. *Saturated FA for wild-type mice, REF vs. LIN (P = .0502).

  6. C18:2ωq6 + C18:3ω3.

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Effect of Diet and Genotype on Hepatic Gene Expression.

Under such low fat intake conditions, we analyzed the expression of genes related to class II NR signaling using INRARRAY 01.2.11 Mean Pearson correlation coefficient was 0.97 between macroarrays (n = 40), indicating good reproducibility and relatively discrete gene expression changes. Table 4 presents the results of the macroarray screening. Thirty-one genes displayed differential expressions between PPARα−/− and wild-type mice. Minimal effects of genotype were observed for the REF diet. For dietary effects, comparisons to the COC and REF diets provided control situations of PUFA deficiency and intermediate FA profile, respectively (Table 4). For wild-type mice, 26 genes displayed at least one significant modulation out of the 7 comparisons performed. In contrast, in PPARα−/− mice, only 8 genes were modulated. Overall, the FISH diet, followed by the LIN diet, were responsible for the majority of gene expression changes observed in wild-type mice. Except for the induction of Cyp3a11 by the FISH diet, all other modulations seen in wild-type mice were absent in PPARα−/− mice. Additionally, the SUN diet reduced the expression of genes in PPARα−/− mice. Table 4 clearly illustrates that dietary effects were more pronounced in wild-type mice than in PPARα−/− mice, establishing that, even under low fat intake, PPARα mediates the effects of dietary FAs on the expression of many genes present on the macroarray.

Table 4. Genotype and Diet Effects on Hepatic Gene Expression Monitored with INRARRAY 01.2
  1. NOTE. Data are expressed as expression ratios (PPARα-deficient/wild-type for genotype effects and diet/COC or REF for diet effects). No significant differences were observed for PPARα−/− mice between the REF, COC, and LIN diets. Shaded areas correspond to transcript ratios that do not reach statistical significance (NS in shaded areas = not significant).

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Analysis of Lipid Metabolism Genes.

To gain insight into the molecular mechanisms underlying the effects observed on hepatic FA content and composition, we further examined genes involved in lipid metabolism. First, we noticed that Lpin1 was induced in the COC diet, suggesting a coregulation with genes involved in FA and cholesterol synthesis (Table 4). Q-PCR confirmed a striking 400-fold increase of Lpin1 messenger RNA under the COC diet in wild-type livers, whereas no effect was observed in PPARα−/− mice (Fig. 2A). Because this effect appeared to be PPARα-dependent, we tested the in vivo effect of fenofibrate on Lpin1 expression. We observed a PPARα-dependent downregulation of Lpin1 (Fig. 2B). The expression of stearoyl-CoA desaturase 1 (SCD1), the main hepatic Δ9-desaturase isoform controlling mono-unsaturated FA synthesis, may also influence hepatic lipid content and composition.13 PPARα−/− mice expressed lower levels of hepatic SCD1 than wild-type mice except when given the REF diet (Fig. 3A), suggesting that altered FA oxidation rather than increased expression of SCD1 (or other lipogenic enzymes) accounts for the hepatic steatosis developed by PPARα knockout mice. Hepatic Δ9-desaturase activity was not significantly lowered in PPARα−/− mice on the REF diet. Whereas all PUFA-containing diets decreased SCD1 expression compared with the COC diet in wild-type mice, only the SUN, LIN, and FISH diets significantly reduced SCD1 expression in PPARα−/− mice. Changes in C16:1ω9 and C18:1ω9 proportions paralleled the diet-induced changes in SCD1 expression within each genotype (Table 3).

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Figure 2. Dietary and pharmacological regulation of Lpin1 messenger RNA levels. (A) Q-PCR quantification of hepatic Lpin1 expression (logarithmic scale) in the diet study. aSignificant difference between the 2 genotypes (same diet). b,cSignificant effect of the diet versus the COC or REF diet, respectively (same genotype). (B) Northern blot analysis of hepatic Lpin1 messenger RNA expression in wild-type and PPARα−/− mice after fenofibrate treatment. Fold changes are indicated. **P < .01.

