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Peroxisome Proliferator-activated Receptor α Deficiency Abolishes the Response of Lipogenic Gene Expression to Re-feeding

RESTORATION OF THE NORMAL RESPONSE BY ACTIVATION OF LIVER X RECEPTOR α*
  • Abdel M. Hebbachi
    Affiliations
    Metabolic Research Laboratory, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LJ
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  • Brian L. Knight
    Affiliations
    Medical Research Council Clinical Sciences Centre, Imperial College School of Medicine, London W12 0NN, United Kingdom
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  • David Wiggins
    Affiliations
    Metabolic Research Laboratory, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LJ
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  • Dilip D. Patel
    Affiliations
    Medical Research Council Clinical Sciences Centre, Imperial College School of Medicine, London W12 0NN, United Kingdom
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  • Geoffrey F. Gibbons
    Correspondence
    To whom correspondence should be addressed: OCDEM, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, UK. Tel.: 44-0-1865-857224; Fax: 44-0-1865-857217
    Affiliations
    Metabolic Research Laboratory, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LJ
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  • Author Footnotes
    * This work was supported by a Medical Research Council (MRC) Programme Grant (to G. F. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      The mRNA expression of lipogenic genes Scd-1 and Fas is regulated partly by the insulin-sensitive transcription factor SREBP-1c and liver X receptor α (LXRα). Compared with normal mice, the increase in the mRNA expression of hepatic Scd-1, Fas, and Srebp-1c was severely attenuated in peroxisome proliferator-activated receptor α (PPARα)-deficient mice during the transition from the starved to the re-fed states. The concentration of the membrane-bound form of SREBP-1c was also lower in the livers of the PPARα-deficient mice during re-feeding but there was little difference in the concentration of the active, nuclear form, or in the abundance of Insig-2a mRNA. The response of plasma insulin to starvation and re-feeding was normal in the PPARα-deficient mice. Rat hepatocytes transfected with an adenovirus encoding a dominant negative form of PPARα were resistant to the stimulatory effects of insulin on Fas and Scd-1 mRNA expression in vitro. When LXRα was activated in vivo by inclusion of a non-steroidal ligand in the diet, the expression of the mRNA for hepatic Srebp-1c, Fas, and Scd-1 was increased severalfold in mice of both genotypes and resistance associated with PPARα deficiency was abolished during re-feeding. However, although re-feeding the LXRα ligand induced the immature form of SREBP-1c equally in the livers of both genotypes, the concentration of the nuclear form remained relatively low in the livers of the PPARα-deficient mice. We conclude that intact PPARα is required to mediate the response of Scd-1 and Fas gene expression to insulin and that this is normally achieved directly by activation of LXRα.
      Insulin-mediated regulation of hepatic lipid metabolism is central to the efficient control of plasma lipid concentration and the avoidance of excessive lipemia. Insulin resistance and its associated hyperinsulinemia are of common occurrence in metabolic diseases such as obesity and Type 2 diabetes (
      • McGarry J.D.
      ), both of which are also associated with excessive hepatic synthesis of fatty acids and triacylglycerol (
      • Ginsberg H.N.
      • Zhang Y.L.
      • Hernandez-Ono A.
      ). Both of these pathways are insulin-dependent processes. Hepatic overproduction of these lipids reflects, both in experimental animals (
      • Shimomura I.
      • Matsuda M.
      • Hammer R.E.
      • Bashmakov Y.
      • Brown M.S.
      • Goldstein J.L.
      ) and in man (
      • Schwarz J.M.
      • Linfoot P.
      • Dare D.
      • Aghajanian K.
      ), a selective hepatic insulin resistance in which lipid synthesis is stimulated by the accompanying hyperinsulinemia, whereas hepatic glucose output is not suppressed. A “worst case” metabolic profile results in which hyperglycemia co-exists with hyperlipidemia (
      • Shimomura I.
      • Matsuda M.
      • Hammer R.E.
      • Bashmakov Y.
      • Brown M.S.
      • Goldstein J.L.
      ). The insulin-inducible transcription factor SREBP-1c (sterol regulatory element-binding protein-1c)
      The abbreviations used are: SREBP
      sterol regulatory element-binding protein
      PPARα
      peroxisome proliferator-activated receptor α
      LXRα
      liver X receptor α
      FAS
      fatty acid synthase
      SCD-1
      stearoyl-CoA desaturase 1
      ACO
      acyl-CoA oxidase
      ChREBP
      carbohydrate-responsive element-binding protein
      GFP
      green fluorescent protein
      GK
      glucokinase
      Adv
      adenovirus.
      2The abbreviations used are: SREBP
      sterol regulatory element-binding protein
      PPARα
      peroxisome proliferator-activated receptor α
      LXRα
      liver X receptor α
      FAS
      fatty acid synthase
      SCD-1
      stearoyl-CoA desaturase 1
      ACO
      acyl-CoA oxidase
      ChREBP
      carbohydrate-responsive element-binding protein
      GFP
      green fluorescent protein
      GK
      glucokinase
      Adv
      adenovirus.
      regulates the expression of a portfolio of glycolytic and lipogenic genes including fatty acid synthase (Fas) and stearoyl-CoA desaturase 1 (Scd-1) (
      • Liang G.
      • Yang J.
      • Horton J.D.
      • Hammer R.E.
      • Goldstein J.L.
      • Brown M.S.
      ,
      • Horton J.D.
      • Goldstein J.L.
      • Brown M.S.
      ). SREBP-1c thus forms an integral part of the mechanism by which insulin co-ordinates hepatic glucose and lipid metabolism. Evidence is also emerging that implicates another transcription factor, peroxisome proliferator-activated receptor α (PPARα), in the regulation of SREBP-1c and its target genes. During starvation, fatty acids, rather than carbohydrates, are the major sources of fuel for body tissues, and PPARα plays a major role in the up-regulation of genes encoding enzymes of fatty acid oxidation in the starved state (
      • Kersten S.
      • Seydoux J.
      • Peters J.M.
      • Gonzalez F.J.
      • Desvergne B.
      • Wahli W.
      ,
      • Ferre P.
      ).
      It has been shown that activation of PPARα induces transcription of Scd-1 (
      • Miller C.W.
      • Ntambi J.M.
      ) and other lipogenic genes (
      • Knight B.L.
      • Hebbachi A.
      • Hauton D.
      • Brown A.M.
      • Wiggins D.
      • Patel D.D.
      • Gibbons G.F.
      ). PPARα deficiency also abolishes the natural diurnal periodicity of Fas mRNA and de novo lipogenesis (
      • Patel D.D.
      • Knight B.L.
      • Wiggins D.
      • Humphreys S.M.
      • Gibbons G.F.
      ) in the fed state. These processes are normally insulin-dependent and the diurnal changes result from the periodicity of plasma insulin concentrations, which reflect the daily pattern of food intake. Although SREBP-1c forms part of the signaling mechanism by which insulin stimulates lipogenesis (
      • Foretz M.
      • Guichard C.
      • Ferre P.
      • Foufelle F.
      ,
      • Shimomura I.
      • Bashmakov Y.
      • Ikemoto S.
      • Horton J.D.
      • Brown M.S.
      • Goldstein J.L.
