Relation between protein metabolism and regulation if energy metabolism

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Amino acid metabolism

Digestion of dietary proteins

After feeding, the dietary protein was digested in the stomach by pepsin which hydrolysed ingested protein at the peptide bonds to cleave the polypeptide chains into a mixture of smaller peptides. The digestion continues in the small intestine by trypsin, chymotrypsin, carboxypeptidase A and B and aminopeptidase. The free AAs or small peptides then enter the blood capillaries in the villi and travel to the liver (Freeman et al. 1979).
The intestine is the primary site of AAs and peptide absorption. AAs are transported into enterocytes by facilitated diffusion or specific transport systems with sodium as a carrier, that take up AAs against a concentration gradient (Stevens et al. 1984; Lerner 1987). The transport systems are specific to groups of AAs rather than to individual AAs. In addition to the AA transporters, enterocytes have an active transport system for di- and tri-peptides, independent of the one for free AAs (Adibi et al. 1976; Webb 1990). AAs arising from dietary protein digestion or from protein degradation are constantly re-synthesized or used in protein turnover. Unrequired or damaged proteins are targeted for destruction. Some AAs are used for protein synthesis in the liver (constitutive or exported plasma proteins). The branched chain amino acids (BCAA) are, along with other AAs, required for the stimulation of total liver protein synthesis (Anthony et al. 2001b). However, when protein intake increased, no change in protein synthesis rate was observed despite an increase in the tissue protein pool (Chevalier et al. 2009). The effect on protein breakdown remained unknown. Furthermore, the AA surplus may be used as metabolic fuel or converted to other compounds. The main fates of the carbon skeletons remaining from AA deamination may be their use for cellular respiration, fatty acids or ketone bodies synthesis, or gluconeogenesis whereas the amino groups are used for the biosynthesis of AAs, nucleotides and biological amines or are converted to urea for excretion through the urea cycle (Figure 1) (Shambaugh 1977; Brosnan 2003). Ureogenesis is an important process to protect the body from potentially toxic ammonium (Shambaugh 1977; Dimski 1994; Ding et al. 1997).

