Endocrinology, doi:10.1210/en.2004-0099
Endocrinology Vol. 145, No. 9 4268-4277
Copyright © 2004 by The Endocrine Society
The Developmental Regulation of Peroxisome Proliferator-Activated Receptor-
Coactivator-1
Expression in the Liver Is Partially Dissociated from the Control of Gluconeogenesis and Lipid Catabolism
Pilar Yubero,
Elayne Hondares,
M. Carmen Carmona,
Meritxell Rossell,
Frank J. Gonzalez,
Roser Iglesias,
Marta Giralt and
Francesc Villarroya
Departament de Bioquímica i Biología Molecular, Universitat de Barcelona (P.Y., E.H., M.C.C., M.R., R.I., M.G., F.V.), 08028 Barcelona, Spain; and Laboratory of Metabolism, National Cancer Institute (F.J.G.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Francesc Villarroya, Departament de Bioquímica i Biología Molecular, Universitat de Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain. E-mail: gombau{at}porthos.bio.ub.es.
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Abstract
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The developmental regulation of peroxisome proliferator-activated receptor-
coactivator-1
(PGC-1) gene expression was studied in mice and compared with that of marker genes of liver energy metabolism. The PGC-1 gene was highly expressed in fetal liver compared with that in adults and remained high in neonatal liver. The regulation of PGC-1 gene expression during the fetal and early neonatal periods was dissociated from that of gluconeogenic genes, i.e. the phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. Only under the effects of starvation was PGC-1 gene expression induced in parallel to phosphoenolpyruvate carboxykinase and G6Pase mRNAs during the perinatal period. Furthermore, the PGC-1 gene was not regulated as part of the developmental program of gene expression associated with the maturation of hepatic gluconeogenesis, as revealed by the impaired PEPCK and G6Pase gene expression but unaltered PGC-1 mRNA levels in CCAAT/enhancer-binding protein--null fetus and neonates. Regulation of the PGC-1 gene and that of mitochondrial 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase, acyl-coenzyme A oxidase, and long-chain acyl-coenzyme dehydrogenase, marker genes of lipid catabolism, were dissociated in fetuses and neonates. The expression of lipid catabolism genes was down-regulated in fasted neonates, whereas PGC-1 was oppositely regulated. The independent regulation of PGC-1 and lipid catabolism genes was also found in peroxisome proliferator-activated receptor--null neonates, in which PGC-1 mRNA levels were unaffected whereas gene expression for 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase and acyl-coenzyme A oxidase was impaired. Thus, regulation of the PGC-1 gene is partially dissociated from the patterns of regulation of hepatic genes encoding enzymes involved in gluconeogenesis and lipid catabolism during fetal ontogeny and in response to the initiation of lactation.
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Introduction
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PEROXISOME PROLIFERATOR-ACTIVATED receptor- coactivator-1 (PGC-1) is a transcriptional coactivator involved in the control of biological responses linked to energy homeostasis in several tissues. It participates in determination of the divergence between white adipocytes (energy storage function) and brown adipocytes (energy expenditure), regulates mitochondrial biogenesis in skeletal muscle, and has recently been reported to play a major role in the control of fuel homeostasis in the liver (1). The action of PGC-1 as a master regulator on liver energy metabolism was revealed by its role in the metabolic response to fasting, where it is induced and leads to a coordinate induction of genes involved in hepatic gluconeogenesis. PGC-1 coactivates transcription factors and nuclear hormone receptors that control the transcription of genes encoding enzymes involved in this metabolic pathway. PGC-1 activates some gluconeogenic genes via its capacity to coactivate hepatic nuclear factor-4 (HNF4) and the glucocorticoid receptor (2, 3). Moreover, PGC-1 requires the forkhead transcription factor FOXO1 to activate the gluconeogenic genes in a pathway that is also critical for the inhibitory regulation of gluconeogenesis by insulin (4). It was proposed that the role of PGC-1 in the metabolic adaptation of the liver to fasting extends to lipid oxidation pathways, because PGC-1 can induce several key genes encoding enzymes of fatty acid oxidation and ketogenesis when overexpressed in hepatic cells (3). The capacity to coactivate peroxisome proliferator-activated receptor- (PPAR) (5), a master regulator of lipid catabolism, is expected to mediate the effect of PGC-1 to induce those genes in the liver in response to fasting, although direct evidence for this is still lacking.