      ), details of the signaling pathway remain unclear. It appears, however, that insulin may act by enhancing the production of a natural ligand for another transcription factor, LXRα, which then activates transcription of Srebp-1c, one of its target genes (
      • Chen G.
      • Liang G.
      • Ou J.
      • Goldstein J.L.
      • Brown M.S.
      ). However, LXRα ligands activate lipogenic genes such as Fas and Scd-1 in mice lacking SREBP-1c, suggestive of direct LXRα-mediated activation in a non-SREBP-1c dependent manner (
      • Liang G.
      • Yang J.
      • Horton J.D.
      • Hammer R.E.
      • Goldstein J.L.
      • Brown M.S.
      ). LXRα has also been shown to interact directly with the Fas promoter (
      • Joseph S.B.
      • Laffitte B.A.
      • Patel P.H.
      • Watson M.A.
      • Matsukuma K.E.
      • Walczak R.
      • Collins J.L.
      • Osborne T.F.
      • Tontonoz P.
      ) and to directly activate the transcription of Chrebp (carbohydrate-responsive element-binding protein), another lipogenic transcription factor, in a non-SREBP-1c manner (
      • Cha J.Y.
      • Repa J.J.
      ). In the present work, we have sought to provide answers to three important questions regarding the role of PPARα in the insulin regulation of lipogenic gene expression. First, does PPARα deficiency interfere with the normal insulin-mediated up-regulation of lipogenic genes during the starved to re-fed transition? Second, if so, does this abnormality reflect defective insulin action resulting directly from the abolition of PPARα at the level of the hepatocyte? Finally, is PPARα involved in insulin signaling through LXRα and, if so, does this occur via SREBP-1c-dependent or non-SREBP-1c-dependent pathways?

      EXPERIMENTAL PROCEDURES

      Animals–PPARα-null mice bred onto a SV/129 genetic background were kindly provided by Dr. J. Peters and Dr. F. J. Gonzalez (National Institutes of Health, Bethesda, MD). Wild-type SV/129 mice were used as controls. All mice were male, and were used between the ages of 16 and 25 weeks. Animals were maintained in a temperature-controlled cabinet (22–24 °C) on a 12-h light/12-h dark cycle (lights on: 15.00 h) and were fed a commercial diet (Special Diet Services, Witham, Essex, UK) containing (by weight), 4.3% fat (0.02% cholesterol), 51.2% carbohydrate (mainly starch), 22.3% protein, and 7.7% ash. When required, the diet was supplemented with TO-901317 (0.01% w/w) obtained from Sigma. In some experiments, mice of both genotypes were starved for 24 h (food removed at 09.00 h) and were then either killed or re-fed with the normal diet or with the TO-901317-supplemented diet for periods of 6, 12, or 24 h. Mice were killed by cervical dislocation at the end of each re-feeding period. PPARα deficiency had no effect on food intake during either the dark phase or the light phase of the diurnal cycle (
      • Patel D.D.
      • Knight B.L.
      • Wiggins D.
      • Humphreys S.M.
      • Gibbons G.F.
      ), nor did dietary supplementation with TO-901317. Mean (± S.E.) values for food intake for 4 mice (g/24 h) were 16.8 ± 2.1 (wild-type fed chow), 14.9 ± 1.7 (wild-type fed TO-901317), 15.2 ± 2.0 (PPARα-deficient fed chow), and 17.0 ± 3.6 (PPARα-deficient fed TO-901317). All procedures performed in this study were approved by the Local Ethics Review Committee of the University of Oxford and were carried out under the appropriate Home Office (UK) personal and project licenses in accordance with the Home Office (UK) Animals (Scientific Procedures) Act 1986.
      mRNA Expression–Livers were freeze-clamped immediately after killing the mice and ground to a powder under liquid nitrogen. mRNA concentrations were determined by reverse transcription followed by real-time PCR (
      • Knight B.L.
      • Hebbachi A.
      • Hauton D.
      • Brown A.M.
      • Wiggins D.
      • Patel D.D.
      • Gibbons G.F.
      ) using an ABI PRISM Sequence Detection System (PE Applied Biosystems, Foster City, CA) or a Rotor-Gene 2000 Real Time Cycler (Corbett Research, Sydney, NSW, Australia). Primers and probes for mouse Srebp-1c, Scd-1, Fas, Aco, Lxrα, β-actin, and Insig-2a were prepared as described previously (
      • Patel D.D.
      • Knight B.L.
      • Wiggins D.
      • Humphreys S.M.
      • Gibbons G.F.
      ,
      • Trickett J.I.
      • Patel D.D.
      • Knight B.L.
      • Saggerson E.D.
      • Gibbons G.F.
      • Pease R.J.
      ) and for glucokinase and Chrebp as given by Dentin et al. (
      • Dentin R.
      • Pegorier J.-P.
      • Benhamed F.
      • Foufelle F.
      • Ferre P.
      • Faveau V.
      • Magnuson M.A.
      • Girard J.
      • Postic C.
      ). Reactions were carried out in triplicate in 30 μl of TaqMan Universal PCR Master Mix (
      • Patel D.D.
      • Knight B.L.
      • Wiggins D.
      • Humphreys S.M.
      • Gibbons G.F.
      ) with β-actin as internal standard. All values were related to a curve generated by a standard liver preparation and were corrected for β-actin mRNA content.
      Preparation of Liver Fractions Containing Nuclei and Microsomes–Liver microsomes were obtained using a modification of the method of Pawar et al. (
      • Pawar A.
      • Botolin D.
      • Mangelsdorf D.J.
      • Jump D.B.
      ). Briefly, livers were excised and most of the blood was removed by rapidly perfusing with ice-cold phosphate-buffered saline containing protease inhibitors (pepstatin, 5 μg/ml; leupeptin, 5 μg/ml; aprotinin, 2 μg/ml) via the vena cava. The perfused livers were placed in ice-cold 0.25 m sucrose solution containing protease inhibitors (
      • Dentin R.
      • Pegorier J.-P.
      • Benhamed F.
      • Foufelle F.
      • Ferre P.
      • Faveau V.
      • Magnuson M.A.
      • Girard J.
      • Postic C.
      ) and minced with scissors. The minced livers were homogenized in the same buffer (6 ml per 0.5 g) using a Potter-Elvehjem Teflon pestle. The homogenate was centrifuged at 500 × g for 5 min to obtain a crude pellet containing nuclei and a supernatant fraction. A fraction containing microsomal membranes was obtained by re-centrifuging the 500 × g supernatant at 105,000 × g for 1 h at 4 °C. The microsomal pellet was re-suspended in Hepes buffer pH 7.6 (
      • Azzout-Marniche D.
      • Becard D.
      • Guichard C.
      • Foretz M.
      • Ferre P.
      • Foufelle F.
      ). Nuclei were prepared from the 500 × g pellet obtained after the first centrifugation using the method of Gorski et al. (
      • Gorski K.
      • Carneiro M.
      • Schibler U.
