metabolism of macro- and micronutrients topic 1 metabolism...

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Metabolism of Macro- and Micronutrients Topic 1 Module 1.2 Metabolism of amino acids and proteins

Luc CYNOBER

Learning objectives • To define the general principles of protein turnover; • To learn how protein metabolism is controlled by hormones and mediators; • To accurately determine how and why amino acid pathways adapt to protein supply; • To understand why protein starvation ultimately leads to morbidity and mortality. Content 1. Protein turnover 2. Regulation of protein and amino acid metabolism 3. Metabolism according to prandial phase 4. From fasting to starvation 5. Adaptation to a low protein intake 6. Summary 7. References Key messages • Whole-body protein balance is a dynamic process for equilibrium in healthy adults; • Nutritional state and specific tissue balance combine with a number of other factors

(including hormones and mediators) to generate a positive or negative protein balance in a number of physiological or pathological situations;

• Given that each protein in the body has a specific function, proteins cannot be considered as a form of amino acid storage;

• This is the reason why protein wasting is responsible for morbidity in malnutrition states.

1. Protein turnover Proteins represent the largest pool of amino acids (AAs) in the body (Figure 1). In contrast, free amino acids represent only a small fraction, 85% of which are intracellular with 20% in the plasma and the interstitium. Exchanges between these three pools are tightly regulated, with the result that in a number of circumstances, net protein synthesis is dependent upon plasma amino acids (i.e. amino acid availability) (1, 2).

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Protein-bound AAs= 114 000 mmoles

Free amino acids

35 mmoles 150 mmoles 185 mmoles

Plasma

+ interstitial

AAs

Intracellular AAs

Total free AAs

Bound amino acids

Figure 1. Pools of amino acids in the body Indeed, free AA and protein pools are balanced, on a 24-hour basis, with the whole body protein synthesis of 300 g of proteins and an equivalent degradation (Figure 2). Food provides ≈ 80 g of proteins which is in equilibrium with a similar loss in urine (88%) or by other routes (12%).

Food intake80 g

Amino Acids Proteins

Muscle75 g

Colagen, elastin5 g

Synthesis300 g

Catabolism300 g

Elimitation80 g

Intestinal secretion

70 g

Feces10 g

Urine 70g

Skin2 g

Liver, lung, brain, intestin

120 g

- Albumin 12g- Fibrinogen 2 g- Globulin 2 g- Leucocytes 20 g- Hemeglobin 8 g

Absorption 60 g

Figure 2. Quantitative sources and utilizations of amino acids This leads to the concept of protein balance (B), which is the difference between synthesis (S) and catabolism (C) (Figure 3). By definition, the balance is at equilibrium in healthy adults (i.e. S = C = 0.34 g N/Kg).


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• By definition: in equilibrium

B = S - C

S = C = 0.34 g N/Kg

• But a dynamic equilibrium

Figure 3. Protein balance in adults

However, protein balance is a dynamic equilibrium with an anabolic phase in the post-prandial state and a catabolic phase between meals. Whether these variations result from variations in synthesis rate, catabolism rate or a combination of both remains to be elucidated. The various possibilities are illustrated in (Figure 4).

Figure 4. Protein balance in adults over a day

Net balance may be the result of variations of both protein synthesis and breakdown

The whole-body net balance is equal to the sum of the net balances of individual tissues. It is important to remember that the rate of protein synthesis (and catabolism) varies strongly from one tissue to another (Table 1). Of course, the mass of a given tissue has to be taken into consideration when calculating its contribution to whole-body net balance. For example, protein turnover is lower in muscle than in other tissues but the mass is much higher, resulting in the high contribution of muscle to protein turnover. In addition, net tissue balance may vary in opposite ways in certain situations; e.g. negatively in muscle and positively in the liver in inflammation states (Figure 5).


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Table1. Protein synthesis according to tissue

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WHOLE BODY

TURNOVER

LIVER

MUSCLE

Net balance = ∑ fractional balances

Figure 5. Whole body protein turnover may not reflect balances in individual

tissues. Here are presented balances during an inflammation state The evidence presented so far thus explains why whole-body net balance can be either positive or negative in various physiological or pathological situations, as shown in (Table 2).


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Table 2. Physiopathological modifications of protein turnover

2. Regulation of protein and amino acid metabolism by hormones and mediators Insulin exerts actions at every level of AA metabolism (3): a) it increases the cell transport of numerous AAs, especially in the muscle and liver. b) it favors net protein anabolism by decreasing protein breakdown. c) It inhibits gluconeogenesis by both decreasing the availability of precursors and

inhibiting key enzymes in this pathway. In addition, insulin exerts a synergistic effect in the control of protein turnover with amino acids. Following a protein-rich meal, some AAs (i.e. arginine, ornithine and leucine) stimulate insulin secretion, which in turn facilitates AA transport in muscle; insulin and muscle leucine then exert a synergistic effect on protein synthesis (4) (Figure 6).


