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The peroxisome proliferator-activated receptor regulates amino acid metabolism

SANDER KERSTEN¹, STÉPHANE MANDARD, PASCAL ESCHER^*, FRANK J. GONZALEZ, SHERRIE TAFURI^*, BÉATRICE DESVERGNE and WALTER WAHLI

Institut de Biologie Animale, Université de Lausanne, CH-1015, Lausanne, Switzerland;
^* Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105, USA; and
Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

¹Correspondence: Nutrition, Metabolism and Genomics Group, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. E-mail: sander.kersten{at}staff.nutepi.wau.nl

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The peroxisome proliferator-activated receptor is a ligand-activated transcription factor that plays an important role in the regulation of lipid homeostasis. PPAR mediates the effects of fibrates, which are potent hypolipidemic drugs, on gene expression. To better understand the biological effects of fibrates and PPAR, we searched for genes regulated by PPAR using oligonucleotide microarray and subtractive hybridization. By comparing liver RNA from wild-type and PPAR null mice, it was found that PPAR decreases the mRNA expression of enzymes involved in the metabolism of amino acids. Further analysis by Northern blot revealed that PPAR influences the expression of several genes involved in trans- and deamination of amino acids, and urea synthesis. Direct activation of PPAR using the synthetic PPAR ligand WY14643 decreased mRNA levels of these genes, suggesting that PPAR is directly implicated in the regulation of their expression. Consistent with these data, plasma urea concentrations are modulated by PPAR in vivo. It is concluded that in addition to oxidation of fatty acids, PPAR also regulates metabolism of amino acids in liver, indicating that PPAR is a key controller of intermediary metabolism during fasting.—Kersten, S., Mandard, S., Escher, P., Gonzalez, F. J., Tafuri, S., Desvergne, B., Wahli, W. The peroxisome proliferator-activated receptor regulates amino acid metabolism.

Key Words: fasting • microarray • PPAR • SABRE

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
IN DEVELOPED SOCIETIES, diseases of metabolic origin such as hyperlipidemia, diabetes, and obesity have become increasingly prevalent. These disorders have a complex etiology involving genetic and nutritional factors. Intense research over the past decade has yielded evidence that a group of nuclear hormone receptors, called peroxisome proliferator-activated receptors (PPARs), are attractive target for pharmaceutical intervention of these diseases (1) .

The molecular mode of action of PPARs is analogous to that of many nuclear hormone receptors. They can be activated by certain ligands and modulate DNA transcription by binding to specific nucleotide sequences in the promoter of target genes. Three PPAR isotypes are known: , ß, and (NR1C1, NR1C2, NR1C3) (2) . The PPAR isotype is mostly expressed in liver and is the cellular target for fibrates. Fibrates, which include gemfibrozil, bezafibrate, and fenofibrate, are potent hypolipidemic drugs widely used to treat cardiovascular disease. The PPAR isotype is mainly expressed in the adipose tissue, where it promotes the differentiation of preadipocytes into adipocytes. It is the target for a group of drugs called thiazolidinediones, such as troglitazone and rosiglitazone, used to treat type II diabetes. Last, PPARß is expressed in many tissues and has been proposed to mediate the inhibitory effects of NSAIDs on colorectal cancer (reviewed in ref 1 ).

Of the three PPAR isotypes known to date, the PPAR isotype has been comparatively well characterized. The list of known PPAR target genes encompasses an array of genes that participate in various aspects of lipid metabolism such as fatty acid uptake through membranes, fatty acid binding in cells, fatty acid oxidation (both peroxisomal and mitochondrial), and lipoprotein assembly and transport. Both positive and negative target genes of PPAR are known. Many functional effects of fibrates can be explained by the activation or repression of some of these genes via PPAR (3) .

Inasmuch as most of our knowledge about the importance of PPAR is directly coupled to the function of its target genes, the identification of genes regulated by PPAR may aid in further deciphering the role of PPAR in vivo. In addition, the identification of new PPAR targets may help us comprehend the pharmacological effects of fibrates, whose action in vivo is only partially understood.