      ). Briefly, the pellet was suspended in 2 m sucrose containing protease inhibitors (aprotinin, 2 μg/ml; leupeptin, 2 μg/ml; benzamide 1 mm; dithiothreitol, 1 mm; and phenylmethylsulfonyl fluoride, 1 mm) and layered over a 2 m sucrose cushion. The nuclear fraction was pelleted by centrifugation at 80,000 × g for 35 min at 4 °C. A nuclear extract containing mature SREBP-1c was obtained following lysis of the nuclear fraction and ammonium sulfate precipitation (
      • Gorski K.
      • Carneiro M.
      • Schibler U.
      ).
      Western Blotting–Samples containing 20 μg of protein from either the nuclear extract or the microsomal fraction were loaded onto a 12% polyacrylamide gel. Following electrophoresis and transfer to nitrocellulose paper, the resulting blots were probed with a mouse monoclonal anti-SREBP-1c antibody (IgG 2A4, Neomarkers, Fremont, CA). The second antibody was sheep anti-mouse IgG conjugated with horseradish peroxidase (Amersham Biosciences). SREBP-1c-containing bands were detected by ECL and visualized by radioautography. Band densities were determined by scanning with a Bio-Rad “Gel-Doc” scanner.
      Hepatocyte Transfection–Rat hepatocytes were prepared and cultured as described previously (
      • Bartlett S.M.
      • Gibbons G.F.
      ). After 3 h in culture, during which time the cells formed a monolayer, the medium was removed and the hepatocyes were transfected with adenovirus expressing either green fluorescent protein (Adv-GFP) or a dominant negative form of PPARα (Adv-null) (
      • Semple R.K.
      • Meirhaeghe A.
      • Vidal-Puig A.J.
      • Schwabe J.W.
      • Wiggins D.
      • Gibbons G.F.
      • Gurnell M.
      • Chatterjee V.K.
      • O'Rahilly S.
      ). Control dishes contained the adenoviral storage buffer only. After 3 h, the virus-containing medium was removed and replaced with virus-free medium containing either 100 nm of the PPARα agonist GW7647 (
      • Semple R.K.
      • Meirhaeghe A.
      • Vidal-Puig A.J.
      • Schwabe J.W.
      • Wiggins D.
      • Gibbons G.F.
      • Gurnell M.
      • Chatterjee V.K.
      • O'Rahilly S.
      ), or 78 nm insulin. In view of the extremely rapid degradation of insulin by hepatocytes, high initial concentrations were required to ensure that insulin levels approximated to physiological values during most of the incubation period (
      • Bourgeois C.S.
      • Wiggins D.
      • Hems R.
      • Gibbons G.F.
      ). Control culture dishes and those containing the PPARα agonist alone received a much lower concentration of insulin (0.1 nm). Dishes were incubated for 18 h, the cells were harvested, and the RNA was extracted using RNAzol (Tel-Test, Inc., Friendswood, TX). mRNA concentrations were determined using reverse transcriptase-PCR in an identical manner to that described above for whole liver samples.
      Other Methods–Plasma and tissue lipid concentrations were assayed as described previously (
      • Patel D.D.
      • Knight B.L.
      • Wiggins D.
      • Humphreys S.M.
      • Gibbons G.F.
      ). Liver microsomal SCD-1 activity was assayed as described by Kim et al. (
      • Kim H.J.
      • Miyazaki M.
      • Ntambi J.M.
      ). Tissue and subcellular fraction protein contents were measured by the method of Lowry and colleagues (
      • Lowry O.H.
      • Rosebrough N.J.
      • Farr A.L.
      • Randall R.J.
      ). Plasma glucose was determined enzymically in duplicate in neutralized perchloric acid extracts (
      • Bulow J.
      • Simonsen L.
      • Wiggins D.
      • Humphreys S.M.
      • Frayn K.N.
      • Powell D.
      • Gibbons G.F.
      ). Plasma insulin concentrations were measured in duplicate using a commercial mouse insulin ELISA enzyme immunoassay (Mercodia AB, Uppsala, Sweden).

      RESULTS

      Re-feeding following Starvation Fails to Restore Srebp-lc, Fas, and Scd-1 mRNA Expression in PPARα-deficient Mice–When normal mice were deprived of food for 24 h, the mRNA expression of hepatic Srebp-lc declined, as expected (
      • Horton J.D.
      • Goldstein J.L.
      • Brown M.S.
      ) to about 25% of that observed in the fully fed state (Fig. 1A). Re-feeding the starved animals for 6 h reversed this decline and expression was restored to that observed in the fully fed state after re-feeding for 24 h. The response of Srebp-lc mRNA expression to starvation and re-feeding in the livers of the PPARα-deficient mice differed markedly from that observed in the wild-type. First, in the PPARα-deficient mice fed ad libitum, the expression of Srebp-lc was somewhat lower than that in the wild-type (Fig. 1A) as observed previously (
      • Patel D.D.
      • Knight B.L.
      • Wiggins D.
      • Humphreys S.M.
      • Gibbons G.F.
      ). This differential expression was retained after 24 h starvation (Fig. 1A). The most striking feature, however, was the almost complete lack of response of Srebp-lc mRNA expression to re-feeding in the livers of the PPARα-null mice. The expression of Fas mRNA in each genotype also showed a pattern similar to that of Srebp-1c in response to starvation and re-feeding. Thus, in the PPARα-deficient mice the response of Fas mRNA to re-feeding was blunted compared with that in the wild-type (Fig. 1B). The response of Scd-1 mRNA was somewhat more complex (Fig. 1C) and, in the wild-type, showed an increase in expression after starvation followed by a decline after re-feeding for 6 h. Expression was restored to that observed in the fully fed state after a total of 24 h re-feeding. Nevertheless, similar to that observed for Srebp-1c and Fas mRNAs, Scd-1 mRNA expression was also highly resistant in the PPARα-deficient mice to the normal stimulatory effects of re-feeding for 24 h. Thus, after 24 h, hepatic Scd-1 mRNA concentration in this genotype was less than 20% of that observed in the animals fed ad libitum. There were no significant abnormalities in the insulin response to starvation and re-feeding in the PPARα-deficient mice (Fig. 1D).
      Figure thumbnail gr1
      FIGURE 1PPARα deficiency attenuates the normal response of lipogenic genes in the liver to re-feeding following starvation with little effect on plasma insulin or glucose levels. Mice were fed a standard, low-fat diet ad libitum and killed at the mid-point of the dark phase of the light/dark cycle. Other similar mice had food withdrawn at mid-dark and were fasted for 24 h. Some of these mice were then killed. Others were re-fed the standard diet for periods of 6, 12, and 24 h and were killed at the end of each time period. All livers were freeze-clamped and mRNA abundance (AC) was determined. SCD-1 activity was also measured in the microsomes of the same livers, portions of which were removed prior to freeze-clamping (F). Each point represents the mean ± S.E. of 6 mice fed ad libitum and 3 mice at all other points. D and E, animals were terminally anesthetized and blood samples were withdrawn from the descending vena cava. Each point represents the mean ± S.E. on plasma samples from 6 mice. In some cases the S.E. range lies within the area of the point. ○, wild-type mice; •, PPARα-deficient mice.
      Adenovirus-mediated Attenuation of PPARα Expression in Vitro Blunts the Response of Fas and Scd-1 mRNA to Insulin and to a PPARα Ligand–Rat hepatocytes in primary culture were transfected with adenoviral constructs encoding either a dominant negative form of PPARα, a constitutive transcriptional co-repressor (Adv-PPARα-null), or GFP (Adv-GFP) (
      • Semple R.K.
