Università degli Studi di Trieste

Graduate School in MOLECULAR BIOMEDICINE

PhD Thesis

BIOMARKERS TO DEFINE OPTIMAL

PROTEIN REQUIREMENT

Filippo Giorgio Di Girolamo

XXVII ciclo – Anno Accademico 2013/2014

INDEX
ABSTRACT ............................................................................................................................ 3
1. INTRODUCTION .............................................................................................................. 6
1.1. Protein composition and metabolism .................................................................... 6
1.1.1. Protein synthesis .................................................................................................. 9
1.1.2. Protein breakdown ............................................................................................. 12
1.2. Protein requirement in adults .............................................................................. 14
1.2.1. Recommended dietary allowances for protein in adults .................................... 15
1.2.2. Tracer methods to define amino acid kinetics. .................................................. 17
1.3. Factors influencing protein requirements in different conditions. ................... 20
1.3.1. Sarcopenia ......................................................................................................... 20
1.3.1.1. Anabolic resistance ................................................................................. 25
1.3.1.2. Insulin resistance ..................................................................................... 29
1.3.1.3. Inflammation ........................................................................................... 35
1.3.1.4. Oxidative stress ....................................................................................... 39
1.3.1.5. Hypoxia ................................................................................................... 44
2. AIM ................................................................................................................................... 46
3. METHODS ....................................................................................................................... 47
3.1. Experimental procedures ..................................................................................... 47
3.1.1. Whole body protein kinetics ............................................................................... 47
3.1.2. Post-prandial anabolic resistance ..................................................................... 49
3.1.3. Post-prandial insulin resistance ........................................................................ 50
3.1.4. Amino acids ........................................................................................................ 51
3.1.5. Oxidative stress .................................................................................................. 52
3.1.6. Lipid pattern....................................................................................................... 54
3.1.7. Systemic inflammation ....................................................................................... 55
3.1.8. Membrane fatty acid composition ...................................................................... 55
3.2. Human study protocols ......................................................................................... 57
3.2.1. The PLANHAB study (experimental hypoxia and bed rest)............................... 57
3.2.2. The PANGeA study (bed rest in the elderly) ...................................................... 59
4. RESULTS ......................................................................................................................... 61
4.1. The PLANHAB study (experimental hypoxia and bed rest). ........................... 61
4.1.1. Body composition ............................................................................................... 61
4.1.2. Whole body protein kinetics ............................................................................... 61
4.1.3. Plasma lipid profile............................................................................................ 64
4.1.4. Insulin sensitivity. .............................................................................................. 66
4.1.5. Inflammatory responses. .................................................................................... 68
4.1.6. Oxidative stress .................................................................................................. 69
4.1.7. Plasma and erythrocyte GSH amino acid precursor ......................................... 72
4.1.8. Red blood cell membrane lipid composition. ..................................................... 73
4.2. The PANGEA STUDY (bed rest in the elderly) ................................................. 74
4.2.1. Body composition ............................................................................................... 74
4.2.2. Insulin sensitivity. .............................................................................................. 75
4.2.3. Anabolic resistance ............................................................................................ 78
5. DISCUSSION ................................................................................................................... 82
5.1. Hypoxia decreases protein synthesis ................................................................... 84
5.2. Aging is characterized by anabolic resistance .................................................... 85
5.3. Aging is a protective factor against inactivity mediated insulin resistances?.. 87
5.4. Hypoxia per se improves oxidative stress ........................................................... 88
5.5. Hypoxia-bed rest interaction induces multiple metabolic changes. ................. 91
5.6. Identification of a new biomarker to evaluate anabolic (and insulin) resistance
to define optimal protein requirement. .............................................................. 94
6. CONCLUSIONS .............................................................................................................. 96
7. ACKNOWLEDGMENT ................................................................................................. 97
8. REFERENCES ................................................................................................................. 98

2
 ABSTRACT

BACKGROUD. Dietary proteins are the source of the amino acids required by the
body for tissue growth and maintenance. The Population Reference Intake (PRI) for
proteins, as defined by the European Food Safety Authority (EFSA) for healthy adults,
including the elderly, is 0.83 g/kg body weight/day. This amount is defined on the net
balance of body protein (or “nitrogen balance”, given by the difference between dietary
nitrogen intake and losses) equivalent to 0.66 g/kg/day plus a safety factor for
interpersonal variability and differences in proteins quality of mixed diets. The PRI,
however, is the minimum daily amount of protein needed to maintain the nitrogen
balance and avoid a progressive loss of lean body mass in healthy people with moderate
physical activity. Therefore nitrogen balance may not be adequate to define protein
requirement in adults and especially in ageing characterized by loss of muscle mass and
function (sarcopenia). Furthermore until recently the prevalent idea was that a protein
intake above PRI had no further benefits and on the contrary could impair health. These
believes are now under discussion, diets with higher protein intake have been shown
beneficial in the prevention and treatment of conditions such as sarcopenia, COPD and
type 2 diabetes mellitus. There is a need of more precise methods to define protein
requirement.
AIM. The aim of the present thesis is to investigate in human healthy volunteers new
biomarkers adequate to define optimal protein intake. Recent studies have determined
protein needs by measuring whole-body protein metabolism using stable labeled
isotope-amino acids.
METHODS. Our research group has applied two different metabolic methods based on
the most widely used tracer, i.e. D5-Phe stable isotope, in two experimental bed rest
campaigns (FP7 PLANHAB and INTERREG PANGaA) in healthy volunteers. BR is a
suitable model to investigate physiologic adaptation to inactivity.
MAIN RESUTLTS. FP7 PLANHAB. We applied the stable isotope infusion
technique, to assess the effect of physical inactivity and/or hypoxic condition on whole
body protein turnover as previously described in Biolo et al 2008. Chronic hypoxia has
been associated with an overall reduction in protein synthesis and in total plasma and
skeletal muscle protein content. During the PLANHAB study we investigated, through
a crossover randomization, the net effects of 10 days normobaric hypoxia (4000 mt.),
associated with either ambulatory conditions or BR, in 11 young (age 24±4 yr), healthy
and normal weight male subjects maintained on eucaloric diets. Main results. Hypoxia

3
in ambulatory conditions significantly decreased whole body protein turnover by
reducing both protein synthesis (-8±2%) and protein degradation (-8±3%). Hypoxia
during bed rest did not caused significant changes in protein metabolism.
INTERREG PANGaA. The skeletal muscle loss in aging is caused mainly by the
“anabolic resistance” i.e. the inadequate increase in the rate of protein synthesis in
response to nutritional-metabolic stimuli, including exercise, protein and amino acid
intake as well as insulin and insulin-like growth factor stimulation. As a consequence,
the net protein balance becomes negative leading to sarcopenia. The effects of ageing
on the anabolic resistance induced by inactivity are poorly investigated. During the
PANGeA study we had the opportunity to perform the second documented
experimental BR in in healthy elderly volunteers and the first comparing aged with
young subjects. To evaluate the anabolic resistance associated with ageing and
inactivity, we enrolled 7 young (23±1yr) and 8 elderly (59±1yr) normal weight
individuals, in a 14-d experimental BR protocol. We replaced our previous infusion
method with a new, simpler, safer and quicker technique, by which tracers are given
orally instead of parenterally, the all procedure is completed in two hours, instead of 6,
and only two blood draws versus 7 are sufficient. Main results. At baseline parameters
of anabolic sensitivity were comparable between young and elderly individuals. The
anabolic resistance significantly increased after BR in both groups (bed-rest effect
p<0.01), with a statistically significant bed-rest×group interaction (p=0.01). Anabolic
resistance increased significantly in elderly (18.5%±7.3%) more than in young
(5.2%±9.4%) subjects.
DISCUSSION. In the PLANHAB study, hypoxia in ambulatory conditions reduced by
the same level both protein synthesis and catabolism, as measured by isotope infusions,
suggesting an adaptive mechanism: the lower energy production and availability
induced by hypoxia associated with ambulatory condition. These modifications could
not have been revealed by the use of nitrogen balance method, showing the relevance of
more sophisticated analysis. The direct evaluation of the muscle protein metabolism
through an infusion of stable-labeled isotope tracer, considered the golden standard
methodology, gave us, in the PLANHAB study, reliable results in the early protein
metabolism changes during hypoxia and/or BR. This method however has the limit of
being complex, onerous and invasive, therefore being unsuitable for clinical evaluation.
In the PANGeA study we could confirm the presence of a reduced sensitivity to
anabolic stimuli in the elderly population compared to the young men. The elderly
subjects are therefore, more at risk to develop changes of protein metabolism induced

4
by inactivity. The simpler, timesaving and less invasive method we have developed for
the PANGeA study, on the other hand, could be applied to a wider ranges of
experimental conditions and clinical settings.