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Figure 3. Analysis of Δ9 (SCD1), Δ6 (FADS2), and Δ5 (FADS1) FA-desaturase expression. Q-PCR quantification of hepatic (A) SCD1, (B) FADS2, and (C) FADS1 messenger RNA expression in the diet study is shown. aSignificant difference between the 2 genotypes (same diet). b,c,d,e,fSignificant effect of the diet versus the COC, REF, SUN, LIN, or FISH diet, respectively (same genotype). Enzyme activities measured under the REF diet are illustrated in the respective insets.

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We next studied FADS2 and FADS1, the Δ6 and Δ5-desaturases (reviewed by Nakamura and Nara14) that are involved in rate-limiting steps of long-chain PUFA biosynthesis from C18:2ω6 and C18:3ω3 (Fig. 3B–C). Both desaturases displayed reduced expression under PUFA-containing diets compared with the COC diet in the two genotypes. Furthermore, FADS2 and FADS1 were expressed—under most dietary conditions—at significantly lower levels in PPARα−/− livers. Similarly, Δ6- and Δ5-desaturase activities under the REF diet were significantly reduced in PPARα−/− mice. Finally, we studied enoyl-CoA isomerases, which allow PUFAs to enter the β-oxidation pathway. In accordance with the macroarray data, PECI, PMDCI, and ECI (monitored by Q-PCR only) always displayed lower expression in PPARα−/− livers (Fig. 4). The only consistent dietary effects observed were the increased expression of these genes by the FISH diet in wild-type mice and a decrease in their expression by the SUN diet in PPARα−/− mice.

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Figure 4. Analysis of three hepatic enoyl-CoA isomerases. Q-PCR quantification of hepatic (A) PECI, (B) PMDCI, and (C) ECI messenger RNA expression in the diet study. aSignificant difference between the two genotypes (same diet). b,c,d,e,fSignificant effect of the diet versus the COC, REF, SUN, LIN, or FISH diet, respectively (same genotype).

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Analysis of Genes Related to Xenobiotic Metabolism.

Table 4 and multidimensional exploratory analyses (data not shown) highlighted a major effect of the genotype on the expression of the gene studied. This is well exemplified by the consistent reduction of Cyp3a11 expression in PPARα−/− livers (Table 4), which was confirmed via Northern blot analysis (Fig. 5). Furthermore, the FISH and LIN diets increased Cyp3a11 levels in wild-type and, to a lesser extent, PPARα−/− livers. Interestingly, PPARα−/− livers displayed a modest but significant overexpression of CAR, a master regulator of xenobiotic-metabolizing enzymes (Table 4, Fig. 6A,B [five alternative transcripts studied15]). Two independent experiments using fenofibrate (PPARα activator) (Fig. 6C) or TCPOBOP (CAR activator) (Fig. 6D) confirmed this overexpression. Fenofibrate had no effect on CAR expression, suggesting that CAR overexpression is an indirect consequence of PPARα deficiency. TCPOBOP reduced hepatic CAR expression in both genotypes. Similarly, an intraperitoneal injection of phenobarbital reduced hepatic CAR expression after 3 hours (data not shown). TCPOBOP induced a more pronounced liver enlargement in PPARα−/− (73% increase) versus wild-type mice (47%), as described previously.16 Furthermore, the Mdm2 transcript, a CAR target implicated in TCPOBOP-induced hepatocyte proliferation,17 was significantly more induced by TCPOBOP in the PPARα−/− genotype (Q-PCR, 2.4- vs. 2.0-fold [data not shown]).

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Figure 5. Northern blot analysis of Cyp3a11 and 36B4. Northern blot analysis (15 μg of total RNA per lane) of hepatic Cyp3a11 normalized by 36B4. aSignificant difference between the 2 genotypes (same diet). b,c,dSignificant effect of the diet versus the COC, REF, or SUN diet, respectively (same genotype).