The induction of gene expression by PGC-1 in response to fasting is thought to be mediated by a dramatic increase in the amount of this coactivator. This is due to the sensitivity of PGC-1 gene expression up-regulation through cAMP-dependent pathways, which are activated by glucagon levels, which rise during fasting (6). Glucocorticoids, which are up-regulated by starvation, also induce PGC-1 gene expression (2). Although less studied than fasting, other models of enhanced hepatic gluconeogenesis, such as streptozotocin-induced diabetes and genetic obesity, have confirmed several aspects of the involvement of PGC-1 in the control of gluconeogenesis and lipid catabolic pathways in the liver (2).
During rodent development, the activation of gluconeogenic and lipid catabolic pathways, including ketogenesis, takes place at birth. Fuel consumption by fetuses is mainly based on glucose, but birth leads to a sudden decrease in glucose availability associated with the massive appearance of lipids in blood, coming from milk. Thus, the metabolic adaptation to birth requires the induction of gluconeogenesis to maintain glycemia and the activation of lipid oxidation to use this fuel for energy metabolism (7). Accordingly, genes involved in these metabolic pathways are poorly expressed in fetal liver, but are dramatically induced at birth (8, 9). This metabolic and gene expression response, which resembles that elicited by fasting in adults, is induced in neonates by the fed state, because it results from the sudden imbalance between glucose and lipid availability in the transition from the fetal to the neonatal period. Moreover, the induction of metabolic genes in the liver requires the appropriate development of the prenatal program of liver differentiation. Thus, the whole basal transcriptional machinery that allows gene expression to respond to postnatal induction should develop in the liver during the fetal period by an ontogenically programmed mechanism and in the absence of the hormonal stimuli associated with birth. For instance, mice with targeted disruption of CCAAT/enhancer-binding protein- (C/EBP), a master transcription factor for the differentiation of hepatic cells, show impaired expression of gluconeogenic-associated genes, such as phosphoenolpyruvate carboxykinase (PEPCK), in such a way that this gene cannot be induced after birth, and gluconeogenesis cannot be switched on (10).
In the present study the developmental regulation of PGC-1 gene expression in the liver was determined in relation to regulation of the expression of genes involved in liver metabolism and was studied in murine models of targeted disruption of master transcription factors controlling gluconeogenic (C/EBP) or lipid oxidative pathways (PPAR). We found partial dissociation between gluconeogenic and lipid oxidation pathways and PGC-1 gene expression during the perinatal period.
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Materials and Methods
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The care and use of mice were in accordance with European Community Council Directive 86/609/EEC and were approved by the comitè ètic d’experimentació animal of University of Barcelona. Female Swiss mice were mated with adult males, and the day of gestation was determined by the presence of vaginal plugs (d 0). For studies in fetuses, cesarean sections of pregnant mice were performed on d 17, 18, and 19 of gestation. For studies in neonates, pups were studied at birth (considered to be the time at which pups had been born, but had not yet started suckling) and 8, 16, and 24 h after birth. When the effects of fasting were determined, fetuses at the indicated ages were obtained by cesarean section and placed in a humidified thermostable chamber (30 C) for 16 h. Identical procedures were followed with neonates born spontaneously, which were separated from their mothers for 16 h before initiating suckling. Two-day-old pups were fasted by separating them from lactating dams for 24 h, and fasting in adult (3 month old) mice was achieved by withdrawal of food pellets for 24 h. When indicated, pups not allowed to suckle were given 100 µl triacylglycerol emulsion (Intralipid 3.5%, Amersham Pharmacia Biotech, Piscataway, NJ) or 100 µl of a 0.1 g/ml glucose solution by intragastric gavage. Postmature fetuses were obtained on embryonic d 21 by cesarean sections of pregnant mice that had been treated daily with 3.5 mg progesterone (35 mg/ml castor oil solution) from d 16.5 of pregnancy. Controls were established by injecting pregnant mice with equal volumes of castor oil. For studies in C/EBP-null mice, heterozygous female mice carrying the targeted deletion (10) were mated with heterozygous males. The day of pregnancy was determined as described above, and fetuses were studied on d 17 and 18 of gestation. Neonates were studied 2–4 h after birth, as described previously (11), and were established to have not initiated suckling by inspection of the stomach contents. For studies in PPAR-null mice, heterozygous females carrying the corresponding targeted deletion (12) were mated with heterozygous males. Pups were studied at birth and 16 h after birth in both the fed (allowed to suckle) and fasted (not allowed to suckle) groups. Mice were killed by decapitation, and livers were dissected and frozen in liquid nitrogen. Interscapular brown adipose tissue and whole muscle from the leg were also extracted for comparative purposes. Control and treated animals as well as wild-type animals, compared with homozygous gene-disrupted mice, were taken from the same litter in each experiment, and at least three different litters per experiment were analyzed.