5
 1. INTRODUCTION

1.1. Protein composition and metabolism

Proteins are the major nitrogen components of the protoplasm of animal tissue,
representing 50% of the dry weight of animal cells. They are formed by a sequence of
amino acids (AA), the basic structural units, attached by covalent chemical bonds (i.e.
peptide bond). AA are characterized by the presence of both a carboxyl group (R-
COOH) and an amino group (R-NH3), nitrogen being equivalent to about 16% of
protein weight (Shils ME et al. 2006; Caballero B et al. 2012). There are 20 different
AAs commonly classified in essentials, EAA (or indispensable), which, since their
carbon skeleton cannot be synthetized by the animal body, need to be introduced by
diet, and non-essentials, NEAA (or dispensable), made in the body from carbon and
nitrogen precursors. In humans the EAA include: histidine, valine, isoleucine, leucine,
lysine, methionine, phenylalanine, threonine, tryptophan, and possibly arginine, while
the NEAAs are reported on figure 1. Cysteine and tyrosine are synthesized in the body
from the EAAs methionine and phenylalanine. AAs are further divided in: dibasic
(arginine lysine and histidine), diacidic (aspartic, glutamic acid), neutral-aliphatic
(glycine, alanine, serine, threonine, cysteine, cystine, methionine and the three
branched-chain AAs -BCAA- valine, leucine, isoleucine) and neutral-aromatic
(phenylalanine, tyrosine and tryptophan). In the human body proteins have multiple
roles. From structural to regulatory (e.g. hormonal activities) to functional (e.g.
enzymes, muscle contraction, transport, osmolality regulation, etc.) (Shils ME et al.
2006; Caballero B et al. 2012).
Proteins, consumed with the diet are enzymatically hydrolyzed in the digestive system
and them reach the peripheral circulation as free amino acids. These AAs mix with
those coming from the tissues protein catabolism forming the circulating free AA pool.
Amino acids follow one of the follow three major metabolic ways: a) incorporation into
tissue proteins (protein synthesis); b) catabolism by oxidation and nitrogen excretion; c)
synthesis of other nitrogen compounds, including purine bases, creatine and
epinephrine. The absorbed AAs through the vena porta reach the liver, where, with the
exclusion of the BCAAs, are catabolized at a rate influenced by the body requirements;
therefore when EAA intake is elevated, their catabolism is also increased. Through this
mechanism the liver regulates the amount of EAA obtained from the diet, which will be
available to the rest of the body. A small portion of the absorbed EAAs is used for the

6
synthesis of liver proteins and of visceral plasma proteins, secreted by the liver,
including albumin, transferrin, pre-albumin, etc. Finally, about one quarter of the diet
EAAs reach the general circulation. The BCAAs from the liver are transported to other
tissues, being metabolized mainly by muscle and kidney. On the other hand, NEAA
plasma concentration is not modified by the passage through the liver. The metabolic
pathways of NEAAs are showed in figure 1 (Shils ME et al. 2006; Caballero B et al.
2012).

Figure 1. Metabolic pathways of the dispensable amino acids.

From Shils ME et al. 2006.

7
Skeletal muscle is the major body site of protein metabolism, being the largest tissue in
the body (Shils ME et al. 2006; Caballero B et al. 2012). Muscle protein synthesis
(MPS) is influenced by AA availability after the intake of a meal containing proteins.
The post-prandial anabolic response is followed in the post-absorptive period, by a
catabolic phase (i.e. muscle protein breakdown, MPB) (Figure 2), the magnitude of
these metabolic cycles being influenced by the quantity and quality of protein intake
and the meal nutrient composition and pattern. During fasting the muscles release AAs,
mainly as alanine and glutamine (with a daily loss equivalent to about 50g of protein in
an 70kg individual) (Shils ME et al. 2006; Caballero B et al. 2012). Alanine is derived
by transamination between pyruvate from glucose, and the amino-groups of AAs
originating from MPB. Alanine carries nitrogen to the liver where gluconeogenesis
takes place. Glucose is formed from the carbon skeleton of alanine, while the amino-
group is either converted to urea or transaminated. Glutamine, formed in skeletal
muscle by transamination with glutamate, is transported to the intestine, where 50%
gets transaminated to alanine, thereafter carried to the liver for gluconeogenesis and
urea synthesis (Shils ME et al. 2006; Caballero B et al. 2012). Part of the glucose
produced by gluconeogenesis in the liver returns to the muscles, the all process being
called “glucose-alanine cycle” (Figure 2B).

CHAPTER 1 ■ PROTEINS AND AMINO ACIDS 25

Brain Brain
Muscle Muscle
PROTEIN PROTEIN
Ala Ala
AA’s ENERGY AA’s ENERGY
Gln Gln
CO2 CO2 CO2 CO2
O2 O2

Fat Liver Fat Liver

Glycerol GLYCOGEN Glycerol
TG TG
GLUCOSE GLUCOSE
FFA KETONES FFA KETONES

AA’s GLUCOSE

UREA UREA

Kidney Kidney

A Postabsorptive state B Starvation

Fig. 1.13. Interorgan flow of substrates in the body to maintain energy balance in the postabsorptive state (A) and after adaptation to starvation
(B). The schematic diagrams are patterned after the work of Cahill. In all states, energy needs of the brain must be satisfied. In the postabsorp-
Figure 2. Substrate interorgan flow in the post-absorptive state (A) and in
tive state, glucose from liver glycogenolysis provides the majority of the glucose needed by the brain. After liver glycogen stores have been
starvation (B).
depleted (fasting state), gluconeogenesis from amino acids from muscle stores predominates as the glucose source. Eventually, the body adapts
to starvation by production and utilization of ketone bodies instead of glucose, thereby sparing amino acid loss for gluconeogenesis. AA’s, amino
Fromacids;
ShilsAla,
ME et al. CO
alanine; 2006.
2, carbon dioxide; FFA, free fatty acids; Gln, glutamine; O2, oxygen; TG, triglycerides. (Redrawn with permission from Cahill
GF Jr, Aoki TT. Partial and total starvation. In: Kinney JM, ed. Assessment of Energy Metabolism in Health and Disease. Report of the First Ross
Conference on Medical Research. Columbus, OH: Ross Laboratories, 1980:129–34.)

previously for those amino acids for which their carbon and glutamine from a-ketoglutarate), they are excellent
skeletons can be easily rearranged to form gluconeogenic vehicles to remove waste N from muscle while avoiding
precursors. The remaining amino acids released from NH3 release. Alanine removes one and glutamine removes
protein breakdown and not used for gluconeogenesis may8 two Ns per amino acid. These observations led to the pro-
be oxidized. The amino acid N released by this process posal of a glucose-alanine cycle in which glucose made by
is removed from the body by incorporation into urea via the liver is taken up by muscle where glycolysis liberates
synthesis in the liver and excretion into urine via the kid- pyruvate. The pyruvate is then transaminated to alanine
The plasma levels of amino acids are also affected by dietary carbohydrate through a
mechanism involving insulin secretion. After digestion of dietary carbohydrates and
monosaccharaides absorption, the raising plasma glucose concentration stimulates
insulin secretion, favoring an increased insulin-mediated transport of most plasma
amino acids into the muscle cells (Caballero B et al. 2012). This effect is maximal for
BCAAs, whose plasma levels can fall as much as 40% after glucose intake (Caballero B
et al. 2012).
When amino acids are degraded for energy rather than entering in anabolic pathways,
the ultimate products are CO2 and water, produced through the pathways of
intermediary metabolism involving the tricarboxylic acid cycle, and urea, whose
synthesis allows the removal of toxic metabolites, such as ammonia (Shils ME et al.
2006; Caballero B et al. 2012).
The whole body protein turnover is the result of protein synthesis and breakdown
processes (Antonione R et al. 2008).

1.1.1. Protein synthesis

At the molecular level, regulation of protein synthesis depends both on transcriptional

and translational mechanisms. The concentration of ribosomes in tissues determines the
capacity for protein synthesis, therefore controlling the protein turnover rate. The
concentration of ribosome, inside the cells is influenced by nutrient intake (protein and
energy) and hormones (i.e. insulin, thyroid, growth hormone and glucocorticoids). The
regulation of the translational processes is exerted mainly through initiation, with
reversible phosphorylations known to control at least four separate steps of the initiation
cycle. This allows very rapid changes in protein synthesis. Peptide hormones (insulin
and insulin-growth factor 1, IGF-1), glucocorticoids and amino acids have all been
implicated in such regulation.
Studies performed in experimental cell models have revealed the importance of Akt
factors (also called protein kinase B) in the regulation of protein synthesis in skeletal
muscle. Akt factors are serine/threonine-specific protein kinases playing a critical role
in muscle hyperthrophy (Bodine SC et al., 2001). Activation of signaling cascades
involving IGF-1 and phosphatidylinositol 3-kinase (PI3K) induces Akt phosphorylation
and activation (Bodine SC et al., 2001). Akt, in turn, activates the eukaryotic translation
initiation factor 2B (eIF2B) by stimulation of glycogen synthase kinase-3β (GSK-3β)
(Rhoads RE 1999). In parallel, Akt can activate initiation of protein translation