      • Meirhaeghe A.
      • Vidal-Puig A.J.
      • Schwabe J.W.
      • Wiggins D.
      • Gibbons G.F.
      • Gurnell M.
      • Chatterjee V.K.
      • O'Rahilly S.
      ). When compared with control hepatocytes that received the adenovirus storage buffer only, transfection with the Adv-GFP construct had no significant effect on the mRNA expression of any of the genes studied (see legend to Fig. 2). To establish if introduction of the dominant negative mutant was effective in attenuating PPARα activity, hepatocytes were treated with a potent PPARα ligand, GW7647, and induction of the mRNA for a known PPARα target gene, acyl-CoA oxidase, was determined. Fig. 2A shows that, although GW7647 increased the expression of Aco mRNA about 10-fold in hepatocytes transfected with the construct encoding GFP, this increase was almost totally abolished in the hepatocytes transfected with the construct containing the dominant negative form of PPARα. Treatment of the Adv-GFP hepatocytes with the PPARα ligand also led to a substantial induction of the mRNA encoding Fas, Scd-1, and Srebp-1c (Fig. 2), thus providing direct support in vitro for our observation that activation of PPARα enhances hepatic de novo lipogenesis and lipogenic gene expression in vivo (
      • Knight B.L.
      • Hebbachi A.
      • Hauton D.
      • Brown A.M.
      • Wiggins D.
      • Patel D.D.
      • Gibbons G.F.
      ). Induction of Scd-1, and Fas mRNAs by GW7647 was significantly attenuated in the Adv-PPARα-null hepatocytes (Fig. 2, B and C). As expected, insulin enhanced the mRNA expression of all the lipogenic genes in the hepatocytes treated with the Adv-GFP construct. However, the stimulatory effect of insulin on the expression of Fas and Scd-1 mRNA was greatly reduced in the Adv-PPARα-null hepatocytes (Fig. 2, B and C). In contrast the response of SREBP-1c to insulin was not significantly attenuated by repression of the Pparα gene (Fig. 2D).
      Figure thumbnail gr2
      FIGURE 2Inactivation of PPARα in vitro attenuates the stimulatory effects of a PPARα ligand and insulin on lipogenic gene expression. Hepatocytes from rats fed ad libitum were established in primary culture. Cells in some dishes were transfected with an adenovirus encoding a dominant-negative mutant of PPARα (Advnull) and others were transfected with an identical virus encoding GFP (Adv-GFP). After 3 h, the medium was removed and replaced with fresh medium containing 100 nm GW 7647 (black bars) or 78 nm insulin (gray bars). Other dishes received medium containing 0.1 nm insulin only (no additions). Cells were cultured for a further 18 h and mRNA concentrations were determined. Data are expressed as the change resulting from the addition of GW 7647 or of insulin asa%of that observed with 0.1 nm insulin alone (no additions). The basal levels (in the presence of 0.1 nm insulin only with no other additions) of Aco expression in the Adv-GFP and Adv-null hepatocytes were 1.10 ± 0.16 and 0.69 ± 0.12, respectively. Corresponding values for Fas were 1.38 ± 0.45 and 1.11 ± 0.31, for SCD-1 were 1.02 ± 0.07 and 1.17 ± 0.09, and for SREBP-1c were 0.85 ± 0.15 and 1.56 ± 0.26. Each point represents the mean ± S.E. of 3 independent hepatocyte preparations. Values in the columns marked with *, **, and *** are significantly different (p < 0.05, 0.01, and 0.001, respectively) from those obtained in dishes with no additions.
      PPARα Deficiency Affects Neither the Expression nor the Exogenous Activation of LXRα–Because the transcription of Srebp-lc, Fas, and Scd-1 is enhanced by activated LXRα (
      • Kalaany N.Y.
      • Gauthier K.C.
      • Zavacki A.M.
      • Mammen P.P.
      • Kitazume T.
      • Peterson J.A.
      • Horton J.D.
      • Garry D.J.
      • Bianco A.C.
      • Mangelsdorf D.J.
      ) it was of interest to determine whether the effects of genotype, starvation, and re-feeding on the expression of these genes were accompanied by changes in the expression of Lxrα mRNA. In the livers of mice fed ad libitum, PPARα deficiency did not affect Lxrα mRNA expression (Fig. 3). Starvation for 24 h increased expression significantly in the livers of the wild-type mice as noted previously and attributed to a fatty acid-mediated increase in LXRα transcription and mRNA stability (
      • Tobin K.A.
      • Ulven S.M.
      • Schuster G.U.
      • Steineger H.H.
      • Andresen S.M.
      • Gustafsson J.A.
      • Nebb H.I.
      ). Expression also increased, to a similar extent, in the livers of the PPARα-deficient mice, suggesting that the effect of fatty acids on Lxrα was not mediated by PPARα. Re-feeding for a period as short as 6 h was sufficient to reduce the expression of Lxrα mRNA to concentrations similar to those observed in the livers of mice fed ad libitum. Once again, PPARα deficiency had no effect on the extent of this decrease during the re-feeding period (Fig. 3).
      Figure thumbnail gr3
      FIGURE 3PPARα deficiency does not affect the response of Lxrα expression to starvation and re-feeding. Mice were treated in a manner identical to that described in the legend to . Results are expressed as the mean ± S.E. of 3 mice in each group. ○, wild-type mice; •, PPARα-deficient mice.
      To determine whether PPARα deficiency affected the activation of hepatic LXRα by exogenous ligands in vivo, we re-fed a diet containing the potent non-steroidal LXRα ligand TO-901317 (
      • Schultz J.R.
      • Tu H.
      • Luk A.
      • Repa J.J.
      • Medina J.C.
      • Li L.
      • Schwendner S.
      • Wang S.
      • Thoolen M.
      • Mangelsdorf D.J.
      • Lustig K.D.
      • Shan B.
      ) to mice of both genotypes following a 24-h period of starvation. The effects of this treatment on the mRNA expression of the LXRα target genes Fas, Scd-1, and Srebp-1c were determined (Fig. 4). These results show that the patterns of recoveries of mRNA expression during re-feeding under these conditions were indistinguishable in the livers of both the wild-type and PPARα-deficient mice, in marked contrast to those observed following re-feeding of the un-supplemented chow diet (Fig. 1).
      Figure thumbnail gr4
      FIGURE 4Re-feeding with an LXRα-ligand restores the response of lipogenic gene expression in PPARα-deficient mice. Mice were treated in an identical manner to that described in the legend to except that the diet contained 0.01% TO-901317. Results are expressed as the mean ± S.E. of 3 mice in each group. ○, wild-type mice; •, PPARα-deficient mice.
      Fig. 4 also shows that when the diet of the animals fed ad libitum was supplemented with TO-901317 the hepatic abundance of all the lipogenic mRNAs was increased 6–8-fold when compared with those on the un-supplemented diet (Fig. 1). This considerable increase probably resulted from the reinforcement of the direct stimulatory effect of LXRα activation (
      • Liang G.
      • Yang J.
      • Horton J.D.