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA). For Northern blot analysis, 18 µg total RNA were denatured, electrophoresed on 1.5% formaldehyde-agarose gels, and transferred to positively charged nylon membranes (N+, Roche, Indianapolis, IN). Equal loading of gels was checked by ethidium bromide staining. Prehybridization and hybridization were carried out using standard procedures (11). Blots were stripped and rehybridized sequentially, as required in each case. The probes used were the murine cDNA for PGC-1 (13) and the rat cDNAs for PEPCK (14), glucose-6-phosphatase (G6Pase) (10), mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase (HMG-CoA synthase) (9), peroxisomal acyl-coenzyme A oxidase (ACO) (15), long-chain acyl-coenzyme A dehydrogenase (LCAD; ATCC 225775, American Type Culture Collection, Manassas, VA), and the mitochondrial genome-encoded subunit II of cytochrome c oxidase (COII) (16). Autoradiographs were quantified by densitometric analysis (Phoretics, Millipore Corp., Bedford, MA). After individual densitometric analysis of the 6.5- and 5.0-kb transcript signals for PGC-1, no differential changes in their expression were detected in any of the experimental situations studied, and both transcripts were modified in parallel in response to the developmental, nutritional, and genetic modifications studied. Therefore, data are expressed by integrating densitometry signals for both transcripts. The same procedure was followed with LCAD mRNA expression, showing two (2.2 and 1.8 kb) transcripts.
Immunoblot analysis of PGC-1 was performed as reported previously (2). Liver extracts were prepared by homogenization in a buffer containing 100 mM Tris (pH 8.5), 250 mM NaCl, 1% Igepal CA-630 (Sigma-Aldrich Corp., St. Louis, MO), 1 mM EDTA, a mixture of protease inhibitors (Complete-Mini Protease Inhibitor Cocktail, Roche), and 0.1% phenylmethylsulfonyl fluoride. Proteins (100 µg /lane) were separated by 10% SDS-PAGE, transferred to Immobilon-P membranes (Millipore Corp.), and probed with an antibody directed against murine PGC-1 (a gift from B. Spiegelman). As a control, protein extracts of HEK-293 cells infected with an adenovirus driving murine PGC-1 (8 µg/ lane) were run in parallel. Gel loading was checked by immunoblotting using a ß-actin antibody (Sigma-Aldrich Corp.; clone AC-15).
Where appropriate, statistical analysis was performed by t test. Significance is indicated in the text.
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Results
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Tissue-specific developmental regulation of PGC-1 gene expression
PGC-1 is highly expressed in the fetal liver. The expression of PGC-1 mRNA was determined during the fetal development of mice in the tissues known to express this coactivator in adults. The pattern of expression in fetuses compared with adults was highly specific for every tissue, as shown in Fig. 1
. In skeletal muscle, the expression of PGC-1 mRNA was hardly detectable at various stages of the fetal period, whereas it was expressed in adults. In contrast, late fetal brown adipose tissue already expressed relatively high levels of PGC-1 mRNA, close to the levels present in adults. Surprisingly, fetal liver expressed much higher levels of PGC-1 mRNA than adult liver, in which PGC-1 was hardly detectable. This high expression of PGC-1 mRNA resulted in the expression of PGC-1 protein, as shown by Western blot analysis of nuclear extracts from fetal and neonatal liver (Fig. 1C), whereas PGC-1 protein was practically undetectable in adult liver.