9
stimulating p70S6 kinase (p70S6K) by mammalian target of rapamycin (mTOR)
protein-kinase (Terada N et al., 1994). Interestingly, a period of resistance training
exercise induces muscle hypertrophy associated with increases in phosphorylated Akt,
GSK-3b and mTOR (Leger B et al., 2009).
Insulin is a potent anabolic stimulus for MPS (Fujita, S et al. 2006). Insulin deficiency
leads to a protein catabolic state with loss of muscle mass, reversible only by insulin
therapy (Abu-Lebdeh HS & Nair KS 1996). Nonetheless, the mechanisms by which
insulin enhances muscle protein anabolism are still debated (Biolo G et al 1995; Nygren
J & Nair KS 2003; Wolf RF et al 1992; Denne SC et al 1991; Heslin MJ et al 1992;
Moller-Loswick AC et al 199). Some studies reported that this effect was due to an
increase in protein synthesis with no major changes, or some reduction, in proteolysis
(Biolo G et al 1995; Nygren J & Nair KS 2003; Wolf RF et al 1992); other studies
found a significant reduction in protein degradation with no significant changes in
protein synthesis (Denne SC et al 1991; Heslin MJ et al 1992; Moller-Loswick AC et al
1994).
A stimulatory effect of insulin on protein synthesis has been demonstrated in various
tissues, including skeletal muscle (Garlick PJ & Grant I 1988; O’Connor PM et al
2003). Furthermore, recent human experiments have shown that insulin can acutely
stimulate muscle protein synthesis by increasing the initiation of mRNA translation
(Guillet C et al 2004; Kimball SR et al 1997). Insulin can also reduce protein
breakdown by stabilizing lysosomes and reducing the activity of the ubiquitin-
proteasome pathway (Lee SW et al 2004).
Protein intake and amino acid availability are key regulators of muscle protein
synthesis. Acute AA administration up regulates muscle protein synthesis and such
effect is enhanced if the intake is associated with resistance or aerobic exercise (Biolo G
et al., 1997). In contrast to exercise, the anabolic efficiency of amino acid
administration is decreased during inactivity. The stimulatory effect of an amino acid
load on whole body protein synthesis in healthy volunteers at the end of a period of
experimental bed rest was reduced (-20%), when compared to individuals with normal
physical activity (Biolo G et al., 2004). Furthermore during bed rest, the rate of protein
turnover, in the fasting state, was decreased both at skeletal muscle and whole body
levels (Biolo G et al., 2004). Other studies showed that dietary protein restriction (i.e.,
0.6g protein/kg/d) led to 23% suppression of whole body protein turnover, in the fasting
state, when compared to adequate levels (i.e., 1.0g protein/kg/d) of protein intake
(Stuart CA et al., 1990). Other Authors, maintained leg mass and ameliorated the

10
muscle strength losses following 4 weeks bed rest by a daily supplementation of about
50g of essential amino acids (Paddon-Jones D et al., 2004).

11
 1.1.2. Protein breakdown

Several proteolytic systems can contribute to the degradation of muscle proteins, among
these, the most relevant are the following:
• The lysosomal-autophagic system which is present in all cells and involves acid
proteinases (i.e. cathepsins), active within a vacuolar structure capable of engulfing and
degrading complete organelles, ribosomes, as well as intracellular proteins and proteins
entering the cells via endocytosis.
• The ubiquitin-proteasome ATP-dependent system, widely distributed among
tissues, is characterized by relative broad protein specificity for the hydrolysis of
proteins and peptides. It involves two components: a) the recognition system
responsible for targeting the protein substrates toward proteolysis and b) the
multifunctional proteasome, causing the proteolysis. In the proteasome system,
degradation is carried out by the 26S subunit (Grune T et al., 2003; Grune T & Davies
KJ 2003), which is composed by the 20S and by the regulatory 19S complex, the latter
playing an important role in adenosine triphosphate (ATP)-dependent degradation
(DeMartino GN & Ordway GA 1998). Protein substrates are marked for degradation in
the 26S proteasome pathway by covalent addition of ubiquitin. This requires the
ubiquitin- activating enzyme (E1), specific ubiquitin-conjugating enzymes (E2), and
ubiquitin protein ligase enzymes (E3). The ubiquitin E3 ligases, atrogin1 and muscle
ring finger-1 (MuRF-1), are involved in skeletal muscle atrophy (Bodine SC et al. 2001;
Gomes MD et al., 2001). MuRF-1 was shown to be directly and indirectly upregulated
by Forkhead family of transcription factors (FoXO) (Stitt TN et al., 2004) and by the
NF-kB transcription factor (Sandri M et al., 2004). Ubiquitinated proteins are
recognized and bound by the 19S regulators of the 26S proteasome, that removes the
polyubiquitin chain and unfolds the substrate protein for final degradation into the 20S
core proteasome (Grune T et al., 2003).
• The calcium-activated calpain and calpastin pathway is responsible for the
initiation of proteolysis. In vitro and animal studies, showed that Ca2+-activated
proteases (Calpain) and the proteasome system play important roles in muscle protein
breakdown during muscle atrophy (Furuno K & Goldberg AL 1986; Ikemoto M et al.,
2001; Purintrapiban J et al., 2003). Moreover, caspase-3 may also contribute to selected
forms of muscle atrophy (Du J et al., 2004). Actomyosin complexes represent 50–70%
of muscle proteins (Tidball JG & Spencer MJ 2002). The proteasome system can
degrade only monomeric contractile proteins (i.e. actin and myosin) (Goll DE et al.,

12
2003), which, therefore, must be released from actomyosin complexes to be degraded
by the proteasome (Goll DE et al., 2003). Both calpain and caspase-3 can play a key
role in producing actomyosin disassociation (Du J et al., 2004; Goll DE et al., 2003;
Tidball JG & Spencer MJ 2002). Calpain activity is increased by an elevation in
cytosolic calcium concentrations (Goll DE et al., 2003). Caspases are cascade-activated
proteases triggered by several signaling pathways (Primeau AJ et al., 2002): whose
activation can result in protein breakdown and apoptosis.

13
 1.2. Protein requirement in adults

Daily protein requirement is influenced by multiple factors the most relevant being the
obligatory nitrogen losses which were measured in fasting subjects, with stable body
weight, with modest levels of physical activity (Shils ME et al. 2006; Caballero B et al.
2012; Guarnieri G et al 1998). On the average this value is equal to 4,4 g/day, about 27
g of proteins considering a conversion factor of 6,25 (1g N = 6.25g protein), being
nitrogen about 16 % of proteins by weight. This minimum protein requirement (0.34
g/kg/day) were raised to 0,8 g/day to adjust for the individual variability and for factors
such as the differences in biological values and of the net protein utilization of proteins
from different food sources. The biological value (BV) of proteins is an index of the
body capacity to utilize them in anabolic processes. It is equal to the ratio between the
retained nitrogen, RN and the absorbed nitrogen AN.

VB = RN/AN.

Where

AN = [Nitrogen intake – (nitrogen excreted in the feces after the intake of proteins –
nitrogen excreted on an aproteic diet)]

and

RN= AN – (urinary nitrogen after the intake of proteins – urinary nitrogen excreted on
an aproteic diet).

Proteins from animal sources have higher biological values than those from vegetable
food. The net protein utilization (NPU) defines, besides biological value also protein
digestive efficiency and it is equal to the ratio between the retained and the dietary
nitrogen (nitrogen intake).

NUP=R/I.

The correction factor for the definition of protein requirements considers an NPU of a
mixed diet, with proteins from different sources. The NPU of various proteins present in
the same food may also differ, as in the case of milk proteins, with whey and casein
being considered respectively a fast protein and a slow protein, in reaching the sites of
body protein synthesis (Shils ME et al. 2006; Caballero B et al. 2012; Guarnieri G et al
1998).