      • Hammer R.E.
      • Goldstein J.L.
      • Brown M.S.
      ,
      • Joseph S.B.
      • Laffitte B.A.
      • Patel P.H.
      • Watson M.A.
      • Matsukuma K.E.
      • Walczak R.
      • Collins J.L.
      • Osborne T.F.
      • Tontonoz P.
      ,
      • Cha J.Y.
      • Repa J.J.
      ) by an indirect effect operating by stimulation of SREBP-1c (
      • Liang G.
      • Yang J.
      • Horton J.D.
      • Hammer R.E.
      • Goldstein J.L.
      • Brown M.S.
      ,
      • Horton J.D.
      • Goldstein J.L.
      • Brown M.S.
      ). LXRα activation also gave rise to other detailed changes in the response of Scd-1 and Fas to starvation and re-feeding. For instance, in the wild-type mice, Scd-1 mRNA expression decreased after starvation (Fig. 4C), in contrast to the increase observed after re-feeding with the un-supplemented diet (Fig. 1C). Furthermore, the “overshoot” of Fas mRNA expression observed after re-feeding the wild-type mice with the chow diet (Fig. 1B) was not observed when LXRα was activated in vivo during the re-feeding period. (Fig. 4B). These changes in lipogenic gene expression following activation of LXRα in vivo were not accompanied by any marked changes in the concentration of plasma glucose (Fig. 4E). However, plasma insulin concentrations were considerably decreased in wild-type mice but only marginally in the PPARα-deficient animals (compare Fig. 4D with Fig. 1D).
      PPARα Deficiency Decreases the mRNA Expression of Glucokinase (Gk) and Chrebp in the Fed and Re-fed States–In addition to their regulation by SREBP-1c, glucose directly regulates lipogenic and glycolytic gene function via changes in the expression of Chrebp (
      • Postic C.
      • Dentin R.
      • Denechaud P.D.
      • Girard J.
      ) in a manner that is dependent upon the presence of Gk (
      • Dentin R.
      • Pegorier J.-P.
      • Benhamed F.
      • Foufelle F.
      • Ferre P.
      • Faveau V.
      • Magnuson M.A.
      • Girard J.
      • Postic C.
      ). In view of recent evidence that Chrebp is regulated by LXRα (
      • Cha J.Y.
      • Repa J.J.
      ) in a glucose-dependent manner (
      • Mitro N.
      • Mak P.A.
      • Vargas L.
      • Godio C.
      • Hampton E.
      • Molteni V.
      • Kreusch A.
      • Saez E.
      ) we studied the effects of PPARα deficiency on Chrebp and Gk mRNA expression and their responses to LXRα activation. When mice were fed a normal diet, Gk expression declined during starvation and increased following re-feeding in the livers of the wild-type mice (Fig. 5A). In the PPARα-deficient mice, Gk expression was lower than that in the wild-type mice in the fed state but it neither decreased upon starvation nor increased following re-feeding (Fig. 5A). This pattern meant that Gk mRNA expression was considerably higher in the PPARα-deficient mice than in the wild-type in the starved state and suggested that glycolysis remained active following 24 h of starvation. The mRNA expression of Chrebp was also significantly lower in the livers of the knock-out mice in the re-fed state compared with that in the wild-type (Fig. 5C). However, Chrebp mRNA expression showed a consistent increase following starvation in the wild-type mice, which then declined following re-feeding. This pattern differs from that described previously (
      • Dentin R.
      • Pegorier J.-P.
      • Benhamed F.
      • Foufelle F.
      • Ferre P.
      • Faveau V.
      • Magnuson M.A.
      • Girard J.
      • Postic C.
      ), possibly because the diet used in the present work contained a higher concentration of fat and a lower proportion of carbohydrate than the high-carbohydrate, low-fat diet used in the previous study.
      Figure thumbnail gr5
      FIGURE 5Effect of starvation and re-feeding on Gk and Chrebp mRNA levels. Groups of mice were treated in a similar manner to that described in the legend to , using either the standard diet or the standard diet supplemented with 0.01% of TO-901317. There were no significant differences in the expression of Gk or Chrebp at any of the 6-, 12-, and 24-h re-feeding points in mice of either genotype, so these values have been combined in the re-fed columns. Gray columns correspond to wild-type mice and black columns to PPARα-deficient mice. Each value represents the mean ± S.E. of 3–8 mice in each group. Symbols * and ** indicate a significant (p < 0.05 and 0.01, respectively) effect of starvation, ‡ indicates a significant (p < 0.05) effect of re-feeding, and † indicates a significant difference (p < 0.05) between PPARα knock-out and wild-type animals.
      In the wild-type mice there was little, if any, stimulation of Gk mRNA expression resulting from activation of LXRα with dietary TO-901317 in the fed and re-fed states (Fig. 5B). Gk mRNA in the livers of the PPARα-deficient mice responded to a somewhat greater extent to dietary TO-901317 supplementation. Surprisingly, the greatest stimulatory effect of LXRα activation on Gk mRNA expression in both genotypes was observed following 24 h starvation (Fig. 5B), so that there was little effect of starvation or re-feeding on Gk mRNA abundance in mice of either genotype. Because there is no evidence that Gk is a direct LXRα target gene, any effect of LXRα activation must have arisen indirectly via SREBP-1c. Compared with Gk, Chrebp mRNA expression responded more vigorously to LXRα activation in the fed and re-fed states in both genotypes, so that there was a reversible reduction in Chrebp mRNA abundance upon starvation (Fig. 5D). There was no obvious difference in hepatic ChREBP mRNA abundance between genotypes in mice fed TO-901317 (Fig. 5D).
      Activation of LXRα Modifies the Effects of PPARα Deficiency on the Processing of SREBP-1c during Re-feeding–Western blotting of microsomal membranes from the livers of mice re-fed the chow diet showed a greater concentration of the immature form of SREBP-1c in the wild-type compared with the PPARα-deficient animals (Fig. 6A). This difference reflects the lower expression of Srebp-1c mRNA in the liver of the PPARα-deficient mice following re-feeding the chow diet (Fig. 1A). However, Western blotting of the nuclear extracts from the livers of each genotype (Fig. 6B) showed that these differences were not reflected in similar relative changes in the concentration of the active form of SREBP-1c. Although expression of mature SREBP-1c was significantly lower (p < 0.05) in the nuclei of the PPARα-deficient mice compared with the wild-type, this difference was relatively small in relation to the decrease in mRNA abundance. Li and colleagues (
      • Li Y.
      • Nara T.Y.
      • Nakamura M.T.
      ) have also shown that PPARα deficiency results in little or no difference in the nuclear abundance of SREBP-1c.
      Figure thumbnail gr6
      FIGURE 6Activation of LXRα modifies the effects of PPARα deficiency on SREBP-1c processing during re-feeding. Mice were starved for 24 h and either killed or re-fed the standard or the TO-901317-containing diet for 24 h. Livers were perfused with ice-cold phosphate-buffered saline (containing protease inhibitors), and microsomal membranes and nuclear extracts were obtained. Following SDS-PAGE and transfer to nitrocellulose, the blots were probed for SREBP-1c that was detected by ECL and radioautography. Band intensities were quantified by scanning. Each value represents the mean ± S.E. relative to wild-type (WT) re-fed chow values of between 2 and 6 different preparations. Columns marked with * are significantly different (p < 0.05) from the values for the wild-type re-fed chow. Knock-out (KO) (PPAR-null) columns marked with † are significantly different (p < 0.05) from the corresponding wild-type value.