Hepatic PGC-1 mRNA expression is dissociated from the regulation of metabolic genes in the immediate postnatal period
The expression levels of PGC-1 mRNA were determined in the transition from the fetal to the neonatal period and compared with the regulation of expression of key genes associated with gluconeogenesis (PEPCK and G6Pase), genes associated with lipid catabolism (mitochondrial HMG-CoA synthase, which is responsible for ketogenesis; ACO, which is involved in peroxisomal fatty acid oxidation; and LCAD, which is involved in mitochondrial fatty acid ß-oxidation) and a marker of mitochondrial biogenesis (the mitochondrial genome-encoded COII). PGC-1 mRNA expression hardly varied during the first hours after birth, and PGC-1 mRNA levels remained as high as in fetuses (Fig. 2). In contrast, the expression of mRNAs encoding PEPCK and G6Pase as well as that of the three gene markers of lipid catabolism, HMG-CoA synthase, ACO, and LCAD, were high during the immediate postnatal period compared with those in fetal life. COII mRNA expression showed a slight increase during fetal life and remained almost constant during the perinatal period. When birth was blocked by progesterone treatment of pregnant mice, and postmature fetuses were analyzed, the induction of gene markers for gluconeogenesis and lipid catabolism pathways was partially blunted, whereas PGC-1 mRNA and COII mRNA expression did not vary (see Table 1).
Hepatic PGC-1 mRNA expression is up-regulated during fasting in the perinatal period
To ascertain whether PGC-1 mRNA expression in the perinatal period is sensitive to the known physiological stimuli acting in adults, fetuses and neonates were studied under the stimulus of starvation. Hepatic PGC-1 mRNA was dramatically up-regulated after 24-h starvation in adult mice (Fig. 3), in agreement with a previous report (2). When fetuses at term or even immature fetuses were born by cesarean sections and kept for several hours without milk intake, the expression of PGC-1 mRNA was up-regulated despite the already high basal levels of expression. We obtained the same results in neonates born spontaneously and not allowed to initiate suckling as well as in 2-d-old neonates that had been suckling and were suddenly withdrawn from their mothers (Fig. 3). This response was similar to that of gluconeogenic genes, such as PEPCK and G6Pase, but was the opposite of the behavior of gene markers for lipid catabolism. Whereas the mRNA levels of HMG-CoA synthase, ACO, and LCAD were induced by starvation in adults, the expression of these three mRNAs in the liver was down-regulated in response to fasting during the perinatal period (Fig. 3). COII mRNA levels were not significantly modulated by starvation in fetuses, neonates, or adults.
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FIG. 3. Effects of fasting at various stages of development on PGC-1 gene expression in the liver. Representation of the relative abundance of the mRNAs for PGC-1 (A); the gluconeogenic genes, PEPCK and G6Pase (B); the lipid catabolism genes, HMG-CoA synthase, ACO, and LCAD (C); and the mitochondrial genome-encoded gene COII (D) in fetuses at d 18 (F18) and d 19 (F19) of embryonic life, neonates at birth (N0), 2-d-old neonates (N2), and adults in response to fasting. Bars are the mean ± SEM of the hybridization intensity signals of at least three samples. Data are expressed as a percentage of maximum expression, which was set at 100. ND, Nondetectable. *, Significant differences (P  0.05) between fed and fasted mice.
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We also determined to what extent the changes in gene expression in the liver of neonates due to starvation are specifically blunted by carbohydrates or lipids (Table 2). Pups at birth were withdrawn from their mothers before initiating suckling and were intragastrically infused with equivalent caloric amounts of a glucose solution or a lipid emulsion. PGC-1 mRNA induction due to starvation was partially reduced by both glucose and lipid administration. The metabolic genes responded differentially. PEPCK mRNA and G6Pase mRNA induction by fasting was reduced by both treatments, whereas the down-regulation of HMG-CoA synthase mRNA and ACO mRNA and, to a lesser extent, LCAD mRNA, was specifically attenuated by lipid intake. The model employed for food intake depletion in cesarean-derived fetal and newborn mice also involved fluid restriction, and this may add stress to the strict nutrition depletion, which can contribute to changes in gene expression. However, the differential effects of nutrients on gene expression suggest that fluid restriction, even if potentially involved, is not the main element determining changes in gene expression in this model.
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TABLE 2. Effects of glucose or lipid intake on the hepatic expression of PGC-1 mRNA and metabolic genes in neonatal mice
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C/EBP-null mice show impaired expression of hepatic gluconeogenic genes, but unaltered PGC-1 gene expression during the perinatal period
Because C/EBP is a master regulator of liver differentiation and, in particular, of gluconeogenic gene expression in the liver, we determined the effects of C/EBP disruption in mice on PGC-1 gene expression (Fig. 4). Given the postnatal mortality of C/EBP-null mice (10), only fetuses at several stages of fetal development and recently born neonates were studied. The data indicated that PEPCK mRNA expression, which is only detectable by Northern blotting in the late fetal and neonatal periods of wild-type mice, was blunted in C/EBP-null mice, and the same was observed for G6Pase mRNA, which was dramatically reduced in fetal and neonatal C/EBP-null mice. These observations were in agreement with the already established impairment of gluconeogenic gene expression in these mice (10). PGC-1 mRNA expression was unaffected by C/EBP-targeted disruption. Neither HMG-CoA synthase mRNA levels nor LCAD mRNA levels were significantly altered in C/EBP-null mice. However, the expression of ACO mRNA was significantly reduced in C/EBP-null fetuses and neonates, suggesting that C/EBP may be involved in developmental regulation of gene expression for peroxisomal enzymes. COII mRNA expression was also unchanged in liver from C/EBP-null mice.