14
 DA

URINARY N (
6 AS NITROG
(mg N/kg/d
4 Urine 38
Feces 12
1.2.1. Recommended
2 dietary allowances for protein in adults Cutaneous 3
Other 2
0 Total 54
The latest recommended dietary allowances (RDAs) in the USA, Europe and Italy
0 2 4 6 8 10 12 14
have
Upper limit 70
TIME (days) (12 standard
maintained the indication for a daily protein intake of 0,8 g/kg body weight, in all
deviations)
Fig. 1.7. Time required for urinary nitrogen (N) excretion to stabilize
healthy adults,after
males and from
changing females, excluding
an adequate pregnancy
to a deficient proteinor lactation,
intake in young independently
Data from Munro HN. Amino ac
their relevance to parenteral nut
from age (World Horizontal
men.Health solid and broken
Organization (WHO) lines 2007;
are mean 61 standard
European Fooddevia-
Safety Authority
Nutrition. London: Churchill Livin
tion for N excretion at the end of the measurement period. (Data from
(EFSA) 2012).Scrimshaw
The adaptation of the
NS, Hussein MA,body to aE et
Murray lowal.or zero requirements
Protein nitrogen intake
of is shown in
man: variations in obligatory urinary and fecal nitrogen losses in young
figure 3: urinary N Jexcretion
men. drops dramatically
Nutr 1972;102:1595–604, in response to the protein-deficient
with permission.) N diet
appears in the fece
over the first 3 days and thereafter stabilize a new lower level of N excretion bycompletely day 8. absorb all dieta
placement of healthy subjects on a diet containing a mini- secreted into the gastroint
mal amount of protein. As shown in Figure 1.7, urinary N addition, N is lost from sk
excretion drops dramatically in response to the protein- shedding of dead skin cel
deficient diet over the first 3 days and stabilizes at a new occur through hair, menstr
lower level of N excretion by day 8 (40). so forth. As N excretion in
The N end products excreted in the urine are end prod- of subjects on a minimal pr
ucts not only of amino acid oxidation (urea and NH3) but increasingly important to
also of other species such as uric acid from nucleotide deg- nonurine, nonfecal routes (
radation and creatinine (Table 1.8). Fortunately, most of ious routes is shown in Tab
the nonurea, non-NH3 N is relatively constant over a vari- are not readily measurable
ety of situations and is a relatively small proportion of the loss under conditions of a p
total N in the urine. Most of the N is excreted as urea, but tation has greatly reduced u
NH3 N excretion will increase significantly when subjects discounted by use of a sim
become acidotic, as is apparent in Table 1.8, when subjects nonfecal N losses. Where
have fasted for 2 days (41). Table 1.8 also illustrates how into play is in the finer de
urea production is related to N intake and how the body occurs as a function of die
adapts its adaptation
oxidation oftoamino acids to follow amino acid pose of determining amino
Figure 3. Physiological a low or absent nitrogen intake
supply. In other words, with an ample supply, excess amino As discussed later, small ch
From Caballero B et al. 2012
acids are oxidized and urea production is high, but with an make significant changes
insufficient supply of dietary amino acids, amino acids are requirements using N bala
conserved and urea production is greatly decreased. Although the N balance
Table 1 also illustrates how urea production is related to N intake and how itthe body no information
provides
adapts oxidation of amino acids to their supply. system. An interesting analo
is illustrated in Figure 1.8,
N balance is represented b
TABLE 1.8 COMPOSITION OF THE MAJOR NITROGEN-
Table 1. Physiological nitrogen-related urea production
CONTAINING SPECIES IN URINE in the body is taken between “coins in”
not come to the conclusion
HIGH-PROTEIN LOW-PROTEIN FASTING into gum, however, even
N SPECIES DIET (g N/d) DIET (DAY 2)
to reach with the N balanc
Urea 14.7 (87%) 2.2 (61%) 6.6 (75%)
Ammonia 0.5 (3%) 0.4 (11%) 1.0 (12%) technique fails to provide is
Uric acid 0.2 (1%) 0.1 (3%) 0.2 (2%) within the system (i.e., insid
Creatinine 0.6 (4%) 0.6 (17%) 0.4 (5%) the system is where the cha
Undetermined 0.8 (5%) 0.3 (8%) 0.5 (6%) thesis and breakdown actua
Total 16.8 (100%) 3.6 (100%) 8.7 (100%)
arrows into and out of the
N, nitrogen.
From Caballero B et al. 2012 further illustration of this p
Data from Folin (1905) and Cathcart (1907), cited in Allison JB, Bird Figure 1.8, in which a pos
JWC. Elimination of nitrogen from the body. In: Munro HN, Allison
JB, eds. Mammalian Protein Metabolism. New York: Academic Press, been observed going from z
1964:483–512, with permission. (cases A to D). A positive

15
 In other words, with a high intake, excess amino acids are oxidized and urea production
is high, but with an insufficient supply of dietary amino acids, amino acids are
conserved and urea production is greatly decreased (Shils ME et al. 2006; Caballero B
et al. 2012; Guarnieri G et al 1998). A reduced protein intake increases the efficiency of
nitrogen retention and may therefore not be indicative of an improved tissue protein
anabolism. The validity of nitrogen balance in the definition of protein requirement
have been therefore questioned. Furthermore, any change in protein intake requires time
to reflect metabolic changes. Methods of assessment may also not be precise, collection
of urine may not be complete over the 24 hours and it is difficult to estimate
unmeasurable nitrogen losses through non-urinary, non-fecal routes (Shils ME et al.
C I F I C D I E T A R Y C O M P2006;
O N E N TCaballero
S B et al. 2012; Guarnieri G et al 1998), shown in Table 2.

TABLE 1.9 OBLIGATORY NITROGEN LOSSES BY MEN ON

Table 2. Physiological nitrogen-related urea production in the body
A PROTEIN-FREE DIET

DAILY NITROGEN LOSS

AS NITROGEN AS PROTEIN EQUIVALENT
(mg N/kg/day) (g PROTEIN/kg/day)
Urine 38 0.23
Feces 12 0.08
Cutaneous 3 0.02
Other 2 0.01
Total 54 0.34
6 8 10 12 14 Upper limit 70 0.44
TIME (days) (12 standard
deviations)
r urinary nitrogen (N) excretion to stabilize
Fromintake
equate to a deficient protein Caballero B et al. 2012Data from Munro HN. Amino acid requirements and metabolism and
in young their relevance to parenteral nutrition. In: Wilkinson AW, ed. Parenteral
broken lines are mean 61 standard devia- Nutrition. London: Churchill Livingstone, 1972:34–67, with permission.
end of the measurement period. (Data from
A, Murray E et al. Protein requirements of
ry urinary and fecal nitrogen losses in young
5–604, with permission.)
Recent studies have Nshown appearsthatin nitrogen
the fecesrequirements
because the are gut increased
does not in the elderly or
during acute and completely
chronicabsorb all dietary
illnesses, as protein
being and reabsorb allthrough
confirmed N higher-level
subjects on a diet containing a mini- secreted into the gastrointestinal tract (see Fig. 1.6). In
n. As shown in Figure methodologies.
1.7, urinary N addition, N is lost from skin via sweat as well as through
atically in response to the protein- shedding of dead skin cells. Moreover, additional losses
e first 3 days and stabilizes at a new occur through hair, menstrual fluid, nasal secretions, and
tion by day 8 (40). so forth. As N excretion in the urine decreases in the case
s excreted in the urine are end prod- of subjects on a minimal protein diet (Fig. 1.7), it becomes
o acid oxidation (urea and NH3) but increasingly important to account for N losses through
uch as uric acid from nucleotide deg- nonurine, nonfecal routes (42). The loss of N by these var-
ne (Table 1.8). Fortunately, most of ious routes is shown in Table 1.9. Most of the losses that
are not readily measurable are minimal (,10% of total N
3 N is relatively constant over a vari-
s a relatively small proportion of the loss under conditions of a protein-free diet in which adap-
Most of the N is excreted as urea, but tation has greatly reduced urinary N excretion) and can be
increase significantly when subjects discounted by use of a simple offset factor for nonurine,
apparent in Table 1.8, when subjects nonfecal N losses. Where the assessment of losses comes
s (41). Table 1.8 also illustrates how into play is in the finer definition of where zero balance
ated to N intake and how the body occurs as a function of dietary protein intake for the pur-
f amino acids to follow amino acid pose of determining amino acid and protein requirements.
, with an ample supply, excess amino As discussed later, small changes in N balance corrections
urea production is high, but with an make significant changes in the assessment of protein
dietary amino acids, amino acids are requirements using N balance.
roduction is greatly decreased. Although the N balance technique is useful and simple,
it provides no information about16 the inner workings of the
system. An interesting analogy for the N balance technique
is illustrated in Figure 1.8, in which the simple model of
N balance is represented by a gumball machine. Balance
 1.2.2. Tracer methods to define amino acid kinetics.

Tracers are compounds, chemically identical to the endogenous metabolites, but with
one or more atoms substituted by its nonradioactive stable isotope, thus allowing a
“labeling” useful to follow the metabolic fate of the compound (the tracee) being
evaluated (Wolfe RR, 2002). This substitution makes the tracers distinguishable and
measurable from the normal metabolites (Wolfe RR, 2002). Because isotopes differ
only in the number of the constituent neutrons they can be identify by mass
spectrometry (GCMS). The molar ratio of the amount of tracer isotope divided by the
amount of unlabeled material is called “tracer-to-tracee ratio” (TTR) or enrichment
(Wolfe RR, 1992). Isotopic methods are frequently used to study protein and amino
acid metabolism. Amino acids are constantly exchanged between the intracellular and
the extracellular pools, through the action of specific transporter. Amino acids from
intracellular protein catabolism enter the intracellular pool and can then follow three
paths: protein synthesis, catabolism inside the cell or passage into the plasma. Amino
acids have a Rate of Appearance (Ra) in the intracellular pool, since they can either
derive from plasma and/or protein catabolism, and a Rate of Disappearance (Rd) due to
their incorporation in a protein or degradation. Generally it is assumed that during the
study period every amino acid entering in the protein pool will be, either metabolized or
incorporated in to a protein, with an irreversible loss (Wolfe RR, 1992).
Furthermore it is also assumed that when the enrichment of the tracer in the pool is at
the plateau (i.e. isotopic equilibrium) the Ra of the compound (endogenous production
+ exogenous infusion) is equal to its Rd:

Ra=Rd

If the tracer is administered through a constant infusion, the kinetics is described by the
single-pool model. When the isotopic equilibrium is reached, the Ra of the tracee can be
calculated as the ratio between the amount of the infused tracer and the isotopic
enrichment at its plateau. A priming dose (bolus), at the start of the infusion, allows to
reduce the time required to reach the isotopic equilibrium (Wolfe RR, 1992).