Interorgan amino acid metabolism

The functions of interorgan AA traffic are to maintain the relatively constant extracellular AA concentration in which tissues are bathed and to provide AAs for protein synthesis and those used in specific functions. AAs serve as the building blocks for proteins and some of them that exceed the body’s needs for protein synthesis undergo oxidative degradation through transamination and desamination (Brosnan 2003). The liver is the main organ where many different proteins are synthesized (Brosnan 2003). For example, albumin is synthesized in the liver of a healthy adult human and 20g/day is secreted. This albumin is catabolized in the peripheral tissues, suggesting that about 20g of AAs are made available each day as a result of albumin catabolism (Maxwell et al. 1990).
The rate of AA uptake by tissues or organs depends on the activity of several transporters. In mammals, there are different AA transporters which are referred to as transport systems. Free AAs are transported across membranes through Na+-independent (facilitated transport) or Na+-dependent (secondary active transport) systems. Nomenclature of AA transport systems have letter designations based on their preferred AA substrates and the presence or absence of the requirement for sodium ion activation and co-transport (Mailliard et al. 1995), as shown in Table 1. The interconversion of AAs trough transamination is an important process in the transport of ammonium and to maintain acid-base balance (Wu 2009). These processes converge on the central catabolic pathways, with the removal of the -amino groups from the carbon skeleton. This was catalyzed by enzymes called aminotransferases or transaminases and then the carbon skeletons of most AAs found their way to the citric acid cycle (Metzler et al. 1982). Almost all AAs can be metabolized in the liver and it is the organ with urea cycle (Brosnan 2003).
Several non-essential AAs, including glutamine, glutamate and aspartate, are oxidized by epithelial cells in the mammalian small intestine and they do not enter the portal vein (Stoll et al. 1998; Wu 1998). The small intestine uptakes the glutamine as the major fuel and nitrogenous products derived from glutamine metabolism are released into the portal vein. These include the alanine and proline which are metabolized by the liver. Moreover, the output from the small intestine also includes citrulline which is taken up and converted to arginine in the kidney (Wu 1997; Wu and Morris 1998) (Figure 2). The kidney plays a major role in the interorgan metabolism of citrulline, arginine, glycine, and glutamine. It takes up glycine and releases serine. In addition, the kidney uptakes glutamine which is the substrate for urinary ammonia production and it contributes in this way to the maintenance of acid-base homeostasis (Brosnan 2003) (Figure 2).
Glutamate and glutamine play critical roles in these transaminations. In the cytosol of hepatocytes, the amino groups from most AAs are transferred to -ketoglutarate to form glutamate. The glutamate serves as an amino group donor for biosynthetic pathways or excretion pathways that lead to the elimination of nitrogenous products. Glutamate is then transported from cytosol into mitochondria, where the amino group is removed to form NH4+ via oxidative deamination promoted by L-glutamate dehydrogenase (Figure 3). The -ketoglutarate formed from glutamate deamination can be used for energy production in the citric acid cycle and for glucose synthesis in the liver and kidney (Brosnan 2003). The ammonium ion is converted to urea for excretion through the urea cycle, which is distributed between the mitochondrial matrix and cytosol of hepatocytes (Shambaugh 1977).
The AAs in the liver can be transaminated and degraded to other citric acid cycle intermediates and acetyl CoA or oxaloacetate which can in turn be oxidized for energy supply or converted to glucose or fat (Figure 4). However, the oxidation of AAs produces much more ATP than the liver could actually use. Therefore, it seems that the carbon skeletons of these AAs are not completely oxidized in the liver and are converted to glucose via gluconeogenesis even in the fed state (Jungas et al. 1992). During starvation, hepatic gluconeogeneis plays an important role in the production of glucose for the brain and other glucose dependent organs and the AAs from the muscle proteolysis are the major precursors for this process (Brosnan 2003). The liver also synthesizes glutathione from glutamate, glycine, and cysteine for use by extrahepatic cells (including immunocytes) and tissues (Figure 2) (Wu 2009).
Given that the bulk of the body’s protein is in the form of muscle proteins, this tissue will apparently play a critical role in the interorgan AA metabolism. The skeletal muscle is the major organ for the catabolism of BCAA and released both alanine and glutamine from BCAA and -ketoglutarate. This alanine is taken up by the liver and converted to glucose (the glucose-alanine cycle). Thus alanine is one of the important molecules in the transport of amino groups to the liver without increasing blood ammonia concentrations (Figure 2 and 4) (Brosnan 2003; Wu 2009)