In recent years, technologies aimed at identifying differentially expressed genes have improved considerably, allowing for a systematic comparison of gene expression between two or more samples. One of these methods, entitled ‘selective amplification via biotin- and restriction mediated enrichment’ (SABRE), has proved useful in identifying genes that display a pronounced circadian rhythm in expression levels (4) . Another technique, called oligonucleotide microarray (Affymetrix), permits the simultaneous expression monitoring of several thousands of genes (5) . This latter technique is increasingly used to study overall changes in gene expression (expression profiling) (6) . To search for new genes regulated by PPAR, we applied the SABRE and microarray technology to the PPAR null mutant model by comparing liver mRNA of wild-type and PPAR null mice (7) . We show here that PPAR influences the expression of numerous genes implicated in amino acid metabolism.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals
Mice were housed in a temperature controlled room (23°C) on a 10 h dark, 14 h light cycle. Pure-bred wild-type or PPAR null mice on a sv129 background were used. Animal experiments were approved by the Animal Authorization Commission of the Canton of Vaud (Lausanne, Switzerland). Fasted animals were deprived of food for 24 h, starting at the beginning of the light cycle. WY14643 was administered by dissolving it in the drinking water (0.1%) for 3 wk.

Oligonucleotide microarray
Total RNA was prepared from mouse livers using Trizol reagent (Gibco BRL, Grand Island, NY); 10 µg of total liver RNA pooled from four mice was used per oligonucleotide microarray experiment. cRNA synthesis, hybridization, washing, and scanning were performed according to standard Affymetrix protocols. Fluorometric data were generated by Affymetrix Software and the gene chips were globally scaled to all the probe sets with an identical target intensity value.

Affymetrix software translates the results of the hybridization into numerical values (average difference, or Avg Diff). The Avg Diff is a measure of the intensity of hybridization to perfect match oligonucleotide probes compared with mismatch probes and is an indicator of the absolute expression level of a gene. The data set was filtered according to the following criteria. Only genes with an absolute expression level (Avg Diff) above the threshold of 30 were kept. All genes that showed a less than 1.5-fold difference in Avg Diff values between wild-type and PPAR null mice were eliminated from the analysis. Genes with a less than twofold difference in expression were eliminated if the Avg Diff values were below 200. Genes with a difference in expression between two- and threefold were eliminated if the Avg Diff values were below 100.

SABRE
SABRE uses selective streptavidin-biotin affinity and restriction enzyme site reconstitution to enrich for cDNA species more abundant in one population than in another. One male sv129 wild-type mouse and one male PPAR null mouse of 8 months of age were killed in the middle of the light cycle. The liver was sampled and total RNA prepared using guanidinium thiocyanate. PolyA RNA was prepared using a kit from New England Biolabs (Beverly, MA). mRNA was reverse transcribed using a Promega cDNA synthesis kit (Madison, WI). cDNA was digested with Sau3AI and ligated with adapters. Ligation products were purified on low melting point agarose, taking a range from 150 bp to 1 kb. Products were amplified by PCR with primers complementary to the adapter; 10 µg of driver PCR products was hybridized by PERT hybridization with 0.33 µg of tester PCR products (0.33 µg driver PCR products for control reaction). The hybridized solution was digested with S1 nuclease and bound to streptavidin beads. Hybrids were eluted with BamHI and used for a new round of PCR. For details, see ref 4 .

The following primers were used:

primer T1: biotin-CCAGGATCCAACCGATC;

primer T2: biotin-CTGGGATCCAACCGATC;

primer D1: GGTCCATCCAACCGATC;

primer D2: CTGCCATCCAACCGATC.

Bands that were enriched in the wild-type vs. PPAR -/- selection and PPAR -/- vs. wild-type selections were excised from the dried denaturing gel and eluted by boiling in 40 µl H₂O for 10 min. The eluted fragments were amplified by PCR and subcloned into a cloning vector for sequencing.

RNA preparation and Northern blots
Total RNA was prepared from frozen livers by RNeasy midikit (Qiagen, Chatsworth, CA) or Trizol reagent (Gibco-BRL); 20–30 µg of total RNA was loaded per lane. Electrophoresis, blotting, and hybridization were according to standard protocols. DNA probes were obtained by RT-PCR of mouse or rat liver total RNA and verified by sequencing. The cDNA of the ribosomal protein L27 was used as a control probe. Labeling was carried out by High Prime kit (Roche, Nutley, NJ). Northern blots were quantified using an Elscript400 quantitative scanner.

Metabolite assays
Blood was drawn by retro-orbital puncture. Blood was collected into heparinized tubes, kept on ice, and spun for 10 min to collect plasma. Plasma was kept at -70°C. Plasma urea and ammonia were measured with a Sigma kit. Plasma beta-hydroxybutyrate was determined using a Boehringer kit (Boehringer-Mannheim, Mannheim, Germany).

