Protein

Amino acidsbreak down synthesis

Amino acidsMuscle

Blood

de novo

synthesis

Oxidation

Transamination

AlanineGlutamineGlutamate

Schematic protein turnover and metabolic fates

Fed-state gains and fasted state lossesin muscle protein balance

Skeletal muscle mass is maintained by normal protein feeding.Feeding refreshes muscle protein to improve muscle function, permitting more physical activity.

Fed state gains are enhanced, fasted state losses are less

Improvement inimmune functionstimulation ofprotein synthesis

Muscle will not improve with protein feeding alone!

Overfeeding of protein increases insulin resistance-muscle proteolysis

blood kidney urine

Degradation of amino acids

purine nucleotide cycle

IMP

P

biosyntheticreactions

excreted directly in the urine

(liver)

rich protein diet:Catabolised for energy,postprandial gluconeogenesis stored as liver glycogen

Starvation, catabolicstates: Gluconeogenesis

PEPCK Glucose-6P-ase

PEPCK Glucose-6P-ase

Amino acid catabolism I: Fate of the nitrogen

amino acid

keto acid

transamination

-keto-glutarate

Glutamate

NH4oxidative deamination

Ser

Thr

His

Asp

Gln

- Amino group from the majority of amino acids is collected by glutamate (by transamination) in the hepatocytes.

- Liberation of the amino group in the formation of NH+4 by GDH.

Central role of glutamate in nitrogen metabolism

L-glutamate dehydrogenase reaction

glutamate

glutamine

intestinal bacteria

other amino acidsNH4+

Glutamate in amino acid synthesis, degradation and interconversion

Allosteric regulation of glutamate dehydrogenase

Glutamate + oxaloacetate (OAA) <---> -ketoglutarate + aspartate

Glutamate + pyruvate <---> -ketoglutarate + alanin

GOT ASAT

GPT ALAT

Catabolism of L-amino acidsTransaminases (aminotransferases)

Coupled transamination reaction

                                                        

  

Pyridoxal phosphate (PRP) and PRP in aldimine linkage to the lysine residue of the transaminase (Schiff-base)

PRP

                                                                                                           

Different forms of pyridoxal phosphate during a transamination reaction

R1-

R1

pyridoxamine phosphate

R2-

E

R2

Specific pathways for the deamination of amino acids (minor routes)

cystein desulphhydrase

D-amino oxidases (FAD), L-amino acid oxidase (FMN)

Serine dehydratase

Metabolism of serine for gluconeogenesis

Transport of ammonia

Concentration of ammonia in the systemic blood is very low (25-50μmol/L), toxic to the brain.

Transport: glutamine and alanine (muscle) glutamine ( brain)Glutamine: non-toxic carrier 0.5-0.8mM in arterial plasma, 20-

25% of circulating free amino acids precursor for synthesis of many nitrogen containing

compounds metabolic fuel for rapidly dividing cells generates glutamate and GABA in the brain

Glutamine - principle non-toxic carrier of nitrogen

Intracellularly – muscle pool – released in response to stress, hypercatabolic states brain – glutamine-glutamate cycle- GABA liver – catabolised - substrate for ureagenesis and gluconeogenesis kidney – catabolised - ammoniagenesis and gluconeogenesis

muscle, lung, adipose - major sites of glutamine release to blood

Glutamine in diet

low glutaminase

glutaminase glutamine synthetasecompartmentalised

de novo synthesis: L-Glutamate + NH4+ + ATP L-Glutamine + ADP+ Pi

Glutamine transport, interorgan metabolism of glutamine

Plasma Glutamin

carbon sceleton:

glycogenglucose

acid-basebalance

Glutamin proline,

ornithine,citrulline, alanine

GutNH+

4 urea

NH3

portal vein liver

glutamine+H2O glutamate + NH3 glutaminase

Muscle

Muscle Lung/adipose

release

uptake

glutamine synthetase

Kidney

Liver

Glutamate-glutaminecycle

Brain

Scource of ammonia in different tissues: 1. degradation of amino acids transdeamination (transamination+GDH) minor patways 2. deamination of other compounds N-containing side chains of nucleotides neurotransmitters 3. ammonia production in the large intestine by bacteria portal vein, direct transport of ammonia.

Urea cycle

Function: 1. prevents ammonia levels from rising too high when large amounts of amino acids are catabolized 2. urea cycle enzymes: extrahepatic arginine synthesis

The liver receives both amino acids and ammonia from circulation

Biosynthesis of urea in the liver

ORNT1

ORNT1

55-100g protein/day

The liver receive both ammonia and amino acids from the circulation

GDH and major aminotransferases catalyze reactions close to equilibrium

Quantitative aspects of nitrogen incorporation, regulation?