Isotopic methods are applied through the following techniques:

1. Precursor incorporation
2. Tracer dilution

17
The precursor incorporation technique requires a constant infusion of a tracer, usually
after priming bolus, to measure, when reaching the isotopic equilibrium, anabolism
from its incorporation rate into a protein (Wolfe RR, 1992). The tracer dilution
technique is applied to measure the Ra and the Rd of a metabolite in a given pool. Since
the tissues do not separate the tracer form the trace, the Rd does not modify the tracer
substrate ratio, therefore any isotopic enrichment change is the consequences of the
dilution effects of the unlabelled substrate (Wolfe RR, 1992).
The most frequently used stable-labelled isotopes for the investigation of protein
metabolism are: 15N-Glycine, 13C-Leucine, 15N-Leucine, L-[ring-2H5]-Phenylalanine.
L-[ring-2H5]-Phenylalanine (D5-Phe) is very useful in the study of protein metabolism.
This essential amino acid is not peripherally catabolized, therefore, in the fasting state,
its irreversible disappearance from the pool can be due only to protein synthesis or
irreversible hydroxylation to tyrosine. On the other side, the appearance of the
unlabelled phenylalanine can only derive from protein catabolism. Thus the
administered D5-Phe produces an amount of 2H4-Tyrosine (D4-Tyr) indicative of net
protein catabolism. If together with the labelled phenylalanine a labelled tyrosine is
administered (e.g. 2H2-Tyr), it is possible to determinate the rate of phenylalanine
hydroxylation (Figure 4) (Matthews DE 2007).

Figure 4. Phenylalanine and tyrosine kinetics in human

These methods, when requiring intravenous infusions of an extemporarily-made

solutions, administered through two catheters, imply the preparation of apyretic and
aseptic infusate, a process which needs a dedicated area and controlled conditions, not
always readily available. Furthermore, the infusion protocols are usually time-
consuming needing up to 6 to 7 hours. All these factors can reduce the compliance of
the examined subjects and increase costs and efforts.
There are also other methods limitation that should be taken in to account. The model
shown in the figure 4 assumes that all proteins have a slow turn-over and therefore will
18
not modify the amino acids entering and living the pool, from catabolism and synthesis
respectively. However some proteins have a very short half-life (e.g. enzymes) (Wolfe
RR, 1992).

19
 1.3. Factors influencing protein requirements in different conditions.

As argued above, the guidelines for dietary protein need traditionally consider a similar
requirement for all adults, regardless of age or sex (0.8g/kg/day of protein) (WHO
2007; EFSA 2012). However, new evidences (Bauer J et al. 2014) have shown that a
higher dietary protein intake is effective to support good health, promote recovery from
illness and preserve functionality in elderly subjects (aged over 65 years) (Walrand S et
al 2011; Gaffney-Stomberg E et al 2009; Kurpad AV& Vaz M 2000; Morse MH et al
2001; Chernoff R. 2004; Morley JE et al 2010). An increased consumption of proteins
with diet can overcome the decline of the anabolic response to dietary protein, and
offset inflammatory and catabolic states associated with chronic and acute disease.
These conditions occur often in elderly individuals (Walrand S et al 2011), whose
generally consume less protein than young adults (Bauer J et al. 2014). An insufficient
protein provision can also lead to loss of muscle mass and strength and, as a
consequence, older people are at higher risk for conditions such as sarcopenia and
osteoporosis than are younger individuals (CederholmTE, et al 2011; Cruz-Jentoft AJ et
al 2010; De Souza et al 2010).

1.3.1. Sarcopenia

Sarcopenia from the Greek σάρξ, "flesh" and πενία, "paucity" defines a condition
characterized by reduced muscle mass, associated with loss of strength or performance.
The European Working Group on Sarcopenia in Older People (EWGSOP) has recently
defined sarcopenia as a “syndrome characterized by progressive and generalized loss of
skeletal muscle mass and strength, with a risk of adverse outcomes such as physical
disability, poor quality of life and death.” (Cruz-Jentoft AJ et al. 2010). Moreover, The
EWGSOP group classified sarcopenia as primary (or age related, associated with the
physiological changes induced by the aging process), and secondary (caused by other
factors such as inactivity, illnesses and nutritional problems, including malabsorption or
other gastrointestinal disorders or medications, causing appetite loss). Recently,
however, some authors disagreed with this etiological classification since in most cases,
muscle loss is induced by multiple factors, difficult to identify separately (Biolo G et al.
2014). This is particularly true in the ageing population, characterized by a higher
prevalence of chronic and acute diseases.

20
 Causes and consequences
Sarcopenia and the associated body composition changes are caused by multiple factors
including the so called “anorexia of aging”, i.e. reduced food intake, seen with
advancing age, hormonal changes, such as reduced synthesis of growth and sex
hormones, resistance to leptin, insulin and thyroid hormones, and neurodegenerative
processes. Other contributing factors include hereditability, intake of proteins (type and
quantity) and energy, vitamin D status, physical inactivity, increased adiposity and
chronic and acute diseases (Bauer J et al. 2013) (Figure 5).
Aging is characterized by changes in protein metabolism: protein turnover is reduced
with lower protein synthesis and increased catabolism, leading to a net decline in
protein synthesis. These changes are sustained by a higher splanchnic extraction of
amino acids, that impairs their availability, a blunted response to the anabolic stimulus
(anabolic resistance) of protein feeding and a reduced anticatabolic effect of insulin.

G. Biolo et al. / Clinical Nutrition 33 (2014) 737e748 741

CHRONIC AGING
DISEASES

SARCOPENIA
SKELETAL MUSCLE LOSS AND DYSFUNCTION
MASS CONTRACTILE METABOLIC MYOKINE
DEPLETION INSUFFICIENCY IMPAIRMENT DYSREGULATION

Anabolic Altered energy

resistance expenditure
Decreased
Insulin
Glutamine antioxidant
resistance
depletion capacity

MORBIDITY
Dynapenia, fatigue, disability and falls, impaired ventilation, osteoporosis,
bone fractures, dyslipidemia, metabolic syndrome, type 2 diabetes,
increased cardiovascular risk, impaired immunity, infections, etc.

MORTALITY
Fig. 3. Skeletal muscle loss and dysfunction characterize sarcopenia of aging and chronic diseases. Sarcopenia increases morbidity and mortality in aging as well as in cancer,
chronic obstructive pulmonary disease, chronic kidney disease, liver cirrhosis, heart failure, rheumatoid arthritis, HIV infection. /, causeeeffect relationships; [, increase; Y,
Figure 5. Loss of skeletal muscle mass and function characterize sarcopenia of
decrease.

both aging and diseases.

4.2.From
Energy expenditure
Biolo et al 2014and muscleefat interaction gain, is frequently observed in some patients with chronic diseases
and cancer as well as in sedentary lifestyle and in aging. In fact, total
Skeletal muscle mass is a major determinant of daily energy energy expenditure decreases substantially in aging resulting from
expenditure at rest and during physical activity. In physiological parallel changes in resting metabolic rate and activity.62 Resting
conditions, the metabolic rate of skeletal muscle accounts for about metabolic rate may be increased in selected chronic diseases.
20% of whole body resting energy expenditure.51 When measured Amyotrophic lateral sclerosis is characterized by severe disuse at-
by indirect calorimetry, resting metabolic rate is strictly propor- rophy, while resting energy expenditure is increased leading to
tional to lean body mass. In 6-week experimental bed rest studies 21 weight loss and cachexia.63,64 Hypermetabolism may also occur in
in healthy young volunteers, muscle disuse atrophy was associated sub-groups of stable patients with LC or COPD.65,66 Hypermetabolic
with 38% decrease in energy expenditure for physical activity52 and patients suffering from chronic diseases have a poor prognosis. The
with 6% decrease in resting energy expenditure, i.e., about 30e origins of such hypermetabolism need to be elucidated.
40 kcal/day for each kg of lean body mass lost throughout the
Besides amino acid availability, physical activity, with muscle loading, also exerts
anabolic effects, both in muscles and in bones. Inactivity is common in the elderly,
caused by loss of strength, overweight, balance and locomotion disturbances, fear of
falls, low motivation and presence of diseases (Biolo G et al. 2014). This sedentary
lifestyle may contribute to the decline of mass and function in muscle and bone tissues.
Bed rest, that is a condition of prolonged and total muscle unloading, in experimental
condition, causes a loss of 3-5% of lean body mass in young healthy subjects (Biolo G
et al. 2014).
The loss of muscle mass in sarcopenic subjects causes a reduction in the basal metabolic
rate and in physical activity energy requirements (Biolo G et al. 2014). These changes,
if not compensated by lower energy intake, or by a heightened energy expenditure,
through increased physical activity, as often is the case with ageing, may lead to an
increased fat deposition (Biolo G et al. 2014). Furthermore physical inactivity itself
leads within a two weeks, short term period to an accumulation of visceral fat, and
insulin resistance, as shown in young subjects who reduced experimentally their level of
physical activity to about 15% of the basal level (Biolo G et al. 2014). Sarcopenia
therefore is generally associated with an increase in the percentage of body fat which
can lead to a condition called sarcopenic obesity (Biolo G et al. 2014). This rise in fat
mass is observed in both sexes and at any given BMI, being present even in normal or
underweight individuals. Two different pathways can lead to sarcopenic obesity, the
first is that associated to conditions of positive energy balance, from overfeeding, or
long-term inactivity, in otherwise healthy individuals (Biolo G et al. 2014), the second
is observed in selected inflammatory states such as COPD, rheumatoid arthritis, chronic
kidney disease or chronic hearth failure (Prado CM et al. 2012). In these conditions the
increased level of cytokines and the hormonal changes are not associated with appetite
loss, thus fat mass is preserved or even increased (Biolo G et al. 2014). The so called
“obesity paradox” may be explained on the basis of a larger skeletal muscle mass in
overweight subjects induced by their higher body mass (Kalantar-Zadeh K et al. 2005).
Fat tissue is mostly expanded at the visceral or abdominal level, with an increase in
waist circumference, observed more frequently in females. Furthermore there is an age
related deposition of ectopic fat at the intra-muscular, intra-hepatic and intra-pancreatic
levels. Enlarged visceral adipocytes and activated macrophages, attracted in the adipose
tissue by adipocyte secreted chemokines, release hormones and cytokines such as
adiponectin, leptin, tumor necrosis factor and interleukin 6 (IL-6), giving rise to an
inflammatory response and to insulin resistance. A higher release of free fatty acids