	RESULTS

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Oligonucleotide microarray
Oligonucleotide microarray allows for the simultaneous expression monitoring of several thousands of mouse genes in a quantitative fashion (5 , 6) . To identify genes not yet known to be regulated by PPAR, the mRNA population from livers of fasted wild-type and PPAR null mice were compared using murine 6500 gene oligonucleotide microarrays (Affymetrix). Fasted mice were used because the effects of PPAR deletion are much more apparent during fasting (8 9 10 11) .

Of a total of 6519 genes, 2300 were found to be present according to Affymetrix software. Using this software, the abundance of each gene is scored as a numerical value (Avg Diff) that reflects the intensity of hybridization to perfect match oligonucleotide probes compared with mismatch probes. It is an indicator of the absolute expression level and can be compared between two separate microarray hybridizations. Genes whose Avg Diff values differed less than 1.5-fold between wild-type and PPAR null mice were eliminated, as well as genes with Avg Diff values below a certain threshold. This threshold was adjusted based on the magnitude of the fold difference in expression between wild-type and PPAR null mice (see Materials and Methods for details).

To facilitate the interpretation of the data, analysis was confined to a small subset of genes up-regulated in the livers of PPAR null mice. Analysis of the remainder of the genes will be reserved for a future publication.

Disruption of the PPAR gene resulted in up-regulation of genes connected with amino acid metabolism and synthesis of amino acid-derived products. This included, among others, arginase, cytosolic aspartate aminotransferase, branched chain keto acid dehydrogenase, and spermidine synthase (Table 1 ). These data suggest that PPAR may decrease the expression of genes involved in amino acid metabolism. Although a preliminary link between fibrates and aspartate aminotransferase and alanine aminotransferase has already been made (12) , the overall effect of PPAR on amino acid metabolism remains to be established. To confirm the microarray data, Northern blots were performed for two genes: arginase and cytosolic aspartate aminotransferase. Both genes were indeed more highly expressed in fasted PPAR null mice than in fasted wild-type mice (Fig. 1 ). Quantitation of the Northern blot suggested that the oligonucleotide microarray underestimated the fold difference in expression (3.8- vs. 11.6-fold for cytosolic aspartate aminotransferase and 1.6- vs. 2.1-fold for arginase).

View this table: [in this window] [in a new window]   Table 1. Genes involved in amino acid metabolism up-regulated in PPAR null mice according to oligonucleotide microarray

View larger version (44K): [in this window] [in a new window]   Figure 1. Expression of cytosolic aspartate aminotransferase and arginase are increased in PPAR null mice. Northern blot was performed with liver total RNA from 24 h fasted wild-type and PPAR null mice. Quantification of the Northern blots is shown on the right.

Subtractive hybridization (SABRE)
In addition to microarray, a recently developed PCR-based subtractive hybridization assay—SABRE (4) —was used to identify genes influenced by PPAR. Liver mRNA from one wild-type sv129 and one PPAR null mouse was prepared. After several rounds of subtractive hybridization, 12 cDNA fragments were isolated (Fig. 2 ). Two of these cDNA clones corresponded to known PPAR target genes (Cyp4A10=fatty acid hydroxylase and short chain acyl-CoA dehydrogenase). One cDNA clone corresponded to arginino succinate lyase and another to hydroxypyruvate/glyoxylate reductase, both of which are implicated in amino acid metabolism. Northern blot confirmed their differential expression between wild-type and PPAR null mice (Fig. 3 ). Whereas arginino succinate lyase was more highly expressed in PPAR null mice, hydroxypyruvate/glyoxylate reductase mRNA was reduced in these mice. These data provide additional evidence that disruption of the PPAR gene influences the hepatic expression of genes involved in amino acid metabolism.

View larger version (30K): [in this window] [in a new window]   Figure 2. Results of SABRE after 0 and 3 rounds of subtractive hybridization. cDNA from liver of one wild-type and one PPAR null mice was digested with Sau3AI and ligated with a linker oligonucleotide. cDNA fragments were amplified by PCR using primers hybridizing to the linker sequence. PCR was carried out in the presence of 32PdCTP and loaded on a 5% sequencing gel.

View larger version (40K): [in this window] [in a new window]   Figure 3. Expression of arginino succinate lyase and hydroxypyruvate reductase/glyoxylate reductase are altered in PPAR null mice. Northern blot was performed with liver total RNA from 24 h fasted wild-type and PPAR null mice. Quantification of the Northern blots is shown on the right.