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Figure 6. Amino acids stimulate insulin secretion which in turn stimulates amino

acid incorporation into proteins Growth hormone stimulates protein synthesis, increases cellular AA uptake and stimulates IGF-1/Sm-C release. IGF-1/Sm-C release in turn promotes protein synthesis. Testosterone simulates protein synthesis in muscle. Cortisol and glucagon coordinate to modulate protein turnover, leading to a unidirectional flux of nitrogen from the muscle to the liver: in muscle, cortisol increases protein breakdown and favors free AA release in the bloodstream (1) (Figure 7). In the liver, glucagon stimulates AA uptake and favors their use in gluconeogenesis.

urea

Figure 7. Coordinated action of cortisol and glucagon for gluconeogenesis from amino acids


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Catecholamines are traditionally considered as catabolic hormones. This is true for the metabolism of lipids and carbohydrates. However, for proteins, catecholamines are actually anabolic, promoting proteosynthesis and inhibiting protein breakdown in muscle and possibly the liver (1). Pro-inflammatory cytokines: Physiologically speaking, pro-inflammatory cytokines play an only minor role (except, perhaps, in the elderly). In disease, however, pro-inflammatory cytokines like tumor necrosis factor α and interleukins 1 and 6 are overproduced and act synergistically with glucagon and cortisol on AA metabolism. This is the cytokine network (Figure 8), which makes it extremely difficult to accurately determine the precise role of each cytokine.

Figure 8. The cytokine network An example of cytokine interactions is given in (Figure 9): after skin injury, cell-to-cell interactions lead to a net production of TNFα by the wound. TNFα binds to Kupffer cells in the liver which then elicit interleukin-6 (IL-6) production. In turn, IL-6 modulates protein synthesis (↑ acute-phase proteins, ↓ nutritional proteins) in hepatocytes.


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[slide 11]

Wound

Blood

Liver

Acute phase proteins

IL-6

TNFα

Figure 9. Paracine and endocrine effects of cytokines

3. Metabolism according to prandial phase In the fed state, after digestion and transport into enterocytes, AAs are used for neosynthesis (e.g. nucleotide synthesis) and energy supply (mainly glutamine). The intestine does not actually play a regulatory role in AA disposal. This role is taken on by the liver, which uses AAs for protein and glycogen synthesis and oxidizes the surplus in order to avoid intoxication (note: as a matter of fact, most AAs or their metabolites may act as mediators in the brain, and excess intakes should therefore be avoided). The splanchnic area retains around 50% of the total AAs ingested. The remainder is made available at the periphery, mainly for protein synthesis (2) (Figure 10).


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Food intake

CO2

Purins, pyrimidins

Protein synthesis

Intestine

CO2

Glycogen

Protein synthesis

Liver

Proteinsynthesis

Peripheral tissues

Glycogen

Glc oxidation

neuroendocrinological effects

Figure 10. Sequential use of amino acids arising from food intake In the post-absorptive state, glycogen stores are rapidly depleted, and so gluconeogenesis is activated to ensure an adequate glucose supply to glucose-dependent tissues. Theoretically (Figure 11), most AAs are glucose precursors. However, in vivo, AAs are handled in multiple metabolic pathways, and the ability to produce glucose is in fact mainly the fate of alanine, glutamine, glycine and proline. Gluconeogenesis mainly occurs in the liver, whereas amino acids are located (in the form of proteins) in the muscle. Therefore, gluconeogenesis from AAs implies 1) the transfer of AAs from muscle to the liver, and 2) the removal of the amine moiety from the AAs. This latter process is achieved by ureagenesis (Figure 12). Glucose produced by the liver is then taken up by the muscle, where it is degraded anaerobically into pyruvate which is transaminated into alanine, again released into the bloodstream. This forms the glucose-alanine cycle, also called the Cahill cycle (5).

HisGln

Glu Orn Arg

α-cetoglutarate

Fumarate

Phe

Thr

Gly

Ser CysAla

Piruvate

Oxaloacetate

Asp

Asn

Glucose

G6P

PEP Succinyl CoA

Met

Val

Ile

Figure 11. Glucose synthesis from amino acids


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LIVER BLOOD MUSCLE

Glucose

GlucoseGlucose

Pyruvate Pyruvate

Alanine Alanine

Alanine

- NH2

UREA

- NH2

AMINO ACIDS

Figure 12. Cahill cycle (alanine-glucose cycle) The transamination of pyruvate into alanine implies the conversion of glutamate into α-ketoglutarate, with glutamate needing to be regenerated in order to allow the cycle to continue to work. This is the task of branched-chain amino acids (BCAA) (6) (Figure 13). However, BCAAs cannot be synthesized de novo in humans, and their sole source in the post-absorptive state is protein breakdown. In other words, gluconeogenesis is the result of a transfer of AAs from the muscles to the liver at the expense of muscle breakdown, with the irreversible loss of nitrogen in liver ureagenesis. In terms of energy, the balance is extremely weak at the whole-body level, but at the organ level the alanine-glucose cycle allows the transfer of energy from an energy-rich organ (i.e. the liver, through oxidation of free fatty acids) to an energy-depleted tissue (i.e. the muscle). Note that the kidney also contributes to gluconeogenesis. Recent findings suggest that the intestine also has some gluconeogenic activity in certain situations, where the major precursor is glutamine (7).