22
further contributes to the insulin resistance. TNF-α has direct inhibitory effects on
insulin signaling and also increases the release of free fatty acids (FFA) from adipose
tissue. These changes increase the risk of developing type II diabetes, metabolic
syndrome and cardiovascular complications (Biolo G et al. 2014).
Recently it has been shown that, besides fat tissue, also skeletal muscle can be
considered an endocrine organ, producing peptides both anabolic (insulin-like growth
factor, IL-15) and catabolic (myostatin) and cytokines, called myokines including IL-6,
IL-8 and IL-15 (produced during muscle contraction) (Biolo G et al. 2014). Physical
activity increases the muscle synthesis of IL-6 with useful metabolic consequences such
an increased local glucose uptake and fat oxidation, a higher gluconeogenesis in liver
and lipolysis in adipose tissue. IL-15 plays anabolic activity in skeletal muscle and has a
role in lipid metabolism.
Sarcopenia, by reducing strength and function, leads to poorer balance and higher risk
of falls and fractures, decreased autonomy and lower quality of life, metabolic
complications and higher morbidity and mortality. Therefore adequate countermeasures
need to be taken (Biolo G et al. 2014).
Currently proposed criteria for sarcopenia assessment in a clinical setting include:
evaluation of muscle mass, determination of strength and assessment of physical
performance (Miller MD et al 2002; Cruz-Jentoft AJ et al. 2010; Malmstrom TK &
Morley JE. 2013). Muscle mass can be measured by many methods including
anthropometry (Miller MD et al 2002), bioimpedance analysis (BIA) (Cruz-Jentoft AJ
et al. 2010; Vermeeren MA et al. 2006; Norman K et al. 2012; Mijnarends DM et al
2013), dual energy X-ray absorptiometry (DXA) (Fearon K et al. 2011; Coin A et al.
2012), computed tomography (CT) scan (Mourtzakis M et al. 2008; Fearon K et al.
2011), and magnetic resonance imaging (MRI). BIA cannot reliably assess skeletal
muscle mass in patients with body fluid abnormalities, as liver cirrhosis (LC), chronic
kidney disease (CKD), chronic heart failure (CHF) or some cancer (Norman K et al
2012). Ultrasonography is another reliable imaging method that can be applied to the
analysis of the musculoskeletal system, based on computerized analysis of ultrasonic
acoustic echoes. Ultrasound imaging can allow proper estimation of muscle thickness
changes as direct evidence of muscle alterations. Reliability of ultrasound imaging was
validated versus MRI as a gold standard (Arbeille P et al., 2009) and moreover, this
technique allows a proper determination of muscle fiber orientation, with respect to
aponeurosis, i.e. pennation angle (Narici M & Cerretelli P 1998). For measurement of
muscle strength the hand-held dynamometer is a reliable tool o assess strength in upper

23
extremities (Mijnarends DM et al 2013). This method is widely used and has been
validated in many physiological and pathological conditions. Several tests of physical
performance are available such as the gait speed, the Timed Up and Go (TUG) and the
Short Physical Performance Battery (SPPB) which includes standing balance, gait
speed, and chair rises (sit-to-stand) (Mijnarends DM et al 2013). The EWGSOP criteria
for sarcopenia diagnosis are described in figure 6.

Figure 6. EWGSOP criteria for sarcopenia diagnosis

The diagnosis requires: the presence of low muscle mass by BIA, plus low muscle strength, or low physical performance;

conversely, the presence of low muscle mass, normal muscle strength, and physical performance is defined as pre-sarcopenia. From

Biolo G et al. 2014

24
 1.3.1.1. Anabolic resistance

The reduced response to an anabolic stimulus, such as dietary protein intake, is defined
“anabolic resistance” (Burd NA et al., 2013). With the onset of this condition, the MPS
response to hyperaminoacidemia is impaired, independently from the insulin
availability (Burd NA et al., 2013).
In physiologic condition, in the post-prandial state, protein synthesis overcome the
protein breakdown in order to equalize the protein loss of the fasting state. The anabolic
resistance impairs the post-prandial protein synthesis and causes muscle mass loss
(Deutz NE et al., 2011). In a previous study (Biolo G et al., 2004), our research group
investigated the effects of 14-d of experimental immobilization in nine healthy young
subjects. The study consisted in a constant infusion of both the stable isotopes L[1-13C]-
leucine, to assess the anabolic response, and of an amino acid solution to simulate the
post-prandial condition. Our results showed a significant reduction of protein synthesis
in the fed state, due to reduced anabolic sensitivity to the amino acids stimulation. Thus
anabolic resistance is one of the major mechanisms of muscle atrophy during inactivity
(Biolo G et al., 2004). On the other hand, a bout of exercise before the dietary protein
intake, determines an improved amino acid utilization, allowing a post-prandial muscle
muss gain (Burd G et al, 2013).
Physical exercise associated to an adequate dietary protein intake showed an anabolic
effect on muscle protein balance (Biolo G et al., 2005). The combined effect of physical
exercise and increased availability of amino acids on the regulation of muscle protein
kinetics was evaluated on healthy young subjects through an intravenous infusion of a
balanced amino acids mixture, during rest and after a resistance exercise (Biolo G et al.,
1997). The results obtained, from muscle protein kinetics with the infusion of stable
isotopes, suggested that the MPS amino acids stimulatory effects is increased by the
exercise session. These evidences (Figure 7) are probably due to the augmented blood
flow promoted by physical activity. Accordingly, the amino acids derived from dietary
proteins may have a greater anabolic effect if administered immediately after physical
activity (Biolo G et al., 1997).

25
Figure 7. Muscle protein synthesis level (F(o,m)) assessed during an amino
acid infusion in conditions of rest and after resistance training.
MPS is expressed as percent variation compared to basal values (*,p<0,05) (Biolo G et al., 1997).

In elderly there is a reduced dose-response relationship between the EAA availability

and the myofibrillar protein synthesis (Burd NA et al, 2013). Moreover, the anabolic
resistance in aging involves also the insulin anabolic effect, with a reduction of the
proteolysis inhibitory effect promoted by insulin after a meal (Rennie MJ 2009), and the
exercise anabolic effect (Russ DW et al., 2012). This resistance to anabolic stimuli,
such as nutrition, exercise or hormones (e.g. insulin), is the basis of the impaired aging
muscle protein turnover (Russ DW et al., 2012).
Cuthbertson and colleagues were the first to compare the levels of protein synthesis,
following the oral administration of EAA, in young and elderly individuals. They
showed that the basal levels of MPS in fasting state, were comparable in both young
and elderly groups, but after the assumption of EAA the MPS response in elderly
individuals was lower than that showed by the young subjects (Cuthbertson D et al.,
2005). Another study demonstrated that in elderly individuals the ability of insulin and
amino acids, especially branched (BCAA), to stimulate the protein transduction
processes is reduced compared to the young subjects (Guillet C et al., 2004).
These results were sustained by other findings showing that the reduced vasodilator
response to insulin, in aging muscles, can play a key role in the onset of the anabolic
resistance, possibly because of a lower muscle availability of nutrients (Rasmussen BB
et al., 2006). Breen et al. also confirms the detrimental effect of aging on anabolic
sensitivity. They have demonstrated that after a single bout of endurance training, the
physiological acute alterations of the MPS are compromised (Breen L. & Phillips SM
2011). Moreover it was observed that even after 3/6 h after the physical activity, an

26
elderly subject does not reach the normal level of MPS stimulation (Drummond MJ et
al. 2008). The detrimental effects of physical inactivity on the levels of protein
synthesis are confirmed by a recent study showing that two weeks of sedentary lifestyle
is associated with loss of gravitational muscle mass, involving mainly the lower limbs
(Krogh-Madsen R et al.,2010). An active lifestyle in aging can be critical to maintain
adequate sensitivity to an anabolic stimulus (Burd NA et al. 2013). The anabolic
resistance in aged is represented in figure 8.