Protein metabolism and regulation by nutritional conditions

Protein and AA metabolism is a large, dynamic and regulated process that accomplishes a variety of physiological functions. In adult humans, some 300 g of new protein is synthesized per day for maintenance, and an equivalent amount of protein is degraded to their constituent AAs. In eukaryotes, the half-lives of proteins vary from minutes to many days (Goldberg and St John 1976; Mayer and Doherty 1986). For example, in the rat liver, proteins might turn over once every one to two days, while some regulatory enzymes have half-lives only 15 minutes. Furthermore, the more stable proteins, such as actin and myosin in skeletal muscle, might turn over once every one or two weeks. In human, hemoglobin can remain for the entire lifetime of an erythrocyte (3 months) (Lecker et al. 1999). The overall process of protein synthesis and protein degradation is referred to as protein turnover. The rates of protein turnover may vary depending on the intracellular and extracellular environmental conditions, including the availability and balance of nutrients to which cells are exposed, and the hormones and the peptide factors that bind to receptors on cell surfaces or within the cell. It has been established that alterations in dietary macronutrient intake greatly affected the balance between tissue protein synthesis and protein degradation (Darmaun 1999). Since AAs serve as the currency of protein metabolism, they are hydrolyzed from protein via proteolysis systems and serve as the building blocks for new protein synthesis. Therefore, protein cell homeostasis is maintained by a precise balance between the overall rates of synthesis and degradation (Lecker et al. 1999).
In mammals, changes in nutrient availability induce changes in the levels of hormones to adapt the metabolism. Protein synthesis requires both AAs, both as precursors, and a substantial amount of metabolic energy. Maintaining the essential AA supply is necessary to maintain the optimal rate of protein synthesis in both the liver and skeletal muscle. Deprivation of even a single essential AA causes a decrease in the cellular protein synthesis by inhibition of the initiation phase of mRNA translation (Kimball 2002).
There is evidence that protein synthesis was stimulated in muscle and in liver by 38 and 41%, respectively when rats were fed a diet containing 20% protein whereas no change was observed in rats fed no added protein (Yoshizawa et al. 1998). Moreover, plasma insulin concentrations were the same in rats fed either diet, suggesting that feeding-induced changes in plasma insulin are not sufficient to stimulate protein synthesis. Both dietary protein and insulin may be required to stimulate translation initiation (Yoshizawa et al. 1998). However, Kimball reported that insulin alone can activate the translation at the initiation step (Kimball and Jefferson 2006a). Moreover, AAs, especially BCAA, stimulated the protein synthesis in primary hepatocytes (Dubbelhuis and Meijer 2002; Ijichi et al. 2003) whereas in the livers of rats fed a high protein diet for 2 weeks, the protein synthesis rate was decreased (Chevalier et al. 2009) A slight inhibition of synthesis rates after the high protein diet was observed in the kidney while protein synthesis rates were significantly increased in stomach and skin. These results suggested that the adaptation to high protein diet was tissue specific (Chevalier et al. 2009). Furthermore, the reduction of protein levels in diets (20.7%, 16.7% or 12.7%) decreased the protein synthesis in the pancreas, liver, kidney and muscle in piglets receiving these diets for 2 weeks (Deng et al. 2008). Muscles play a role as a protein reservoir. Skeletal muscle is also the main organ of BCAA catabolism. There is evidence that carbohydrate restricted-with high protein diet, during 7 days, stimulated muscle protein synthesis (Harber et al. 2005). In humans, increasing protein ingestion resulted in an increase in protein synthesis (up to 20%) and a decrease in protein breakdown after adaptation for 7 days to higher protein intake (Motil et al. 1981; Hoerr et al. 1993; Gibson et al. 1996; Fereday et al. 1998; Forslund et al. 1998; Harber et al. 2005). However, the acute protein intake resulted in only slight increase of protein synthesis (around 8%) and greater decrease in its breakdown (Gibson et al. 1996; Forslund et al. 1998; Cayol et al. 1997; Fereday et al. 1998). Essential AAs and BCAA (especially leucine) specifically modulate protein synthesis by activating the initiation of translation (Anthony et al. 2001a; Anthony et al. 2001b; Yoshizawa 2004; Crozier et al. 2005). In rats in vivo, infusion of the BCAA stimulated muscle protein synthesis and essential AAs maintained this effect (Kobayashi et al. 2006).
Protein degradation is also regulated by nutrition (Kettelhut et al. 1988). High amino acid concentrations and insulin are the main inhibitors of protein degradation, whereas glucagon and low concentrations of amino acids are the principal stimulators (Gelfand and Barrett 1987; Flakoll et al. 1989; Mortimore et al. 1989; Kadowaki et al. 1992; Blommaart et al. 1997; Boirie et al. 1997; Balage et al. 2001; Kanazawa et al. 2004; Waterlow 2006; Capel et al. 2008). Under acute feeding, proteolysis is inhibited while a chronic increase in protein intake, induced proteolysis in the fed state (Price et al. 1994; Forslund et al. 1998). In the post-absorptive state, whole body protein degradation varies only very slightly (Price et al. 1994; Forslund et al. 1998). In specific tissues, only a few studies have examined the response of protein degradation to increased protein intakes (Taillandier et al. 1996; Bolster et al. 2002; Harber et al. 2005). Liver proteolysis is known to be inhibited by insulin (Duckworth et al. 1994; Hamel et al. 1997; Bennett et al. 2000; Bennett et al. 2003; Kanazawa et al. 2004) and stimulated by glucagon (Schworer and Mortimore 1979; Mortimore et al. 1989). AAs also act as a negative feed- back regulator for proteolysis in the perfused rat liver (Poso et al. 1982; Mortimore et al. 1989; Kadowaki et al. 1992; Miotto et al. 1992) and isolated hepatocytes (Mortimore and Schworer 1977; Seglen et al. 1980). In muscle, numerous publications have described the stimulation of muscle protein breakdown in response to fasting, followed by an inhibition after re-feeding with a normal diet. Muscle protein breakdown is activated in response to one or more days of starvation (Medina et al. 1991; Wing and Banville 1994; Wing et al. 1995). Several AAs also have a direct regulatory affect on proteolysis: Leu, Gln, Tyr, Phe, Pro, Met, Trp and His in the liver and Leu in the skeletal muscle (Kadowaki and Kanazawa 2003; Meijer and Dubbelhuis 2004; Oshiro et al. 2007).