ACTIN-MYOSIN BCKA

BCAA

Amino acids

proteolysis

Carbon chains

α - kg

GLU

GLN

NH4+

Pyr

Ala

glycolysis

Glucose

Figure13. Source of nitrogen for alanine synthesis in the muscle


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4. From fasting to starvation With time, the organism adapts to fasting. Ketogenesis increases as a result of the accumulation of free fatty acids in the liver. Some AAs (e.g. leucine) also contribute to ketogenesis. As a consequence, glucose utilization decreases, thus freeing-up certain AAs (and therefore proteins). However, with time, this adaptive process becomes limited, and gluconeogenesis is able to restart, leading to muscle depletion and, ultimately, death (Table 3). Table 3. Protein metabolism and starvation

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Protein metabolism and starvation

J1 Protein phase

Requirements covered by

gluconeogenesis*

↓ prot. synthesis N balance

J3 Ketonic phase

FFA leucine

↓ prot. synthesis ↓ prot. catabolism N balance ≈ 0

Jx Ketone bodies

Jn → death gluconeogenesis ↑↑ proteolysis

*200 g proteins →120 g Glc

[slide 16]

5. Adaptation to a low protein intake The regulation of ureagenesis is very efficient in adapting to excess nitrogen and removing it. This is the result of the effects of arginine activating N-acetylglutamate (NAG) synthase producing NAG, which in turn allosterically activates carbamoyl-phosphate synthase (CPS) (8) (Figure 14). Conversely, in the case of low protein intake, this regulatory mechanism is less efficient, and another mechanism is at work: i.e. a decreased supply of arginine to the liver due to the activation of conversion of arginine into citrulline in the intestine. Indeed, citrulline is neither taken up nor released by the liver. In turn, citrulline is taken up by the kidneys which possess the enzymes allowing production of the arginine required to sustain tissue needs (4, 9) (Figure 15).


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ARG

ARG

urea

ORN

NH4+ HCO3

–

CPS

OCT

CIT

Carbamoylphosphate

NAG

NAGS

GLN

+

GLU

GLNase

GLN

NH4+

⊕

⊕

⊕

Figure 14. The ureagenesis pathway

urea

Intestine Liver

ARG ureagenesisARG

Kidney

CIT CIT CIT

ARG

ARG

Figure 15. The arginine-citrulline-arginine interorgan cycle 6. Summary In the event of starvation or disease, AAs are used as an energy source at the expense of proteins. Now, proteins are not a form of AA storage (contrarily to glycogen for glucose or triglycerides for free fatty acids), since all proteins have specific functions (Table 4). This is why protein wasting (occurring in response to inadequate supply, whatever the reason) is responsible for morbidity, as described in (Table 5).


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Table 4. Nutrient Macro-molecular functions

Nutrient Macro-molecular FunctionGlucose Glycogen StorageFAA Triglycerides Storage

Amino acid Proteins

Thus: protein degradation → AAs for energy use= functional loss

Hormone carrier

Enzymes

Function (muscle...)

Mediators

Precursors of hormones

or mediators

Table 5. Net proteolysis leads to morbidity and mortality

7. References

1. Cynober L. Amino acid metabolism. In: Encyclopedia of Biological chemistry. Vol 1. New-York: Elsevier Inc. 2004;90-95. 2. Cynober L. Plasma amino acid levels with a note on membrane transport: Characteristics, regulation, and metabolic significance. Nutrition 2002;18:761-766. 3. Millward DJ. The hormonal control of protein turnover. Clin Nutr 1990;9:115-126. 4. Cynober L. Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition. Boca Raton: CRC Press 2004; 746p. 5. Felig P. Amino acid metabolism in man. Annu. Rev. Biochem. 1975;44:933-955. 6. Harper AE, Miller RH, Block KP. Branched-chain amino acid metabolism. Annu. Rev. Nutr. 1984;4:409-454. 7. Mitthieux G. New data and concepts on glutamine and glucose metabolism in the gut. Curr. Opin. Clin. Nutr. Metab. Care 2001;4:267-271. 8. Meijer AJ, Lamers WH, Chamuleau RA. Nitrogen metabolism and ornithine cycle function. Physiol. Rev. 1990;70:701-748. 9. Husson A, Brasse-Lagnel C, Fairand A, Renouf S, Lavoinne A. Argininosuccinate synthetase from the urea cycle to the citrulline-NO cycle. Eur. J. Biochem. 2003;270:1887-1899.


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