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Figure 6. Overexpression of CAR in PPARα−/− liver and regulation by fenofibrate and TCPOBOP. (A) Quantification of five CAR transcripts (normalized by 36B4). aSignificant difference between the two genotypes (same diet). b,c,d,eSignificant effect of the diet versus the COC, REF, SUN, or LIN diet, respectively (same genotype). (B) Representative blots of hepatic CAR. (C, D) Q-PCR quantification of hepatic CAR expression in wild-type and PPARα−/− mice after fenofibrate (C) or TCPOBOP (D) treatment. aSignificant difference between the two genotypes. bSignificant effect of the treatment.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Our current knowledge on FA molecular signaling in physiology relies mostly on high-fat diet exposures. This study was designed to investigate, under conditions of low fat intake, the role of PPARα in mediating the effects of dietary FA composition on hepatic gene expression, lipid content, and FA profile. Overall, our study, which fully complied with mouse dietary requirements (4%–6% fat intake) and used contrasted dietary FA profiles, highlighted a physiological role of PPARα as a sensor of the quality of the ingested fat.

Beneficial effects of dietary PUFAs, especially ω3-PUFAs, on hepatic steatosis have been reported in the context of high fat input (59%).18 This condition triggers the simultaneous transcriptional upregulation of hepatic FA oxidation through PPARα activation and downregulation of hepatic lipogenesis through the inhibition of sterol regulatory element binding protein-1c.19, 20 Accordingly, under the COC diet in wild-type mice, we observed a higher hepatic FA content than under any other PUFA-containing diet (Table 2, Fig. 1). Consistent with previous reports,9 we found that PPARα deficiency led to higher hepatic triglyceride accumulation under all diets. However, in PPARα−/− mice fed SUN, LIN, and FISH diets, TG accumulation was reduced, which is consistent with the beneficial effect of PUFAs on hepatic steatosis reported in other rodent models.19, 21, 22 The absence of an effect of the REF diet in PPARα−/− mice further suggests that when the hepatic FA sensor PPARα is compromised, some PUFAs can achieve their beneficial effect only when they have reached a critical threshold rate in the diet. Remarkably, in our study, the replacement of 15% of the fat with fish oil (FISH vs. REF) led to a significant reduction of hepatic FA accumulation in both genotypes. This suggests that physiologically relevant levels of dietary FAs present in fish oil have a beneficial impact on hepatic FA accumulation independently of PPARα. Together, these results further support the hypothesis that fish oil can be used as a therapeutic approach in the treatment of nonalcoholic fatty liver disease.23 Unexpectedly, our macroarray study revealed that sterol regulatory element binding protein target genes involved in cholesterol and FA synthesis were downregulated by PUFA-containing diets in wild-type mice only (Table 4). Many of these genes display significant circadian variations in expression, some of which are altered in PPARα−/− mice.24, 25 A role for PPARα in the dietary downregulation of these genes cannot be excluded but requires additional investigation, including a time-course study. SCD1 plays an important role in hepatic cholesterol ester and triglyceride biosynthesis.13 Its disruption reduces hepatic steatosis in PPARα−/− mice.26 In our study, all the diets that reduced SCD1 expression (Fig. 3A) in a given genotype also reduced hepatic fat stores (Table 2) and proportion of C18:1ω9, the major hepatic mono-unsaturated FA (Table 3). Thus, downregulation of SCD1 may contribute to the reduction of hepatic lipid stores in both genotypes. Moreover, we report the regulation of lipin expression by dietary FAs. The mouse null mutation of lipin accounts for the fatty liver dystrophy phenotype,27 which combines metabolic disorders, lipodystrophy, and neuropathy.28 Lpin1 has been related to adipocyte differentiation,29 obesity, energy expenditure, and fuel partitioning,30 and very recently has been identified as a phosphatidate phosphatase,31 the penultimate enzyme in TG synthesis. The Lpin1 hepatic transcript is negatively regulated by PUFA-containing diets or fenofibrate in wild-type mice only (Fig. 2). Its coregulation with cholesterol and FA synthesis enzymes (Table 4) is consistent with its enzymatic function.31 Recently, Lpin1 was described as an enhancer of the PGC-1α/PPARα pathway.32 Adenoviral overexpression of Lpin1 upregulated PPARα, increased FA oxidation and decreased lipogenesis while increasing liver TG content.32 The elevated Lpin1 expression under our COC diet (wild-type) (Fig. 2) was consistently associated with increased hepatic TG stores. However, we observed a different gene expression pattern than Finck et al.32 This discrepancy may be due to the specific hepatic FA profile of COC-fed mice, which may not provide appropriate PPARα ligands and/or differentially affect other transcriptional pathways. Further studies are required to understand the interplay between Lpin1 biochemical and transcriptional functions and its potential as a drug target.