Protein synthesis

Protein synthesis is one of the most complex biosynthetic processes. In eukaryotes, almost 300 macromolecules cooperate to synthesize polypeptides. These macromolecules consist of over 70 different ribosomal proteins, 20 or more enzymes to activate the AA precursors, a dozen or more auxiliary enzymes and other protein factors for the initiation, elongation and termination of polypeptides, perhaps 100 additional enzymes for the final processing of different proteins and 40 or more kinds or transfer and ribosomal RNAs.
First, the production of polypeptides follows the process of transcription, the production of messenger RNA (mRNA) from a gene’s nucleotide sequence which involves several steps. It consists of transcription initiation, elongation, RNA–processing reactions, e.g. capping and splicing, and termination. Second, the transcription is followed by the transport of mRNA to the cytosol where mRNA is decoded into protein by the translation. The mRNA transported the genetic code into the cytosol in the form of codon which is a triplet of nucleotides that codes for a specific AA. A specific first codon in the sequence of mRNA establishes an open reading frame. The reading frame is set when translation of an mRNA molecule begins and is maintained as the synthesis machinery reads sequentially from one triplet to the next. Several codons serve special functions such as the initiation codon, AUG, which signals the beginning of a polypeptide in all cells, in addition to coding for methionine residues of polypeptides. Moreover, there are the termination codons (also called stop codons or nonsense codons), UAA, UAG and UGA, which normally signal the end of polypeptide synthesis.
The three major stages of translation are:
1. initiation
2. elongation
3. termination

Initiation (Figure 5)

In eukaryotes, the initiation begins by the assembly of a complex from initiator methionyl-transfer RNA (met-tRNAi), 40S and 60S ribosomal subunits, with the aid of eukaryotic initiation factors (eIFs), into an 80S ribosome at the initiation codon of mRNA.
In the first step of translation initiation, the eukaryotic initiation factor 2 (eIF2) binds GTP and met-tRNAi, selected from the pool of tRNAs, to form the ternary complex (eIF2●GTP●met-tRNAi) and then binds to the 40S ribosomal subunit with other eIFs (eIF1, eIF1A, eIF3 and eIF5) to form the 43S preinitiation complex (Kapp and Lorsch 2004) (Figure 5). The eIF1, 1A and 3 promote the dissociation of 80S ribosomes (Kapp and Lorsch 2004). The eIF4F, including eIF4A, eIF4B, eIF4E and eIF4G, bind mRNA by a mechanism involving the initial recognition of the m7G cap at 5’-end of mRNA by eIF4E (Gingras et al. 1999). Then, mRNA binds to the 43S complex by the association of eIF3 and eIF4G (Gross et al. 2003; Prevot et al. 2003). The 43S complex scans along the mRNA in a 5’ to 3’ direction towards the initiation codon, base-paired with the anti-codon of met-tRNAi (Lopez-Lastra et al. 2005). Moreover, eIF1 interacts with the eIF1A to promote scanning of the start codon (Pestova and Kolupaeva 2002). There is another factor, the poly (A) binding protein (PABP), facilitating mRNA binding to the 43S complex. PABP interacts with eIF4G to circularise mRNA by linking the 5’ cap and poly(A) tail in a “closed loop” (Figure 5). This association stimulates mRNA binding to the 43S complex, by enhancing eIF4F binding to the capped 5’ end of mRNA (Kahvejian et al. 2005). Initiation ends when the initiation factors are released from the complex and the 60S ribosomal subunit joins to form the 80S ribosomal (initiation complex) and leave met-tRNAi in the ribosomal P site (Lopez-Lastra et al. 2005).