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FIG. 4. PGC-1 gene expression in liver of fetal and neonatal mice with C/EBP gene-targeted disruption. Representation of the relative abundance of the mRNAs for PGC-1 (A); the gluconeogenic genes, PEPCK and G6Pase (B); the lipid catabolism genes, HMG-CoA synthase, ACO, and LCAD (C); and the mitochondrial genome-encoded gene, COII (D). Bars are the mean ± SEM of the hybridization intensity signals of at least three samples. N, Neonate. Data are expressed as a percentage of maximum expression, which was set at 100. ND, Nondetectable. *, Significant differences (P 0.05) of 18-d-old fetuses and neonates compared with 17-d fetuses; #, significant differences between wild-type and C/EBP-disrupted mice for every developmental stage.
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PPAR disruption impairs hepatic expression of genes of lipid catabolism during the perinatal period without altering PGC-1 mRNA expression
To further explore the link between PGC-1 mRNA expression and lipid catabolism in perinatal liver, neonatal mice with targeted disruption of PPAR were studied (Fig. 5). The expression of PPAR mRNA in control mice was high at birth, as previously reported (8), and was not essentially affected in the first hours after birth in either the 16-h fed (135 ± 14% vs. 0-h-old pups) or 16-h fasted (98 ± 8% vs. 0-h-old pups) condition. Disruption of PPAR caused a marked down-regulation of mitochondrial HMG-CoA synthase mRNA and ACO mRNA expression in pups at birth, after 16 h of suckling, and after 16 h of postnatal starvation. LCAD mRNA was not significant reduced, in agreement with previous findings on the expression of this gene in adult PPAR-null mice (17). This confirmed that PPAR acts a master regulator of several genes of mitochondrial and peroxisomal lipid catabolism after birth and can contribute to the switch-on of lipid oxidation pathways during development. PEPCK mRNA, G6Pase mRNA, and COII mRNA were essentially unaffected, as was PGC-1 mRNA, pointing to a dissociation between PGC-1 gene expression and the PPAR-regulated pathways of gene expression in the liver during this period.
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Discussion
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Our results indicate that PGC-1 gene expression is under powerful tissue-specific developmental regulation. The high levels of PGC-1 mRNA expression in fetal brown adipose tissue are consistent with the postulated role for this coactivator in brown fat differentiation and the fact that brown adipocyte differentiation in vivo takes place during the late fetal period in rodents (11, 18). Moreover, the progressive induction of PGC-1 mRNA in skeletal muscle, with very low levels during the fetal period that become much higher in adults, is also in agreement with a major role for PGC-1 in this tissue, i.e. the control of mitochondrial biogenesis (19), which develops postnatally in mice (20). In contrast, the high expression of PGC-1 in fetal liver was a surprising finding. The up-regulation of PGC-1 in liver has been attributed to cAMP-dependent activation due to the action of glucagon or the effects of glucocorticoids (2, 6). These mechanisms are responsible for the high levels of expression of PGC-1 in the liver of fasted adults, but they can hardly be involved in the high expression in the fetal liver, when stress signaling elicited by birth and mediated by glucagon or glucocorticoids is still not active. However, the PGC-1 gene in late fetal development appears to be already sensitive to those pathways when they are suddenly activated, as revealed by the up-regulation elicited by delivery plus starvation even in immature fetuses. This is in agreement with the reported capacity of fetal liver to activate gluconeogenesis in response to glucocorticoid infusion (21).