Figure 8. Protein metabolism trend after an anabolic stimulus (physical activity

and/or amino acids intake) in young and elderly subjects.
The shaded sections mark the difference between elderly and young people in the MPS after an anabolic stimulus (from Breen &
Phillips, 2011).

The age-related sarcopenia has a multifactorial etiology and the mechanisms associated
to the anabolic resistance development require further investigation, however, the
reduction of physical activity levels (i.e. sedentary lifestyles) and/or the potential
presence of acute or chronic diseases could have a role.
Accordingly, some studies showed that both protein turnover and anabolic sensitivity
could be altered by the development of an inflammatory condition, associated with
aging (Breen L & Phillips SM, 2011). The role of inflammation on the alteration of
protein metabolism has been extensively studied. In humans an association was found
between MPS levels and plasma concentrations of several inflammation markers (Toth
MJ et al., 2005).
Many cytokines, and particularly the TNF- α could alter MPS though the inhibition of
the phosphorylation of the proteins involved in the mTOR pathway. It was

27
demonstrated that mTOR signaling is crucial to stimulate the MPS after endurance
training and furthermore, the phosphorylation of the proteins involved in this pathway
seems to be enhanced by the availability of amino acids (Breen L & Phillips SM, 2011).
Consequently, the muscle loss and the reduced sensitivity to anabolic stimuli, observed
during inflammation could be associated with an altered protein phosphorylations of the
mTOR signaling pathway. This mechanism could affect the elderly individuals. The
development of a low-grade inflammation may reduce their sensitivity of this signaling
pathway to a load of amino acids, thus determining anabolic resistance and reduced
levels of MPS (Breen L & Phillips SM, 2011)
Recent studies suggest an impact of the oxidative stress on the inactivity-related muscle
mass loss, probably determining an unbalance between protein synthesis and
degradation (Pellegrino MA et al., 2011). However further investigation are needed to
better understand the pivotal mechanism of such influence.

28
 1.3.1.2. Insulin resistance

Insulin is a peptide hormone secreted by the β-cells of the pancreatic islets of

Langerhans that maintains normal blood glucose levels by facilitating cellular glucose
uptake. Besides carbohydrate metabolism, insulin regulates also lipid and protein
metabolism and cell division and growth (Bailey CJ et al 2010).
The term “insulin resistance” refers to a condition in which, insulin is correctly
produced by the pancreas, but the target tissues (i.e. muscle, liver and adipose tissue)
show a reduced sensitivity to its actions (Berg JM et al., 2012). Consequently, the tissue
uptake of the circulating glucose is impaired in both fasting and fed state, leading to
hyperglycemia and to detrimental effects in lipid and glucose metabolism (Silverthorn
DU, 2010). Insulin resistance is an important T2DM risk factor and may be detectable
even years before the appearance of hyperglycemia and T2DM. Essentially, the first
stages are characterized by a hyperinsulinemic response to glucose that ensures a proper
glucose metabolism (Hunter SJ & Garvey WT, 1998). In advanced stages, on the
contrary, the even greater amount of insulin produced by the β-cells is insufficient to
compensate the insulin resistance of the tissues, leading to hyperglycemia and T2DM
(Reaven G, 2004). This mechanism could be one of the reasons for the development of
T2DM during aging (Silverthorn DU, 2010).
The cellular mechanisms responsible of the altered insulin functions may involve
detrimental modifications for the insulin receptor, signal transduction and glucose
transport. The major cause of the altered insulin sensitivity seems to be the missed or
reduced translocation of the GLUT4 transporteron the cellular membrane of the skeletal
muscle and adipose tissue, in response to the hormone; consequently, glucose
absorption by these cells is lowered (Hunter SJ & Garvey WT, 1998). The insulin
resistance effects vary in relation to the type of tissue and its insulin dependence for
metabolic process regulation. The organs and tissues most affected by the altered
insulin sensitivity are: skeletal muscle, adipose tissue, liver, endothelium, brain,
pancreas, pituitary gland, kidney, gonads and bone (Wilcox G, 2005). The impaired
glucose tolerance and the reduced insulin sensitivity are two common phenomena in the
elderly population. The association between aging and insulin resistance was deeply
investigate, since there is an increased prevalence of T2DM with ageing (Karakelides H
et al., 2010). The progressive age-related glucose tolerance derangement depends
mainly on the decreased tissue sensitivity to insulin and the resulting reduced ability of
tissues to metabolize glucose (DeFronzo RA, 1981) (Figure 9).

29
Figure 9. Plasma glucose (A), insulin (B) and C peptide (C) concentrations in
young (○) and elderly (■) subjects in fed state.
from Basu R et al, 2003.

This altered glucose tolerance has a multifactorial etiology and it involves mainly the
skeletal muscle (Jackson RA, 1990). Among other factors, the reduced physical activity
and body composition changes are critical contributors to this progressive condition
during aging. In the elderly subjects there is a significant modification of the body
composition with a reduction of the lean mass in favor of enhanced fat mass. Since one
of the main actions of insulin is promoting the muscle glucose uptake, a reduced muscle
mass could lead to insulin resistance (DeFronzo RA, 1981). An inverse relation was
found (Srikanthan P et al. 2011) between skeletal muscle mass and insulin resistance
and the risk of developing pre-diabetes, while an increased muscle mass was associated
with additional protection against insulin resistance and pre-diabetes. The protective
association was stronger in individuals with overt diabetes (Srikanthan P et al. 2011.).
Moreover the reduced physical activity associated to aging could contribute to the
impaired glucose metabolism (DeFronzo RA, 1981). Some authors argue that aging per
se has a harmful effect on tissue sensitivity to insulin and consequently on glucose
metabolism (DeFronzo RA, 1981). On the other side, recent studies suggest that the

30
age-related insulin resistance is associated to body composition changes and the
lowered physical activity rather than aging per se (Karakelides H et al., 2010). For
example, the increased abdominal fat mass is a key factor in the onset of insulin
resistance (Karakelides H et al., 2010).
Aging is also associated to a low-grade systemic inflammation and to an increased
reactive oxygen species production (i.e. oxidative stress) (Karakelides H et al., 2010;
Csiszar A et al., 2008). Inflammation and oxidative stress are considered two of the
major mechanisms in the onset of the insulin resistance most likely due also to the
higher cardiovascular risk associated to a sedentary lifestyle. The individuals affected
by T2DM or metabolic syndrome, frequently show a low-grade systemic inflammation
and are characterized by higher concentration of proinflammatory fatty acids in the cell
membranes (Mazzucco S et al., 2009). Oxidative stress and low-grade systemic
inflammation are common alterations in obese or diabetics but in aged persons they
seems independent from body composition and may directly affect the onset of T2DM
and cardiovascular diseases (CVD) (Karakelides H et al., 2010; Csiszar A et al., 2008).
A good way to prevent or at least improve insulin resistance is physical activity. A
study aiming to analyze the distinct effects of age, physical activity and fat
accumulation on glucose tolerance and insulin sensitivity, demonstrated that regular
physical activity can prevent the age-related insulin resistance (Seals DR et al., 1984).
Insulin sensitivity decreases in sedentary subjects, while it is enhanced in trained
individuals (DeFronzo RA, 1981). The detrimental effect of inactivity on insulin
sensitivity was showed in many studies. Furthermore, experimental immobilization in
healthy young subjects (Figure 10) caused an altered glucose tolerance and a higher
insulin secretion. Stuart et al. showed an impaired glucose metabolism after just 7 days
of bed rest in healthy subjects (Stuart CA et al, 1988)

31
Figure 10. Effect of 5 days of bed rest insulinaemia (upper figure) and glycaemia
(lower figure) levels after an oral glucose load in 20 healthy subjects.
Bed rest is associated to a significant augmented insulin (p<0,001) and glucose (p=0,03) response and thus to insulin resistance

(from Hamburg NM et al., 2007).