Elongation (Figure 6)

The elongation phase involves three distinct steps that are repeated many times during the formation of a polypeptide chain. The order of AAs is specified by the sequence of codons in the mRNA. Moreover, each AA is specific to its cognate tRNA to form amino acyl-tRNA (aa-tRNA) for which there is one aa-tRNA synthase per aa-tRNA pair (Guth and Francklyn 2007). The elongation cycle requires the eukaryotic elongation factors (eEFs), including eEF1and eEF2, to catalyse this process. First, the eEF1A picks the aa-tRNA in the presence of GTP, and then the aa-tRNA●eIF1A●GTP complex enters the empty A-site on a ribosome. The anticodon of the incoming aa-tRNA needs to be matched against the mRNA codon positioned in the A-site. As the three bases in the codon can be arranged in 64 different combinations, the translational machinery must be able to select the aa-tRNA carrying the matching anticodon. When the correct three-base anticodon forms a complementary base pair with the codon on mRNA, the GTP is hydrolyzed leading to eEF1A●GDP dissociating from aa-tRNA. The resulting of eEF1A●GDP binds to eEF1B complex which facilitates exchange of GDP to GTP on eEF1A. The eEF1A●GTP now is ready to accept the next aa-tRNA. In the second step, a peptidyl transferase reaction catalysed by the ribosome itself occurs immediately after the accommodation of the correct aa-tRNA in the ribosomal A-site. The growing polypeptide in the ribosomal P-site is linked to the new AA in the A-site via a peptide bond. The reaction leaves an empty tRNA in the ribosomal P-site and the new peptidyl-tRNA in the A-site. The last step is the translocation, which promotes the ribosome’s translocation along the mRNA by the length of one codon. Translocation is catalysed by the eEF2 and subsequent GTP hydrolysis. After the translocation, the ribosome is in the position of having an empty tRNA in the E site, the peptidyl-tRNA in the P site, and the next codon of mRNA in the A site, available for interaction with a new aa-tRNA. These reaction steps are repeated until the ribosome encounters an in-frame stop-codon. At this point, the translation is terminated (Kasinath et al. 2006; Frank et al. 2007; Groppo and Richter 2009; Ling et al. 2009).

Termination (Figure 6)

The final step is termination which involves the release of the polypeptide chain from mRNA. The three stop codons (UAA, UAG and UGA), the eukaryotic release factors (eRFs) and one GTP are required. The eRF1 recognizes one of three stop codons and binds to the ribosome in the place of a tRNA (Kisselev et al. 2003). This event along with binding of the eRF3, facilitates eRF1 stop codon recognition and stimulates GTP hydrolysis to release the polypeptide chain (Salas-Marco and Bedwell 2004; Fan-Minogue et al. 2008).