A second aspect to be considered is how the high PGC-1 expression in the fetus is compatible with the low expression of gluconeogenic genes. Two facts should be taken into account. First, fetuses are hyperinsulinemic (7), and insulin is known to behave as an inhibitor of the gluconeogenic gene expression program mediated by PGC-1 (1). Second, it was recently reported that HNF4 is a key transcription factor required for the coactivation of gluconeogenic genes by PGC-1 (3). HNF41, the major isoform of HNF4, is hardly expressed in fetal liver, and it is first expressed around birth (22). Moreover, when HNF4 expression is reduced by targeted gene disruption, PGC-1 expression is dramatically up-regulated, suggesting that HNF4 is not only a target of this coactivator, but is also a negative regulator of PGC-1 gene expression (3). The low hepatic expression of HNF41 in fetuses may mediate the high PGC-1 gene expression and the dissociation between high PGC-1 levels and low gluconeogenic gene expression during the fetal period. Moreover, several reports have recently identified negative regulators of PGC-1 action, such as sterol regulatory element-binding protein-1, which interacts with HNF4 and interferes with PGC-1 recruitment to suppress hepatic gluconeogenic genes (23), or p160 Myb-binding protein, which inhibits the mitochondriogenic action of PGC-1 (24). Additional studies are required to test the involvement of these transcription factors and coregulators in the dissociation between the expression of PGC-1 and that of target genes in the late fetal development.
In contrast, the potential of PGC-1 as a mitochondriogenic coactivator has been stressed in muscle cells and brown adipocytes, but a recent report also indicates that overexpression of PGC-1 in hepatic cells up-regulates mRNA expression for mitochondrial components (25). High PGC-1 gene expression may have been involved in the high COII mRNA expression in the fetal liver. However, there were no signs of marked regulation of COII mRNA levels even in situations such as perinatal starvation, in which PGC-1 expression is induced.
In addition to the fetal period, the early neonatal period revealed a dissociation between the regulation of PGC-1 gene expression and the activation of gluconeogenic and lipid oxidation pathways. After birth and initiation of suckling, glucagon- and glucocorticoid-mediated pathways of regulation are rapidly activated, and insulinemia declines suddenly; thus, metabolic target genes involved in gluconeogenesis and fat oxidation are dramatically induced in the first hours after birth (7). This is associated with the sudden appearance of fatty acids in milk as the major source of metabolic energy and the requirement of lipid oxidation and glucose synthesis to maintain glycemia. However, again in this situation, PGC-1 expression is high, but not higher than in fetuses despite the induction of gluconeogenesis. Only if neonates are not allowed to suckle, starvation leads to a parallel overinduction of PGC-1, PEPCK, and G6Pase expression. This indicates that the regulation of PGC-1 expression is not associated with the naturally occurring induction of gluconeogenesis after delivery, although it can be involved in the overinduction of this pathway in response to perinatal starvation, as in adults. Apart from starvation, the dissociation between PGC-1 gene expression and the modulation of gluconeogenesis during the perinatal period is also evidenced in postmature fetuses, in which the up-regulation of PEPCK and G6Pase is blunted, but PGC-1 remains unaltered. Although progesterone may affect the expression of metabolic genes in fetuses, the absence of the stress and nutrient shift associated with delivery was probably the cause of the impaired PEPCK and G6Pase induction in postmature fetuses. Additional evidence of the dissociation between PGC-1 gene expression and the gluconeogenic program during the perinatal period comes from C/EBP-null mice, in which blockage of the whole program of hepatic gluconeogenic gene expression in the late fetal and early neonatal periods does not modify PGC-1 gene expression. All of these findings are consistent with a recent report indicating that PGC-1 acts a transcription amplifier, but is not essential for basal and hormone-induced PEPCK gene expression (26), and support the idea that in a particular physiological situation, such as the perinatal period, the extent of activation of target metabolic pathways by PGC-1 does not necessarily require a parallel regulation of PGC-1 gene expression.
Some of the present findings are not in total agreement with a recent report by Lin et al. (25). These researchers also observed a higher expression of PGC-1 mRNA in late fetuses compared with adults, although the differences they found were fewer. They did not observe such an increase in younger fetuses, and they also report a spontaneous induction in neonatal mice after birth. Those studies were performed using mice from the C57BL/6 strain, which is the background strain we used in our experiments with mice bearing targeted disruption of PPAR. We did not observe postnatal induction in wild-type, fed pups from this strain; therefore, additional research is required to establish the reasons for this discrepancy.