1. Short term: NAG an allosteric regulator of CPSI and glutaminase activity

Regulation of the urea cycle

2. Long term: high protein diet: transcriptional regulation. Hepatic glycogen syntesis Caloric restriction: increased protein catabolism – CPSI induction (cAMP responsive element), glucose need.

ORNT1 - increased transcription.

mitochondria

Arginine +

increased amino acid catabolism

increase in NAG

increased flux with constantammonia concentrationincrease in glutamate, more NAG

glutaminase +

Hyperammonemias deffect: carbamoyl phosphate synthetase

deffect: ornithine transcarbamoylase

NH4+

CPSD

OTCD

CP cytosol, pyrimidine synthesis, orotic acid

Inherited urea cycle diseases (+liver failure)

Having no urea cycle, brain relies on glutamine synthetase for the removal of exes ammonia

Hyperammonemia Brain edema, convulsions, coma

Change in astrocyte morphology: cell swelling astrocytosis

acutehyperammonemia

chronichyperammonemia

Changes in expression of glutamate transporters in astrocytes.

NH3

Scriver et.al.The metabolic and Molecular Bases of Inherited Deseases,2001

Hyperammoniemic encephalopathy

Computer axial tomography scan of the head of hyperammonemic encephalopathyin the composite case of ornythine transcarbamoylase deficiency.A. CT within normal limits upon admissionB. CT scan after tonic seizure with bilateral hemispheric edema with effacement of cerebrospinal fluid spaces.

Brusilov: Rev. in Mol. Medicine,2002

The actrocyte demonstrating its relationship with other structures in the brain

Brusilov: Rev.in Mol. Medicine,2002

The glutamate synapse, effect of NH3 on the the Glutamate-glutamine cycle

Felipo et.al.:Progress in Neurobiology,2002

NH3

intracellular Glu depletion

extracellular accumulation

Ca2+

NOBrain injury

glutamine synthetaseglutaminase

Treatment: - limited nitrogen diet - arginine becomes an essential amino acid - detoxification reactions as alternatives to the urea cycle, ATP dependent

Hepatic metabolism of glutamine, zonal distribution of glutaminase and glutamine synthetase

detoxify

high capacitylow affinity

bulk remaining

glutaminase

glutamine synthetasehigh affinity

Sequential synthesis of urea and glutamine – efficient to ensure systemic/nontoxic level of ammoniaAmmonium ion - feed-forward activator of synthesis of glutamate and N-acetyl glutamateHepatic synthesis of glutamine – acid-base balance. Decrease pH – activation of glutamine synthetase –sparing of glutamine

at metabolic acidosis:net producer of glutamine

spare aminonitrogenin starvation

Interorgan metabolism of glutamine during metabolic acidosis

Acut response: plasma glutamine Renal extraction of glutamine

uptake

release

glutamine synthetase

incresased ammonia excretion

increased gluconeogenesis PEPCK pH

The urea cycle – part of the metabolism centered around L-arginine

L-arginine is semiessential amino acid,synthesized in collaboration. The intestinal – renal axis.

urea

circulation

EC, nerve cells,macrophages

Arg

AS, AL

NO+citrulline

arginase

CAT-1

Arg

CAT-1

Arg

Bioavailability of arginine is complex1.Exogenous supply2.Endogenous release3.Arginine resynthesis4.Arginine catabolism, arginase5.Arginine transport

strict carnivorssmall bowel, kidney diseaseconditions with elevatedamino acid catabolism:inflamation, sepsis, recovery.

Insufficient Arg:

Arginine availability: arginases and NOS use a common substrate

Citrulline recycled to Arg, in kidney +other tissues

- cell proliferation repair

Fate of citrullin: intercellular citrulline-NO cycle

inflammatory stimuli

“Arginine paradox”

Km for eNOS: 1.4-2.9 μmol/LIntracellular L-arginine: 0.5-2mmmol/L eNOS should be saturated with substrate

Despite high cellular arginine, and low Km of eNOS:

arginine, citrulline supplementation “in vivo” improves NO function: increased vasodilation decreased leukocyte adhesion decreased platelet adhesion

Possible reasons: altered arginine transport increased arginase activity compartmentalisation of arginine

Arginine is the largest scource for NO production

NO,(EDRF): labile, common gasNO-cGMP-mediated effects: smooth muscle cell relaxation in EC: cGMP-prostacyclin mediated decrease in platelet aggregation decrease in leukocyte adhesion and migration

NO functionality – vascular health/vasculopathy - production of NO – depends on NOS activity

Supplementation:Arg: low bioavailability, increased arginaseCit: Arg synthesis, increased NO levelsGln: major vehicle of transport, Glu-gluthatione reduction of oxidative stressGly: restores NO balance at increased nutrient demands

Meth, Homocys: increased cardiovascular riskLys: decreases Arg transport