Regulation of translation at the initiation step

Translation is an important regulatory step in cellular protein synthesis. It is not only a metabolic pathway, but also a signaling pathway because most regulation of protein synthesis occurs at translation. In addition, the dominant mechanism of control of global protein synthesis occurs via the phosphorylation/dephosphorylation of the translation components, primarily of initiation and elongation factors (Sonenberg and Hinnebusch 2009). It is established that AAs are important factors in the regulation of intracellular signal transduction pathways involved in the control of translation. The essential AAs have been found to regulate signaling which modulate mRNA translation through the binding of met-tRNAi to the 40S ribosomal subunit to form the 43S preinitiation complex and the binding of mRNA to the 43S preinitiation complex (Kimball and Jefferson 2005).
The first regulated step of translation at the initiation step involves the binding of met-tRNAi to the 40S ribosomal subunit to form the 43S preinitiation complex by the phosphorylation of the -subunit of eIF2. In the later step of initiation, the bound GTP of eIF2 is hydrolyzed to GDP, and the eIF2●GDP binary complex is released from the ribosome. To reform the active ternary complex, eIF2 binds to met-tRNAi, and the GDP is exchanged to GTP. This guanine nucleotide exchange reaction is catalyzed by a second initiation factor eIF2B. The mechanism for regulating eIF2B activity is through phosphorylation of the – subunit of eIF2. The phosphorylation of eIF2 converts it from a substrate into a competitive inhibitor of eIF2B, effectively sequestering eIF2B into an inactive complex. Because translation of essentially all mRNA begins with met-tRNAi, the phosphorylation eIF2 results in a decline in the synthesis of almost all proteins (Kimball 2002).

Table of contents :

GENERAL INTRODUCTION
PART I: SCIENTIFIC BACKGROUND
I. Amino acid and protein metabolism
1. Amino acid metabolism
1.1. Digestion of dietary proteins
1.2. Interorgan amino acid metabolism
2. Protein metabolism and regulation by nutritional conditions
2.1.Protein Synthesis
2.1.1. Initiation
2.1.2. Elongation
2.1.3. Termination
2.1.4. The regulation of the translation at the initiation step
2.2 Protein Degradation
2.2.1. Lysosomal pathway
2.2.2. Ubiquitin-proteasome system
2.2.3. Other cytosolic proteolytic systems
II. Amino acids and their sensing elements
1. Functions of amino acids
2. Amino Acid Sensing Pathways
2.1. The mTOR Transduction Pathway
2.1.1. The mTOR signaling pathway
2.1.2. Effect of nutritional conditions on mTOR transduction pathway
2.1.3. The Role of mTOR in the regulation of translation
2.1.4. The Role of mTOR in the regulation of translation proteolysis
2.2. The AMPK Transduction Pathway
2.2.1. The AMPK signaling pathway
2.2.2. Effect of nutritional conditions on AMPK transduction pathway
2.2.3. The Role of AMPK in the regulation of translation
2.2.4. The Role of AMPK in the regulation of translation proteolysis
2.3. The GCN2 Transduction Pathway
2.3.1. The GCN2 signaling pathway
2.3.2. Effect of nutritional condition on GCN transduction pathway
2.3.3. The role of GCN2 in the regulation of translation
2.3.4. The role ofGCN2 in the regulation of translation proteolysis
3. Amino acid transporters and their role as nutrient sensors
III. Relation between protein metabolism and regulation if energy metabolism
1. The role of mTOR in the regulation of energy metabolism..
2. The role of AMPK in the regulation of energy metabolism
3. The role of GCN2 in the regulation of energy metabolism
PART II: PERSONAL WORK
I. The effect of amino acid levels on translation and the transduction signaling pathways involved in these effects in the liver
1. Respective role of amino acids, insulin and glucose in the effect of HP diet on translation and the identification of the signaling pathways involved in these effects in rat liver
Publication 1: mTOR, AMPK and GCN2 coordinate the adaptation of hepatic energy metabolic pathways in response to protein intake in the rat
2. Complementary results
2.1. Identification of the amino acid signals
2.2. Effect of AICAR and Rapamycin on the mTOR, AMPK and GCN2 transduction pathways
II.Effect of high protein intake and amino acids on the proteolysis
1. Effect of high protein diet, amino acid and insulin on proteolysis and protein ubiquitination in liver
1.1 Role of mTOR and AMPK signaling pathways in the control of ubiquitin proteasome pathway in response to the increase of amino acid concentrations and insulin
Publication 2: Down-regulation of the ubiquitin-proteasome proteolysis system in response to amino acids and insulin involves the AMPK and mTOR pathways in rat liver hepatocytes.
PART III: DISCUSSION AND CONCLUSION
1. General discussion
2. General conclusion
3. Perspectives
REFERENCES

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