The dissociation between PGC-1 regulation and lipid catabolism in the neonate was even more marked than that for gluconeogenesis. Genes for lipid oxidation and ketogenesis are induced by overexpressing PGC-1 in hepatic cells (3, 25). Because the expression of PGC-1 and lipid oxidation genes is up-regulated in adults in response to starvation, a major role for PGC-1 has been proposed in the control of lipid catabolism, similarly to gluconeogenesis. During the perinatal period, lipid oxidation genes, either peroxisomal or mitochondrial, as well as genes involved in ketogenesis, are poorly expressed in fetal liver and are induced around birth to cope with the massive influx of fat from milk (Refs.8 and 9 and the present results). This pattern is not associated with parallel changes in PGC-1 expression. In contrast to adult starvation, perinatal starvation down-regulates lipid oxidation genes. This differential response may be due to the fact that the fasting of neonatal rodents results in the cessation of fatty acid supplies, which can come only from milk, not from white adipose tissue stores that have not yet developed. In contrast, fasting in adults is linked to the increased availability of free fatty acids from white fat lipolysis. The up-regulation of PGC-1 expression in fasted neonates is the opposite of the down-regulation of fat catabolism genes, which rules out a role for PGC-1 in the control of this pathway.
PPAR acts as a master transcriptional regulator of genes encoding enzymes involved in fatty acid catabolism (27) and has been proposed as the major mediator of coactivation by PGC-1 to induce the genes involved in lipid catabolism (5). Here we show that the targeted disruption of PPAR impairs the expression of HMG-CoA synthase and ACO, marker genes for fatty acid catabolism, during the perinatal period. However, this does not affect PGC-1 gene expression in the fed or fasted state. The depletion of fatty acid-derived metabolites may explain why high PGC-1 levels in association with the high PPAR in neonates (8) did not elicit a high expression of lipid oxidation genes in the liver of fasted neonates. Fatty acids provide not only the fuel supply for energy metabolism, but also the ligand molecules required for the PPAR-dependent activation of target genes (28). In the presence of high PPAR and PGC-1 expression, low availability of lipid ligands in fasted neonates can result in low gene expression of lipid oxidation genes. The preferential reactivation of expression of lipid catabolism genes when fasted pups were treated with a single dose of a lipid emulsion compared with equivalent amounts of glucose supports this possibility.
In summary, PGC-1 gene expression is partially dissociated from the activation of gluconeogenic genes during the perinatal period depending on developmental stage and nutritional status. It appears that developmental regulation of transcription factors such as HNF4, FOXO1, or other targets of PGC-1 coactivation can be essential regulatory elements to build up the appropriate regulatory machinery for the gluconeogenic program in which PGC1 is involved as a coactivator. The regulation of PGC-1 gene expression is completely dissociated from the extent of activation of genes of lipid catabolism during the perinatal period, which is highly dependent on PPAR and nutritional status. Other coactivators, such as PGC-1ß, a recently identified member of the PGC-1 family (25) that shows preferential coactivation activity for PPAR (29), deserve additional research as more preferential candidates to regulate lipid oxidation pathways through transcriptional coactivation. The growing identification of novel coregulators, such as p160 Myb-binding protein (24), or transcription factors and nuclear receptors, such as sterol regulatory element-binding protein-1 (22), estrogen-related receptor- (30, 31), or farnesoid X receptor (32), which act as repressive or activating modulators of the metabolic effects of PGC-1, indicates that the action of PGC-1 on target metabolic genes results from cross-talk with all of these factors. Developmental regulation of the expression of these factors together with PGC-1 gene expression would be essential to determine the activity of PGC-1 at a given stage of development.
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Acknowledgments
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We thank P. Puigserver and B. Spiegelman for providing PGC-1 cDNA and antibody; R. Hanson, F. G. Hegardt, L. Fajas, and N. Glaichehaus for cDNA probes; and G. Darlington for C/EBP-null mice.
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Footnotes
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This work was supported by Grant SAF2002-03648 from the Ministerio de Ciencia y Tecnología and Grant C03/08 from FIS, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain.
Abbreviations: ACO, Acyl-coenzyme A oxidase; C/EBP, CCAAT/enhancer-binding protein-; COII, subunit II of cytochrome c oxidase; G6Pase, glucose-6-phosphatase; HMG-CoA synthase, mitochondrial 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase; HNF4, hepatic nuclear factor-4; LCAD, long-chain acyl-coenzyme A dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1, peroxisome proliferator-activated receptor- coactivator 1; PPAR, peroxisome proliferator-activated receptor-.
Received January 28, 2004.
Accepted for publication May 28, 2004.
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Abstract
Introduction