Collectively, our results suggest that reduction of SCD1 expression may contribute to the reduction of hepatic TG content by PUFA-containing diets. In wild-type mice, additional mechanisms include PPARα-dependent induction of FA catabolism, reduction of lipogenesis, and TG synthesis (including Lpin1). Further studies in PPARα−/− mice are underway to evaluate the possible role of TG secretion and intestinal lipid absorption in these beneficial effects of PUFAs.

In addition, dietary FA composition markedly impacted the hepatic FA profile in both genotypes. Consistent with other reports,33 we observed that PPARα−/− livers accumulate the ω3 (C18:3ω3) and ω6 (C18:2ω6) FA precursors at the expense of long-chain PUFAs (Table 2). First, this may reflect an altered ability to synthesize long-chain PUFAs through the elongase/desaturase pathways. Indeed, PPARα is implicated in the transcriptional control of FA desaturases,10 and Li et al.34 have reported the role of PPARα in the nutritional regulation of PUFA synthesis. We observed reduced expression and activity of Δ6- and Δ5-desaturases in PPARα−/− livers (Fig. 3), which is likely to reduce long-chain PUFA synthesis and contribute to the precursor accumulations. Interestingly, alteration in Δ6- and Δ5-desaturase levels developed with age and were not observed in younger PPARα−/− mice10 (data not shown). Second, PPARα−/− livers displayed reduced expression of three enoyl-CoA isomerases (Table 4, Fig. 4), which may also contribute to precursor accumulations, as enoyl-CoA isomerase have been shown in vivo to be rate-limiting for linoleic and α-linolenic acids to enter β-oxidation.35 Thus, impairments of both PUFA-desaturases and enoyl-CoA isomerases likely play a role in the combined depletion of long-chain PUFAs and the converse accumulation of their precursors in PPARα−/− livers.

To evaluate the safety of a therapeutic intervention with dietary FAs, potential drug–diet interactions involving xenobiotic-metabolizing enzymes should also be investigated. As a key factor of drug metabolism, Cyp3a11 induction under diets containing high ω3-FA levels (Fig. 5) may raise concerns. However, recent studies identified PUFAs as inhibitors of rodent and human CYP3A activities.36, 37 The induction of Cyp3a11 transcript, also reported by others,38 may thus be secondary to the reduction in enzymatic activity and restore a constitutive CYP3A hepatic activity. We also observed an overexpression of CAR in PPARα−/− livers (Fig. 6). Our preliminary results on TCPOBOP-mediated liver enlargement and Mdm2 induction support an increased CAR function in PPARα−/− livers. Interestingly, CAR activation downregulated its own transcript (Fig. 6D), which could represent a negative feedback loop preventing excessive induction of xenobiotic-metabolizing enzymes after xenochemical exposure.

In conclusion, this study further defines the role of PPARα as a hepatic dietary FA sensor under low fat intake conditions and delineates its importance in regulating hepatic FA content and composition.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are grateful to Dr. Daniel Catheline for expertise and assistance in analytical procedures. We thank Colette Bétoulières and Gérard Galy for excellent technical assistance. We thank Talal Al Saati and Florence Capilla (Histopathology core facility of IFR30, Toulouse, France) for histology studies. We are grateful to Dr. Franck Jess Gonzalez for the generous gift of the PPARα−/− mouse line. We thank Dr. Suhasini Kulkarni, Pr. Alain Baccini, and Dr. Christelle Robert-Granié for critical review of the manuscript.

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  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supplementary material for this article can be found on the H EPATOLOGY website ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

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