      Insig-2a is a protein that prevents the activation of immature SREBP-1c (
      • Yabe D.
      • Komuro R.
      • Liang G.
      • Goldstein J.L.
      • Brown M.S.
      ,
      • Engelking L.J.
      • Kuriyama H.
      • Hammer R.E.
      • Horton J.D.
      • Brown M.S.
      • Goldstein J.L.
      • Liang G.
      ) and, consistent with the small decrease in the nuclear concentration of the active SREBP-1c in the PPARα-deficient mice, there was only a small but significant (p < 0.05) difference in the mRNA expression of Insig-2a in the livers of the two genotypes following re-feeding after a 24-h period of starvation (Fig. 7). However, starvation gave rise to a large increase in Insig-2a mRNA expression in the livers of both genotypes compared with that observed when the mice were fed ad libitum. As observed previously (
      • Yabe D.
      • Komuro R.
      • Liang G.
      • Goldstein J.L.
      • Brown M.S.
      ) the increase in Insig-2a correlated with the decreased processing of SREBP-1c in the starved state.
      Figure thumbnail gr7
      FIGURE 7Re-feeding following starvation rapidly suppresses the expression of Insig-2a mRNA in the livers of wild-type and PPARα-deficient mice. Mice were treated in an identical manner to that described in the legend to but were re-fed for only 6 h. Each column represents the mean ± S.E. of the livers from 6 mice. Open bars, wild-type mice; filled bars, PPARα-deficient mice. Values in the columns for the re-fed mice marked * are significantly different (p < 0.01) from those for the starved mice. Values in the columns marked † denote a significant effect (p < 0.05) of PPARα deficiency in the fed and re-fed states compared with the wild-type mice.
      When the non-steroidal LXRα ligand TO-901317 was included in the diet during the re-feeding period there was a large increase in the concentration of the microsomal, membrane-bound form of SREBP-1c compared with that observed following the re-feeding of chow only (Fig. 6A). In this case, however, there was no difference between the genotypes, reflecting the similarity in the expression of Srebp-1c mRNA in the livers of the two groups of mice after re-feeding with TO-901317 (Fig. 4A). However, blotting of the nuclear extracts from the mice fed the exogenous ligand showed a greater concentration of active SREBP-1c in the livers of the wild-type compared with the PPARα-deficient animals. Thus the processing of SREBP-1c during re-feeding with TO-901317 appeared to be retarded in the liver of the PPARα-deficient mice, compared with the wild-type, despite the fact that TO-901317 increased the concentration of the membrane-bound, immature form to the same extent in both genotypes.
      PPARα Deficiency Does Not Affect the Response of Plasma Glucose Concentration during Refeeding after Starvation–There was no difference in the concentration of plasma glucose between the wild-type and PPARα-deficient mice fed ad libitum (Fig. 1E). Fasting for 24 h gave rise to a significant decline in plasma glucose in the wild-type mice and to an even greater extent in the PPARα-deficient mice (Fig. 1E). This relative hypoglycemia in the PPARα-deficient genotype may reflect either a lack of ATP for gluconeogenesis because of defective fatty acid oxidation (
      • Kersten S.
      • Seydoux J.
      • Peters J.M.
      • Gonzalez F.J.
      • Desvergne B.
      • Wahli W.
      ), or a continued whole body reliance on glucose oxidation for energy that cannot be provided by utilization of fatty acids. The impaired up-regulation of PDK4 in the livers of starved PPARα-deficient mice (
      • Sugden M.C.
      • Bulmer K.
      • Gibbons G.F.
      • Knight B.L.
      • Holness M.J.
      ) and the relatively high expression of glucokinase mRNA in these animals (Fig. 6A) suggests that glycolysis remains active, providing some support for the latter explanation. Re-feeding for only 6 h following starvation restored plasma glucose to pre-starvation levels in mice of both genotypes. There was no hyperglycemia in the PPARα-deficient mice compared with the normal mice suggesting that insulin effectively suppressed hepatic gluconeogenesis and hepatic glucose output in both genotypes.

      DISCUSSION

      The results presented here show that, in the liver, PPARα deficiency affects the response of Srebp-1c, Fas, and Scd-1 mRNAs to re-feeding following starvation. The response pattern of the lipogenic genes in the PPARα-deficient mice differed in detail from each other. For instance, whereas re-feeding for 24 h restored Fas mRNA concentration to the pre-starved level in the PPARα-deficient mice, this was not the case for Srebp-1c or Scd-1. Again, Scd-1 mRNA did not increase whatsoever even after 24 h re-feeding. Clearly, feeding for much longer than 24 h is required to restore Srebp-1c and Scd-1 mRNAs to the pre-starvation levels. These distinctive patterns presumably reflect the complexities of regulation of these genes, each of which is subject to control by its own distinct set of regulatory factors. Nevertheless, a common feature of each of these mRNA species to re-feeding in the PPARα-deficient mice was the attenuated response compared with that observed in the wild-type, suggesting that PPARα is normally responsible for transmitting lipogenic signals that mediate the transition from the starved to the fed state.
      In the absence of PPARα, fasting results in hypoglycemia (
      • Kersten S.
      • Seydoux J.
      • Peters J.M.
      • Gonzalez F.J.
      • Desvergne B.
      • Wahli W.
      ) (Fig. 1E) and relative hepatic steatosis (Fig. 8B), which might induce a counter-regulatory response, thereby antagonizing and delaying the re-expression of Srebp-1c. However, normoglycemia is restored in the PPARα-deficient mice following re-feeding for 6 h (Fig. 1E), yet Srebp-1c expression is suppressed at this point and remains so for the next 18 h (Fig. 1A). Moreover, following re-feeding for 6 and 12 h, the relative steatosis was resolved in the PPARα-deficient mice (Fig. 8B) but lipogenic gene expression remained low. Thus there is no evidence that the suppressed response of hepatic lipogenic mRNAs to re-feeding in PPARα-deficient mice is related to fasting hypoglycemia or steatosis.
      Figure thumbnail gr8
      FIGURE 8Effect of starvation and re-feeding on plasma and hepatic lipid concentrations. Mice were treated in a manner identical to that described in the legend to . Plasma samples were assayed for non-esterified fatty acids (NEFA) and liver samples were assayed for triacylglycerol (TAG). Each point represents the mean ± S.E. of 3 mice in each group. ○, wild-type mice; •, PPARα-deficient mice.
      We have previously shown that Pparγ mRNA expression is increased 2-fold in the livers of PPARα-deficient mice fed a low-fat chow diet compared with that in wild-type mice (
      • Patel D.D.
      • Knight B.L.
      • Wiggins D.
      • Humphreys S.M.
      • Gibbons G.F.
      ). However, even this relatively elevated expression of Pparγ remained less than 5% of the concentration of hepatic PPARα in the intact mice. We do not consider that this small change in Pparγ expression would contribute significantly to the effects of PPARα deficiency that we have described.
      The attenuated response of lipogenic gene expression to re-feeding in the livers of the PPARα-deficient mice raised two immediate questions. First, is the decreased mRNA expression of Srebp-1c in the PPARα-deficient mice accompanied by a decreased post-translational processing to give the nuclear, active form and, if so, does this account for the decreased expression of its targets, Scd-1 and Fas? Second, are the different responses of the two genotypes to re-feeding a direct result of the differing PPARα status at the level of the hepatocyte and, if so does this reflect differences in the efficiency of insulin signaling during the re-feeding process? To address the first question, we compared the concentrations of the nuclear and microsomal forms of SREBP-1c in the livers of the two genotypes (Fig. 6). Although PPARα deficiency was associated with a decreased microsomal, inactive form, consistent with the decreased mRNA (Fig. 1A), this decline was not accompanied by a correspondingly large decrease in the concentration of the nuclear, active form. Previous work using isolated hepatocytes (
      • Hegarty B.D.
      • Bobard A.
      • Hainault I.
      • Ferre P.
      • Bossard P.
      • Foufelle F.
      ) has also shown that the expression of Srebp-1c mRNA can be uncoupled from its post-translational processing by activation of LXRα. Thus, irrespective of whether insulin is directly involved, it would appear unlikely that, despite the decreased abundance of Srebp-1c mRNA, the decreased Fas and Scd-1 mRNA expression during refeeding in PPARα-deficient mice resulted exclusively from a lack of active SREBP-1c.
      To answer the second question, PPARα expression was abolished in cultured hepatocytes in vitro by transfection of an adenoviral construct encoding a dominant negative form of PPARα (Adv-PPARα-null hepatocytes) (Fig. 2). Possible nonspecific effects of transcription were accounted for by comparing the response of hepatocyes transfected with a similar adenoviral construct encoding GFP. The mutant PPARα harbored twin substitutions in the ligand binding domain (
      • Semple R.K.
      • Meirhaeghe A.
      • Vidal-Puig A.J.
      • Schwabe J.W.
      • Wiggins D.
      • Gibbons G.F.
      • Gurnell M.
      • Chatterjee V.K.
      • O'Rahilly S.
      ) and repressed endogenous wild-type activity as shown in the present work by abolition of the normal response of Aco, a PPARα target gene, to the potent PPARα ligand, GW7647 (Fig. 2A). PPARα deficiency in these transfected hepatocytes also abolished the normal stimulatory effects of the GW7647 ligand on the mRNA expression of Scd-1, Fas, and Srebp-1c. To determine whether a lack of insulin response contributed to the insensitivity of lipogenic genes to re-feeding in the livers of the PPARα-deficient mice, the Adv-GFP and Adv-PPARα-null hepatocytes were challenged with insulin in vitro for 18 h. As expected, insulin stimulated the mRNA expression of all three lipogenic genes in the control and Adv-GFP hepatocytes. The response of Fas and Scd-1 mRNA to insulin was blunted in the Adv-PPARα-null hepatocytes suggesting that the lack of response of these genes to refeeding in the PPARα-deficient mice was due, at least in part, to a disturbance in insulin signaling. The response of Srebp-1c to insulin was not, however, attenuated significantly in the Adv-PPARα-null hepatocytes suggesting that the lack of a response in vivo resulted from a factor(s) other than defective insulin signaling. Although there is no evidence for such an action, we cannot entirely discount the possibility that the adenovirus nonspecifically interfered with signaling between PPARα and Srebp-1c.
      Insulin forms only a part of the complex integrated response responsible for transmitting information by which the body regulates the physiological transformation between the starved and fed states. Our experiments in vitro cannot, of course, completely rule out the possibility that during the transition between the starved and the fed states in vivo, PPARα deficiency compromises signaling by messengers other than insulin, such as counter-regulatory hormones like glucocorticoids and glucagon. As mentioned above, another possible factor is abnormal fatty acid metabolism, although we were unable to demonstrate any significant elevation of plasma NEFA in the PPARα-deficient compared with the wild-type mice after 6 h of re-feeding (Fig. 8A). Nevertheless, the possibility still remains that PPARα deficiency may also confer an insensitivity of lipogenic gene expression to the changes in lipid metabolism characteristic of the transition from the starved to the fed state.
      The mechanism by which defective PPARα may interfere with the insulin regulation of Scd-1 and Fas remains obscure. However, it has been shown recently that insulin is capable of activating LXRα, probably by increasing the endogenous provision of an activating LXRα ligand (
      • Chen G.
      • Liang G.
      • Ou J.
      • Goldstein J.L.
      • Brown M.S.
      ). Furthermore, in addition to their transcriptional activation by SREBP-1c, the transcription of both Scd-1 and Fas is mediated by activation of LXRα in a non-SREBP-1c-dependent manner (
      • Liang G.
      • Yang J.
      • Horton J.D.
      • Hammer R.E.
      • Goldstein J.L.
      • Brown M.S.
      ,
      • Joseph S.B.
      • Laffitte B.A.
      • Patel P.H.
      • Watson M.A.
      • Matsukuma K.E.
      • Walczak R.
      • Collins J.L.
      • Osborne T.F.
      • Tontonoz P.
      ,
      • Cha J.Y.
      • Repa J.J.
      ). It is possible, therefore, that at least in part, insulin normally regulates FAS and SCD-1 by ligand-mediated activation of LXRα independent of SREBP-1c. If this is the case, then the insulin-signaling pathway may be disturbed under conditions of PPARα deficiency by a lack of endogenous LXRα-ligand. To test this idea, we investigated whether the defects in insulin signaling in the PPARα-null mice could be rescued by providing excess of an exogenous LXRα ligand during the re-feeding process. Fig. 4 shows that when the potent LXRα ligand TO-901317 (
      • Schultz J.R.
      • Tu H.
      • Luk A.
      • Repa J.J.
      • Medina J.C.
      • Li L.
      • Schwendner S.
      • Wang S.
      • Thoolen M.
      • Mangelsdorf D.J.
      • Lustig K.D.
      • Shan B.
      ) was included in the diet during re-feeding after starvation, the responses of Fas and Scd-1 mRNA in the two genotypes were virtually superimposable. Thus the original PPARα dependence of insulin action was abolished under conditions in which the concentration of an LXRα ligand was not limiting. It appears, therefore, that PPARα may be required for the normal, insulin-dependent production of an endogenous LXRα ligand. The almost identical patterns of Lxrα mRNA expression in the livers of the wild-type and PPARα-deficient mice during the fed/fasted/re-fed transitions (Fig. 3) showed that differences in the mRNA expression of Lxrα itself were not responsible for the differing sensitivities of lipogenic gene expression. Fig. 3 also shows that, in the livers of both genotypes, Lxrα mRNA expression increased in starvation. This effect has also been shown previously in the livers of starved wild-type animals, and has been ascribed to a PPARα-dependent mechanism activated by the starvation-induced increase in fatty acids (
      • Tobin K.A.
      • Ulven S.M.
      • Schuster G.U.
      • Steineger H.H.
      • Andresen S.M.
      • Gustafsson J.A.
      • Nebb H.I.