Another study (Lipman RL et al., 1972) showed that 3 days of experimental

immobilization are sufficient to determine an impairment of about 50% of the glucose
uptake from the tissue and an augmented level of insulinaemia and glycaemia after an
oral glucose load. The same authors have observed that physical exercise during
immobilization can improve insulin resistance. The bed rest state has different effects
on trained and inactive individuals. Smorawiński and colleagues have observed the
effects of three days of experimental immobilization on endurance and power athletes
and sedentary subjects. After an oral glucose load, the insulin resistance-derived
hyperinsulinemia affect both groups but the athletes (especially those performing
resistance exercise) were more responsive. This result is probably due to a faster
compensatory response to insulin resistance (Smorawiński J et al., 2000). Recently, a
study has shown the association between five days of bed rest on healthy volunteers
with dyslipidemia and altered blood pressure and vascular function (Hamburg NM et
al., 2007). The authors propose a direct connection among the vascular dysfunction and
the onset of insulin resistance suggesting that a lower peripheral blood flow, especially

32
in the lower limbs, could determine a reduced tissues glucose uptake (Hamburg NM et
al., 2007). Another study comparing athletes and subjects inactive for 7-10 days,
showed an association between physical inactivity and both an impaired glucose
tolerance and the energy intake, while the trained subjects (controls) maintained a
suitable glucose tolerance. This positive effect in athletes is associated to an increased
peripheral blood flow favoring the insulin-mediated glucose uptake by the skeletal
muscle (Arciero PJ et al., 1998; Hayashi T et al., 1997). It was also demonstrated
(Vukovich MD et al., 1996) that the impaired insulin action evidenced after 6 days of
physical activity in runners is associated to a reduced levels of GLUT4, the skeletal
muscle tissues glucose transporter. These data were recently confirmed (Henriksen EJ,
2002), however, further studies are required to verify that such mechanisms (i.e.
impaired blood flow and GLUT4 levels) are responsible of the inactivity-related insulin
resistance. In addition, preliminary data of our research group, showed a development
of insulin resistance after 7 days of bed rest in healthy subjects. We have observed that
after 35 days of experimental inactivity the insulin resistance levels are comparable to
those measured after a week of bed rest, suggesting a rapid and full expression of this
condition maintained in the long-term.
Increased levels of physical activity could have a fast and healthy effect on the
metabolic alterations determined by a sedentary life style or resting from
hospitalization. Recently (Heer M et al., 2014) it was demonstrated that 4 days of
regular physical activity are not sufficient for counteract the insulin resistance
determined by 21 days of experimental inactivity. In healthy individuals 5 to 14 days of
specific training are needed to reach again a suitable glucose metabolism. Moreover
insulin resistance decreases after a single bout of physical exercise, in young subjects
while in middle aged or elderly individuals several workout sections are needed to reach
the same metabolic improvement (Henriksson J, 1995). Nonetheless further
investigations are necessary to select the correct categories and levels of physical
activity that could improve insulin resistance (Heer M et al., 2014).
Insulin resistance is also associated to the so called “metabolic syndrome”, a disorder
characterized by at least three of the following conditions: abdominal obesity, elevated
blood pressure, hyperglycaemia, high serum triglyceride levels and low plasma HDL
concentrations. Metabolic syndrome increases the risk of developing cardiovascular
disease and diabetes (Kaur J. 2014). Besides the high plasma glucose levels, several
studies suggest a direct influence of the insulin resistance on augmented VLDL hepatic
levels, hypertension and atherosclerosis (Biolo G et al., 2005). Together these risk

33
factors are defined “insulin resistance syndrome” (Hunter SJ & Garvey WT, 1998).
Skeletal muscle insulin resistance directly contributes to cardiovascular risk through
induction of dyslipidemia (i.e., decreased HDL cholesterol and increased triglycerides),
as shown in experimental bed rest studies (Mazzucco S, et al. 2010). Prolonged obesity
leads to ectopic lipid accumulation in non-adipose tissues, particularly in skeletal
muscles, inducing metabolic dysfunctions (reduced glucose uptake, mitochondria
dysfunction, etc.) (Masgrau A et al. 2012). The accumulation of lipids within skeletal
muscle, due to a blunted muscle capacity to oxidize fatty acids, is a relevant factor in
the pathogenesis of insulin resistance. Fat infiltration is also associated with muscle
fiber modification, decrease in muscle mass and impairment in muscle strength. Thus,
obesity causes quantitative and qualitative alterations in skeletal muscle. This obesity-
related insulin resistance not only causes defective insulin-stimulated glucose disposal
but has also detrimental consequences on muscle protein metabolism with a reduced
post-prandial anabolic response. Moreover, the fat tissue release pro-inflammatory
cytokines, including Tumor Necrosis Factor-α, interleukines and PAI-1, associated with
the development of insulin resistance (Wilcox G, 2005).
The best way to reduce the detrimental effect of the obesity-related insulin resistance is
the weight loss through physical activity.

34
 1.3.1.3. Inflammation

Inflammation is a multifactorial response mediated by the activity of cytokines, which

can have pro-inflammatory (as interleukin 1, IL-1), anti-inflammatory roles (as
interleukin 10, IL-10) or both (as intreleukin-6, IL-6). IL-6 is also known as
“myokyne”, as it is released by contracting muscle and is up-regulated after high
intensity physical exercise (Petersen AM & Pedersen BK, 2006). Furthermore it has an
immunomodulatory role, decreasing expression of pro-inflammatory cytokines and
down-regulating the activation of factors involved in intracellular inflammatory
response, as NF-kB and related tissue injury (Yoshidome H et al., 1999). Physical
exercise was shown to increase also circulating IL-10 concentrations, thus suggesting
that muscle activity can exert a beneficial anti-inflammatory action (Nunes RB et al.,
2008). Tumor necrosis factor alpha (TNF-α) is an additional important cytokine that
plays a crucial roles in the onset and maintenance of the inflammation process, by
stimulating acute phase reaction factors, in liver, macrophage phagocytosis and
chemoattraction of neutrophils (Tracey KJ & Cerami A, 1990; Tracey KJ & Cerami A
1994). Through the action on two specific cell membrane receptors, TNF-α can activate
several biological responses as activation of NF-kB (Bouwmeester T et al., 2004) and of
intracellular pathways leading to cell differentiation or to apoptosis (Gaur U &
Aggarwal BB 2003). Prolonged elevation of circulating TNF-α can lead to muscle mass
reduction: a wasting condition linked to poor prognosis of patients (Kandarian SC &
Jackman RW 2006). Synthesis of interleukins and TNF-α can trigger the production of
a known marker of inflammation called C-reactive protein (CRP), a factor used in
clinical practice as marker of inflammation (Ho KM & Lipman J 2009).
Inflammation was shown to play a crucial role in muscle mass wasting, occurring in
healthy aging subjects (Jensen GL 2008). In particular IL-1, IL-6 and TNF-α were
previously shown to potentially trigger muscle sarcopenia in elderly subjects (Yende S
et al., 2006). Interestingly, physical exercise training in elderly subjects was shown to
limit the muscle mass wasting and the synthesis of proinflammatory cytokines (Nicklas
BJ & Brinkley TE 2009). Moreover, strength training programs in older volunteers
ameliorated muscle strength and performance and, in parallel, increased levels of anti-
inflammatory interleukin-6 and 10 (Bautmans I et al., 2005).
A previously published work showed in humans that experimental bed rest upregulated
plasma CRP (+143%), the ratio between plasma IL-6 and IL-10 (4 times) and, in white
blood cell, the ratio between IL-6 and IL-10 mRNAs (5 times) (Bosutti A et al., 2008).

35
Cytokines are direct modulators of inflammatory pathways, but other factors are also
deeply involved in the control of inflammation. Eicosanoids, including prostaglandines
tromboxanes and leukotrienes are key mediators and regulators of inflammation (Lewis
RA et al., 1990). Cell availability of polyunsaturated fatty acids (PUFA) of the n-6
series affects production of eicosanoids. Eicosanoids are synthesized from the n-6
PUFA arachidonic acid by the enzymic action of cyclooxygenase. Arachidonic acid is,
in turn, synthesized in separate biochemical steps from the n-6 PUFA linoleic acid,
principally by the action of ∆6-desaturase, elongase and ∆5-desaturase (Figure 11)
(Mazzucco S et al. 2009).

Figure 11. Fatty acid biosynthesis.

Fatty acids are metabolized by desaturases and elongases in the endoplasmic reticulum; only 24:5 n-6 and 24:6 n-3 are b-oxidized
into the peroxisome. From Mazzucco S et al 2010

Linoleic acid is an essential FA, contained in many vegetable oils (Bozan B & Temelli
F 2008), which determines, together with the endogenous synthesis, the availability of
n-6 PUFA (Wertz PW 2009). Elevated availability of n-6 PUFA in cell membranes was
linked to inflammation (Ueda Y et al., 2008) and enhanced gene expression of
proinflammatory cytokines and transcriptional activity of NF-kB (Weaver KL et al.,

36
2009). Thus the n-6 PUFAs have a key role in the stimulation of pro-inflammatory
processes.
The n-3 PUFAs are synthesized from the n-3 PUFA alpha-linolenic acid in a pathway
sharing the same enzymes involved in the n-6 series synthesis, the ∆5-desaturase. This
enzyme leads to eicosapentaenoic acid (EPA) that is converted, by ∆6-desaturase, to
docosahexaenoic acid (DHA) in the peroxisome. n-3 PUFA are well known to play an
anti-inflammatory action (Figure 11). In analogy with n-6 PUFAs, alpha-linolenic acid
and n-3 PUFAs availability are strongly conditioned by dietary intake of alpha-linolenic
acid from vegetable oils (Wertz PW 2009), or EPA and DHA from fish (Pickova J
2009). Fatty acids of the n-3 series are known to have an anti-inflammatory action.
Increased intake of EPA and DHA can affect the cell membrane content of these
PUFAs (Lee TH et al., 1985), thus reducing the fraction of the pro-inflammatory
arachidonic acid. Increased dietary n-6 to n-3 PUFA ratio was shown to increase the
expression of CRP and of other proinflammatory agents, as tumor necrosis factor
(Zhang L et al., 2009).
Phospholipids content in red blood cell membranes can be considered reliable markers
of fatty acid availability in plasma, and of cell membrane composition in the whole
body (Harris WS & Von Schacky C 2004). