**1. Introduction**

Transcriptional regulation of gene expression is essential for development, tissue differentiation, and organisms' responses to changes in their environment. Maintenance of homeostasis would be impossible without regulation of expression of genes that code for enzymes in the carbohydrate, fat, and protein metabolism. Omnivorous mammals, such as humans, mice, and rats, can adapt their metabolism to varying proportions of amino acids, fats, and carbohydrates as sources of energy [1, 2]. Strict carnivores, such as cats, lack such adaptive mechanisms [3]. Diet rich in carbohydrates and fats triggers utilization of these nutrients as sources of energy and storage of excess sugars and fat in the form of glycogen and adipose tissue [1, 4]. On the other hand, a diet rich in proteins imposes changes in nitrogen balance because excess proteins and amino acids cannot be stored [1]. High intake of proteins, combined with low intake of carbohydrates and fats, leads to utilization of amino acids as energy sources and results in increased catabolism of amino acids [1, 4] and increased need to dispose of waste nitrogen, generated in this process. Transcriptional regulation of expression of enzymes in the pathways for degradation of nutrients as well as biosynthesis of molecules that can be stored is necessary for adaptations to these dietary changes.

Urea cycle is a liver metabolic pathway that converts ammonia into urea. Ammonia is a waste product of catabolism of dietary and cellular proteins, and a potent neurotoxin [5]. Defects in any of the enzymes of the urea cycle lead to partial or complete block of urea production and accumulation of ammonia, which damages the brain and can be lethal [5]. Therefore, the physiological role of urea cycle is to protect the brain from the toxic effects of ammonia.

Five enzymes of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL) and arginase 1, catalyze conversion of ammonia into urea. In addition, N-acetylglutamate synthase (NAGS), ornithine/citrulline transporter (ORNT), and aspartate/glutamate transporter (also known as ARALAR2 or Citrin) are required for the normal function of the urea cycle. The pathway is partitioned between mitochondria and cytoplasm of the liver cells. NAGS, CPS1, and OTC are located in the mitochondrial matrix [6] while ASS, ASL, and arginase 1 are in the cytosol [6]. **Figure 1** shows enzymes, transporters, and intermediates of the urea cycle together with their cellular localization. The NAGS (EC 2.3.1.1) uses glutamate and acetyl coenzyme A to produce NAG, an obligatory allosteric activator of CPS1 [6–9]. Upon binding of NAGS, CPS1 (EC 6.3.4.16) consumes two molecules of ATP to catalyze formation of carbamoyl phosphate from ammonia and bicarbonate [6, 10]. Carbamoyl phosphate and ornithine, which is transported into mitochondria by ORNT, are converted to citrulline by OTC (EC 2.1.3.3), followed by the transport of citrulline from mitochondria into cytoplasm by ORNT [6]. ASS (EC 6.3.4.5) catalyzes formation of argininosuccinate from citrulline and aspartate, which is supplied by the Citrin [6]. The argininosuccinate is then converted

#### **Figure 1.**

*Enzymes, transporters, and intermediates of the urea cycle. Urea cycle enzymes are shown in blue typeface: NAGS—N-acetylglutamate synthase, CPS1—carbamoyl phosphate synthetase 1, OTC—ornithine transcarbamylase, ASS—argininosuccinate synthase, ASL—argininosuccinate lyase, and Arg1—arginase 1. Urea cycle transporters ORNT (ornithine/citrulline transporter) and Citrin (glutamate/aspartate transporter) are shown as ovals. The orange arrow indicates activation of CPS1 by N-acetylglutamate (NAG). AcCoA acetyl coenzyme A. CoASH—coenzyme A.*

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*Data Mining Approaches for Understanding of Regulation of Expression of the Urea Cycle Genes*

to arginine by the ASL (EC 4.3.2.1). Arginase 1 (EC 3.5.3.1) completes the cycle by hydrolyzing arginine into urea and ornithine, which is transported into mitochondria to be a substrate for OTC (**Figure 1**). Urea cycle genes and enzymes are not uniformly expressed in the liver; their expression follows a gradient from high in hepatocytes surrounding portal vein to no expression in hepatocytes surrounding central vein [11]. NAGS, CPSI, and OTC are also expressed in the small intestine, where they synthesize citrulline which is then transported to the kidneys for the biosynthesis of arginine by the ASS and ASL [6]. Argininosuccinate synthase and lyase also function in the NO signaling and are present in the tissues that express

Long-term changes in dietary protein intake lead to adaptive changes in expression of urea cycle enzymes. Their expression increases in rats and monkeys fed high-protein diet and decreases upon feeding of low-protein diet [4, 12]. These adaptive changes seem to be mediated, at least in part, by the hormones glucagon, glucocorticoid, and insulin [13–25]. Glucagon and glucocorticoid hormones trigger changes in mRNA and protein levels of all five urea cycle enzymes, but the mechanisms responsible for these changes seem to differ for each enzyme [21]. It is not known whether changes in the dietary protein intake trigger similar changes in the expression of the NAGS gene because it has not been identified at the time. Also unknown are signaling cascades that mediate effects of hormones to regulate expression of urea cycle genes and whether specific amino acids and/or other

Inspection of the regulatory regions of genes for urea cycle enzymes (**Figure 2**) does not reveal a common regulatory element that would bind one or more transcriptional factors to coordinately regulate transcription of all urea cycle genes [13, 26–36]. The studies of expression of urea cycle genes in knockout mice also show a lack of common regulatory mechanism. Ureagenesis is defective in mice lacking hepatocyte nuclear factor 4 α (HNF4α) due to absence of OTC mRNA and protein [37] as well as in mice lacking CCAAT/enhancer binding protein α (C/EBPα) due to lack of CPSI mRNA [38]. However, ureagenesis appears normal in mice lacking C/EBPβ although this transcriptional factor appears to regulate expression of the arginase 1 gene [39]. It is also unknown if short-term increases in nitrogen load following a meal trigger any

This chapter focuses on the regulation of NAGS, CPS1, and OTC expression because their only known functions are protection of the brain from ammonia toxicity through participation in the urea cycle and intestinal biosynthesis of citrulline. The three genes share common expression pattern in the liver, intestine, and during development. Because of the role of urea cycle in protecting the brain from ammonia toxicity, expression of the three genes have been studied in much greater detail in the liver than intestinal cells. Detailed understanding of the transcriptional regulation of the urea cycle genes is important for our understanding of bodies' response to changes in the environment such as dietary changes as well as events that trigger increased catabolism of cellular proteins such as starvation, infections, and invasive medical procedures [6, 40–43]. Because regulation of expression of the mammalian CPS1 and OTC genes has been studied for more than three decades while expression of human NAGS, which was identified and cloned in 2002, took place less than a decade ago the approaches taken in these studies differed greatly. Knowledge of transcriptional regulation of mammalian CPS1 and OTC was gained through cloning of genomic DNA, construction of reporter gene plasmids with various fragments from the CPS1 and OTC regulatory regions and their expression in cultured cells and transgenic mice, whereas regulatory elements of the NAGS

gene have been identified using comparative genomics approaches.

*DOI: http://dx.doi.org/10.5772/intechopen.81253*

metabolites act as sensors of the dietary protein intake.

change in expression of urea cycle enzymes.

nitrous oxide synthase [5].

#### *Data Mining Approaches for Understanding of Regulation of Expression of the Urea Cycle Genes DOI: http://dx.doi.org/10.5772/intechopen.81253*

to arginine by the ASL (EC 4.3.2.1). Arginase 1 (EC 3.5.3.1) completes the cycle by hydrolyzing arginine into urea and ornithine, which is transported into mitochondria to be a substrate for OTC (**Figure 1**). Urea cycle genes and enzymes are not uniformly expressed in the liver; their expression follows a gradient from high in hepatocytes surrounding portal vein to no expression in hepatocytes surrounding central vein [11]. NAGS, CPSI, and OTC are also expressed in the small intestine, where they synthesize citrulline which is then transported to the kidneys for the biosynthesis of arginine by the ASS and ASL [6]. Argininosuccinate synthase and lyase also function in the NO signaling and are present in the tissues that express nitrous oxide synthase [5].

Long-term changes in dietary protein intake lead to adaptive changes in expression of urea cycle enzymes. Their expression increases in rats and monkeys fed high-protein diet and decreases upon feeding of low-protein diet [4, 12]. These adaptive changes seem to be mediated, at least in part, by the hormones glucagon, glucocorticoid, and insulin [13–25]. Glucagon and glucocorticoid hormones trigger changes in mRNA and protein levels of all five urea cycle enzymes, but the mechanisms responsible for these changes seem to differ for each enzyme [21]. It is not known whether changes in the dietary protein intake trigger similar changes in the expression of the NAGS gene because it has not been identified at the time. Also unknown are signaling cascades that mediate effects of hormones to regulate expression of urea cycle genes and whether specific amino acids and/or other metabolites act as sensors of the dietary protein intake.

Inspection of the regulatory regions of genes for urea cycle enzymes (**Figure 2**) does not reveal a common regulatory element that would bind one or more transcriptional factors to coordinately regulate transcription of all urea cycle genes [13, 26–36]. The studies of expression of urea cycle genes in knockout mice also show a lack of common regulatory mechanism. Ureagenesis is defective in mice lacking hepatocyte nuclear factor 4 α (HNF4α) due to absence of OTC mRNA and protein [37] as well as in mice lacking CCAAT/enhancer binding protein α (C/EBPα) due to lack of CPSI mRNA [38]. However, ureagenesis appears normal in mice lacking C/EBPβ although this transcriptional factor appears to regulate expression of the arginase 1 gene [39]. It is also unknown if short-term increases in nitrogen load following a meal trigger any change in expression of urea cycle enzymes.

This chapter focuses on the regulation of NAGS, CPS1, and OTC expression because their only known functions are protection of the brain from ammonia toxicity through participation in the urea cycle and intestinal biosynthesis of citrulline. The three genes share common expression pattern in the liver, intestine, and during development. Because of the role of urea cycle in protecting the brain from ammonia toxicity, expression of the three genes have been studied in much greater detail in the liver than intestinal cells. Detailed understanding of the transcriptional regulation of the urea cycle genes is important for our understanding of bodies' response to changes in the environment such as dietary changes as well as events that trigger increased catabolism of cellular proteins such as starvation, infections, and invasive medical procedures [6, 40–43]. Because regulation of expression of the mammalian CPS1 and OTC genes has been studied for more than three decades while expression of human NAGS, which was identified and cloned in 2002, took place less than a decade ago the approaches taken in these studies differed greatly. Knowledge of transcriptional regulation of mammalian CPS1 and OTC was gained through cloning of genomic DNA, construction of reporter gene plasmids with various fragments from the CPS1 and OTC regulatory regions and their expression in cultured cells and transgenic mice, whereas regulatory elements of the NAGS gene have been identified using comparative genomics approaches.

*Gene Expression and Control*

Urea cycle is a liver metabolic pathway that converts ammonia into urea. Ammonia is a waste product of catabolism of dietary and cellular proteins, and a potent neurotoxin [5]. Defects in any of the enzymes of the urea cycle lead to partial or complete block of urea production and accumulation of ammonia, which damages the brain and can be lethal [5]. Therefore, the physiological role of urea cycle is

Five enzymes of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL) and arginase 1, catalyze conversion of ammonia into urea. In addition, N-acetylglutamate synthase (NAGS), ornithine/citrulline transporter (ORNT), and aspartate/glutamate transporter (also known as ARALAR2 or Citrin) are required for the normal function of the urea cycle. The pathway is partitioned between mitochondria and cytoplasm of the liver cells. NAGS, CPS1, and OTC are located in the mitochondrial matrix [6] while ASS, ASL, and arginase 1 are in the cytosol [6]. **Figure 1** shows enzymes, transporters, and intermediates of the urea cycle together with their cellular localization. The NAGS (EC 2.3.1.1) uses glutamate and acetyl coenzyme A to produce NAG, an obligatory allosteric activator of CPS1 [6–9]. Upon binding of NAGS, CPS1 (EC 6.3.4.16) consumes two molecules of ATP to catalyze formation of carbamoyl phosphate from ammonia and bicarbonate [6, 10]. Carbamoyl phosphate and ornithine, which is transported into mitochondria by ORNT, are converted to citrulline by OTC (EC 2.1.3.3), followed by the transport of citrulline from mitochondria into cytoplasm by ORNT [6]. ASS (EC 6.3.4.5) catalyzes formation of argininosuccinate from citrulline and aspartate, which is supplied by the Citrin [6]. The argininosuccinate is then converted

*Enzymes, transporters, and intermediates of the urea cycle. Urea cycle enzymes are shown in blue typeface: NAGS—N-acetylglutamate synthase, CPS1—carbamoyl phosphate synthetase 1, OTC—ornithine transcarbamylase, ASS—argininosuccinate synthase, ASL—argininosuccinate lyase, and Arg1—arginase 1. Urea cycle transporters ORNT (ornithine/citrulline transporter) and Citrin (glutamate/aspartate transporter) are shown as ovals. The orange arrow indicates activation of CPS1 by N-acetylglutamate (NAG). AcCoA—*

to protect the brain from the toxic effects of ammonia.

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**Figure 1.**

*acetyl coenzyme A. CoASH—coenzyme A.*


#### **Figure 2.**

*Transcriptional regulatory elements of the urea cycle genes. Hooked arrows indicate transcriptional initiation sites. Gray boxes—cisacting regulatory elements. Proteins that bind to cis-acting regulatory elements are shown above each box. C/EBP—CCAAT/enhancer binding proteins; HNF4α—hepatic nuclear factor a; COUP-TF chicken ovalbumin upstream promoter transcriptional factor; FoxA—forkhead box A transcriptional factor/ hepatic nuclear factor 3; GR—glucocorticoid receptor; P3—unidentified protein of approx. 75 kDa; P1, P2—two unidentified proteins; NF-Y—CCAAT—binding factor; AP-2—activator protein 2; Sp1—Sp1 transcriptional factor; CREB—cAMP response element binding transcription factor.*