      ). The present results suggest that, if fatty acids are involved in the increased LXRα mRNA expression, then this effect is independent of PPARα.
      Fig. 4 shows that the presence of TO-901317 during the re-feeding period also abolished the inhibitory effects of PPARα deficiency on the mRNA expression of Srebp-1c. Under these conditions, there was now no difference in Srebp-1c mRNA expression between the two genotypes. Consistent with this, the LXRα-ligand also increased the microsomal form of SREBP-1c to an equal extent in the livers of both genotypes (Fig. 6). Despite this, however, the concentration of the active, nuclear form of SREBP-1c was decreased in the PPARα-deficient mice, compared with the wild-type. Activation of LXRα has been shown to increase Srebp-1c transcription but decrease processing in hepatocytes from normal rats (
      • Hegarty B.D.
      • Bobard A.
      • Hainault I.
      • Ferre P.
      • Bossard P.
      • Foufelle F.
      ). Under these conditions, processing was restored by insulin. In the present work, PPARα deficiency may, therefore, interfere with the insulin-dependent processing of SREBP-1c in the presence of high concentrations of an LXRα-ligand. The relative lack of nuclear SREBP-1c in the livers of the TO-901317-treated PPARα-deficient mice makes it unlikely that the increased Fas and Scd-1 mRNA expression occurs exclusively through the SREBP-1c pathway. In all likelihood, expression of these genes is increased by direct LXRα-mediated activation of Fas and Scd-1, or by their activation by Chrebp, a recently discovered LXRα target (
      • Cha J.Y.
      • Repa J.J.
      ). Consistent with this, in the present work, Chrebp mRNA expression increased by ∼3-fold in the fed state when LXRα was activated in vivo by TO-901317. When mice were fed the normal, un-supplemented diet, Chrebp mRNA expression was decreased by PPARα deficiency in all nutritional states, similar to that observed with Srebp-1c, Fas, and Scd-1. However, activation of LXRα in vivo was unable to completely restore Chrebp mRNA expression during the re-feeding period although the difference between the wild-type and PPARα-deficient mice was no longer significant (Fig. 5).
      The role of PPARα in the development of insulin resistance is controversial. On the one hand, stimulation of PPARα activity by administration of potent ligands such as WY 14,643 in animal models of obesity has been reported to decrease hepatic triacylglycerol concentrations and increase glucose tolerance (
      • Chou C.J.
      • Haluzik M.
      • Gregory C.
      • Dietz K.R.
      • Vinson C.
      • Gavrilova O.
      • Reitman M.L.
      ). PPARα is also required to mediate the lipid-lowering effects of hyperleptinemia in liver and adipose tissue (
      • Lee Y.
      • Yu X.
      • Gonzales F.
      • Mangelsdorf D.J.
      • Wang M.Y.
      • Richardson C.
      • Witters L.A.
      • Unger R.H.
      ). On the other hand, PPARα activation gives rise to an increase in the expression of TRB-3 mRNA, which encodes a protein that decreases insulin sensitivity by inhibition of insulin signaling via Akt/PKB (
      • Koo S.H.
      • Satoh H.
      • Herzig S.
      • Lee C.H.
      • Hedrick S.
      • Kulkarni R.
      • Evans R.M.
      • Olefsky J.
      • Montminy M.
      ). PPARα deficiency has also been reported to protect against insulin resistance induced by fat feeding (
      • Guerre Millo M.
      • Rouault C.
      • Poulain P.
      • Andre J.
      • Poitout V.
      • Peters J.M.
      • Gonzalez F.J.
      • Fruchart J.C.
      • Reach G.
      • Staels B.
      ), although this conclusion has recently been challenged (
      • Patsouris D.
      • Reddy J.K.
      • Muller M.
      • Kersten S.
      ). Finally, although the present work has concentrated on the effects of PPARα deficiency on lipogenic gene mRNA expression, it should be recognized that the overall lipogenic response to re-feeding following starvation occurs at other levels of regulation, particularly those that involve post-translational effects. These, often more rapid responses, can contribute to the overall changes in whole body metabolism. However, we have studied the enzymic activity of SCD-1, which can be rapidly regulated at the level of protein stability (
      • Mziaut H.
      • Korza G.
      • Ozols J.
      ), and found that the patterns of change in activity in the livers of both genotypes closely paralled those of Scd-1 mRNA expression in all nutritional states (Fig. 1F), as did those of SCD-1 protein expression as measured by Western blotting (results not shown).
      In conclusion, therefore, it appears that intact PPARα forms part of the signaling mechanism by which insulin activates the LXRα-mediated transcription of Fas and Scd-1 during re-feeding. That an exogenous non-steroidal ligand of LXRα is effective in rescuing PPARα deficiency suggests that intact PPARα is somehow involved in the generation of an endogenous LXRα ligand. Why a transcription factor, whose primary role is the coordination of fatty acid oxidation in the starved state, is required to facilitate fatty acid synthesis during the starved/fed transition is not clear. Nevertheless, it has been shown previously that PPARα activation stimulates the transcription of Scd-1 (
      • Miller C.W.
      • Ntambi J.M.
      ) and other lipogenic genes (
      • Knight B.L.
      • Hebbachi A.
      • Hauton D.
      • Brown A.M.
      • Wiggins D.
      • Patel D.D.
      • Gibbons G.F.
      ) and it was suggested that the physiological purpose of this response is to compensate for the oxidative removal of structurally important fatty acids during the previous period of starvation (
      • Knight B.L.
      • Hebbachi A.
      • Hauton D.
      • Brown A.M.
      • Wiggins D.
      • Patel D.D.
      • Gibbons G.F.
      ). This interpretation was supported by the observation that abolition of PPARα-mediated fatty acid oxidation during the period of low food intake in the diurnal cycle also abolished the insulin-mediated increase in lipogenic gene expression and lipogenic flux in the liver when food intake increased naturally during the diurnal cycle in vivo (
      • Patel D.D.
      • Knight B.L.
      • Wiggins D.
      • Humphreys S.M.
      • Gibbons G.F.
      ). Interestingly, a recent report has documented a role for newly synthesized fatty acids in the activation of PPARα (
      • Chakravarthy M.V.
      • Pan P.
      • Zhu Y.
      • Tordjman K.
      • Schneider J.G.
      • Coleman T.
      • Turk J.
      • Semenkovich C.F.
      ). Such a role, together with the present results, provides evidence for a symbiotic interdependence of PPARα signaling and newly synthesized fatty acids in the coordinated regulation of hepatic lipid metabolism. We suggest that the physiological purpose of this relationship is to ensure the correct balance between de novo lipogenesis and fatty acid oxidation in the liver, consistent with the energy requirements when food intake of the animal fluctuates over a period of time (
      • Gibbons G.
      ).

      Acknowledgments

      We thank Robert Semple and Antonio Vidal-Puig, Department of Clinical Biochemistry, University of Cambridge, for samples of adenoviruses encoding GFP and mutated PPARα. Dr. J. Peters and Dr. F. W. Gonzalez kindly provided mating pairs of PPARα-deficient mice. Bronwyn Hegarty, MRC Cellular Stress Group, Imperial College School of Medicine, provided invaluable practical help and advice with Western blotting of SREBP-1c.

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