It can be hypothesized that the elevated expression levels observed in fasted PPAR null mice may be a consequence of the severe metabolic perturbations present in these mice (9 , 10) . To exclude this possibility, the effect of direct ligand activation of PPAR was studied by feeding mice the synthetic PPAR ligand WY14643 (Fig. 4 ). Changes in expression greater than or equal to 1.5-fold or less than or equal to 0.6-fold were considered significant. Administration of WY14643 altered expression levels of cytosolic aspartate aminotransferase, arginino succinate lyase, and hydroxypyruvate/glyoxylate reductase in a PPAR dependent manner and in an opposite direction from the changes observed in PPAR null mice. Expression of arginase, which was increased in fasted and fed PPAR null mice vs. control mice, was also increased by WY14643 (to evaluate the effect of PPAR disruption in fed mice, compare lanes 1, 2 with lanes 5, 6). This suggests that regulation of arginase expression by PPAR is more complex and contains both stimulatory and inhibitory components. Taken together, it can be concluded that since ligand activation of PPAR in normal mice results in altered expression of cytosolic aspartate aminotransferase, arginino succinate lyase, hydroxypyruvate/glyoxylate reductase, and arginase, the differential expression between fasted wild-type and PPAR null mice is probably not a consequence of metabolic perturbations accompanying PPAR disruption.

View larger version (53K): [in this window] [in a new window]   Figure 4. Administration of WY14643 influences expression of cytosolic aspartate aminotransferase, arginase, arginino succinate lyase, and hydroxypyruvate/glyoxylate reductase. Northern blot was performed with liver total RNA from wild-type and PPAR null mice administered WY14643. Numbers indicate the fold difference in expression using the wild-type mice NOT administered WY14643 as a reference.

PPAR influences expression of genes involved in transamination and deamination
Amino acids are required for numerous processes, including protein synthesis, energy generation, and the synthesis of specialized products such as neurotransmitters and polyamines. Excess amino acids are either transaminated or deaminated. Via a complex network of metabolic reactions, the amino acid nitrogen is converted to urea, which is excreted by the kidneys. The results presented above provide a preliminary link between PPAR and amino acid metabolism. To more closely examine the effect of PPAR on explicit pathways of amino acid metabolism, mRNA levels of numerous enzymes were assessed by Northern blot (Fig. 5 ). The effect of ligand activation of PPAR was studied by administering WY14643 and the effect of disruption of the PPAR gene was studied by comparing fasted wild-type and PPAR null mice.

View larger version (55K): [in this window] [in a new window]   Figure 5. Expression of numerous enzymes involved in amino acid metabolism is affected by PPAR. Northern blot was performed with liver total RNA from 24 h fasted wild-type and PPAR null mice (left panel) or with liver total RNA from wild-type and PPAR null mice administered WY14643 (right panel).

We first investigated the transamination process by determining the expression of mitochondrial aspartate aminotransferase (expression of cytosolic aspartate aminotransferase was shown above to be decreased by PPAR). Expression of mitochondrial aspartate aminotransferase was unaltered in fasted PPAR null mice and increased by WY14643 in a PPAR-dependent manner. Alanine glyoxylate aminotransferase expression was unaltered between fasted wild-type and PPAR null mice, but was markedly decreased by WY14643 in a PPAR dependent manner.

Oxidative deamination is mainly catalyzed by two enzymes, glutamate dehydrogenase and glutaminase, which liberate ammonia. In the reverse reaction, ammonia can be sequestered into glutamine by glutamine synthase. Glutamine synthase expression was unaltered by WY1643 and in fasted PPAR null mice, but mRNA levels were higher in fed PPAR null mice than in fed wild-type mice (see right panel, compare lanes 1, 2 with lanes 5, 6). Expression of glutamate dehydrogenase was barely higher in fasted PPAR null mice and was slightly decreased by WY1643, but in a PPAR-independent manner. Expression of glutaminase was much higher in fasted PPAR null mice than in wild-type mice and was decreased by WY14643 in a PPAR-dependent manner. Together, the data show that PPAR influences the expression of genes involved in trans- and deamination of amino acids.

PPAR influences urea cycle gene expression
Deamination of amino acids in the liver releases ammonia, which is efficiently converted to nontoxic urea by the urea cycle. The urea cycle is catalyzed by five enzymes: arginino succinate lyase, arginino succinate synthase, carbamoyl phosphate synthase 1, ornithine transcarbamoylase, and arginase. The results of arginase and arginino succinate lyase were shown above. The pattern of expression of arginino succinate synthase greatly resembled that of arginino succinate lyase, with higher expression of the gene in fasted PPAR null mice and PPAR-dependent negative regulation by WY14643. A similar pattern was observed for carbamoyl phosphate synthase 1, except that its expression was elevated in fed PPAR null mice compared with fed wild-type mice. Expression of ornithine transcarbamoylase was also down-regulated by WY14643 in wild-type mice, but expression levels did not differ between wild-type and PPAR null mice in the fasted state. Taken together, the data indicate that PPAR has an inhibitory effect on the expression of urea cycle enzymes in mice, with the exception of arginase, whose regulation is more complex.

Plasma urea is increased in fasted PPAR null mice
The gene expression data indicate that PPAR has an overall suppressive effect on the expression of several genes that participate in ammonia and urea synthesis in the liver. To examine whether this has any functional consequences on the rate of urea formation and release in vivo, we measured the concentration of urea in plasma (Fig. 6A ). In the fed state, the plasma urea concentration was identical between fed wild-type and PPAR null mice. In contrast, plasma urea in the fasted state was significantly higher in PPAR null mice than in wild-type mice. The increased plasma urea concentration agrees with the increased mRNA levels of four out of five urea cycle enzymes in fasted PPAR null mice vs. wild-type mice. Apparently, changes in gene expression caused by PPAR are translated into actual functional changes at the physiological level. No differences were found in plasma ammonia concentration between fasted wild-type and PPAR null mice (Fig. 6B ).

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of PPAR deletion on plasma urea and ammonia concentration. A) Plasma urea concentration in fed and fasted wild-type and PPAR null mice. Error bars are SE. Analysis of variance yielded a significant effect for fasting/feeding (P<0.05), genotype (P<0.01), and the interaction between these two parameters (P<0.01). B) Plasma ammonia concentration in fasted wild-type and PPAR null mice. Error bars are SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Using oligonucleotide microarray, subtractive hybridization, and Northern blot, we provide evidence that PPAR influences the expression of numerous genes implicated in major pathways of amino acid metabolism. This includes transamination (aspartate aminotransferase, alanine glyoxylate transaminase), deamination (glutaminase), urea cycle (all five enzymes), oxidation of alpha keto acids (branched chain keto acid dehydrogenase, hydroxypyruvate/glyoxylate reductase), amino acid inter-conversions (asparagine synthetase, phenylalanine hydroxylase), and synthesis of amino acid-derived products (spermidine N1 acetyltransferase, spermidine synthetase). With the exception of hydroxypyruvate/glyoxylate reductase and arginase, PPAR suppresses the expression of these genes, leading to an overall decrease in amino acid degradation. The functional importance of regulation of amino acid metabolism by PPAR is substantiated by the demonstration that plasma urea concentrations are increased in fasted PPAR null mice vs. wild-type mice.

Differences exist between mice and humans with respect to their metabolic response to fasting. Metabolism in mice is much more rapid. Furthermore, whereas urea production in humans is increased during the first 24–36 h of fasting, our data suggest that the opposite is true in mice. However, there is no good reason to believe that the overall effects of PPAR and PPAR ligands on genes involved in amino acid metabolism would differ between mice and humans.

Contrary to common understanding, oxidation of amino acids contributes significantly to energy production of several organs, including liver and the gut (13) . Amino acid oxidation is dramatically elevated during conditions such as sepsis and cachexia and after severe trauma and burns. These catabolic diseases are characterized by massive net body protein breakdown, leading to a negative nitrogen balance. Despite the clinical importance of amino acid metabolism and unlike lipid and glucose metabolism, little information is available about its regulation at the genetic level. It has been demonstrated that glucocorticoids and glucagon increase expression of urea cycle enzymes (14 15 16) . Recent work has established the important role of the transcription factor C/EBP in the regulation of urea synthesis. Using C/EBP null mice, it was demonstrated that C/EBP stimulates expression of urea cycle enzymes (17) . Our data point toward another global transcriptional regulator of the urea cycle, PPAR, which by inhibiting mRNA expression of transamination, deamination, and urea cycle enzymes has an effect directly opposite to that of C/EBP. Consistent with these data, plasma urea levels are increased in fasted PPAR null mice vs. wild-type mice.

PPAR activates gene transcription by binding to specific nucleotide sequences in the promoter region of target genes. Positive targets of PPAR include an array of genes that participate in various aspects of fatty acid metabolism (1) . In addition to stimulating gene expression, PPAR also inhibits the expression of numerous genes such as apoCIII (18 19 20) , apoAI (in rodents) (20 , 21) , transferrin (22) , fibrinogen (23 , 24) , pyruvate kinase (25) , S14 (26) , Cyp2C family members (24) , and positive acute-phase response genes (24 , 27) . Our microarray analysis revealed that the number of genes up-regulated in livers of PPAR null mice exceeds the number of genes that are down-regulated. Accordingly, negative regulation by PPAR may be responsible for a plethora of effects attributed to PPAR or to PPAR ligands, including its effect on cell proliferation and inflammation (S. Kersten and P. Escher, unpublished observations).

With respect to the mechanism of down-regulation by PPAR, it is not unthinkable that in addition to a positive PPAR response element, there may also be a negative PPAR response element that mediates repression of DNA transcription by PPAR. However, such a promoter element has yet to be identified. Alternatively, it is possible that PPAR stimulates expression of another transcription factor that serves as a transcriptional repressor. For example, down-regulation of the apoA-I gene by PPAR in the rat has been proposed to be mediated by the nuclear receptor rev-erb, whose expression is stimulated by PPAR and is a negative regulator of apoA-I transcription. Rather than being increased, however, we find that Rev-erb expression in mice is slightly decreased by WY14643, in a PPAR-dependent manner (data not shown). A final mechanism by which PPAR may repress transcription is by competing with other transcription factors for a common coactivator protein that is needed for transcriptional activation.

As fatty acids are ligands for PPAR, the suppressive effect of PPAR on urea cycle enzymes may provide a potential explanation for the inhibitory effect of fatty acids on ureagenesis (29) and ammonia detoxification (30) . Fatty acids have also been shown to suppress arginino succinate synthase and carbamoyl phosphate synthase expression in cell culture (31) . This mechanism may account for the abnormal expression of urea cycle enzymes observed in carnitine-deficient juvenile visceral steatosis, a disease characterized by defective fatty acid uptake into mitochondria and associated accumulation of fatty acids in the cytosol (32) .

Why would the same transcription factor that stimulates hepatic fatty acid oxidation suppress amino acid degradation and ureagenesis? During prolonged fasting, fatty acid oxidation becomes the major source of energy for the liver, an effect mediated by PPAR (9 , 10) . At the same time, the relative contribution of amino acid metabolism to hepatic ATP production, which is dominant in the fed state (13) , declines. In mice, this is associated with a decreased expression of several amino acid metabolizing and urea-synthesizing enzymes during fasting (S. Kersten et al., unpublished results). The reciprocal relationship between fatty acid oxidation and nitrogen metabolism is illustrated by comparing the plasma ketone body concentration, which reflects the rate of fatty acid oxidation, and the plasma urea concentration, which (in the absence of changes in renal clearance) is indicative of the rate of amino acid metabolism and subsequent urea synthesis (Fig. 7 ). It is conceivable that the simultaneous increase in ketone body concentration and decrease in urea concentration during fasting in mice are actually due to the action of a single factor, PPAR, which balances the needs of the two pathways by altering the expression of genes involved.

View larger version (16K): [in this window] [in a new window]   Figure 7. Reciprocal relationship between plasma ketone bodies and urea concentration during the course of fasting. Blood was taken from wild-type mice at the beginning of the light cycle (0 h, fed) and after 2.5, 5.5, and 24 h of fasting.

In addition to amino and fatty acid metabolism, recent evidence also implicates PPAR in the regulation of carbohydrate metabolism. Using the oligonucleotide microarray, it was shown that PPAR up-regulates the expression of several genes involved in gluconeogenesis. Overall, this suggests that PPAR acts as a global regulator of energy metabolism in the liver, which coordinates the rates of utilization of the various energy substrates in relation to food availability.

In conclusion, it is shown here that fibrates and PPAR down-regulate hepatic expression of genes involved in amino acid metabolism. Regulation of amino acid metabolism by PPAR is supported by functional data showing increased plasma urea concentrations in fasted PPAR null mice. The data presented add to a growing body of evidence indicating that negative regulation by PPAR may be responsible for an abundance of effects attributed to PPAR or to PPAR ligands.

	ACKNOWLEDGMENTS

 
The authors would like to thank Tim Jatkoe, Steve Madore, Yixin Wang, and Sandra Rojas-Caro for their help in carrying out this study. S.K. was supported by fellowships from the European Molecular Biology Organization, the Roche Research Foundation, and the Royal Netherlands Academy of Arts and Sciences. This work was partially financed by the Swiss National Science Foundation, the Etat de Vaud, and the Human Frontier Science Program.

Received for publication March 7, 2001. Revision received May 15, 2001.

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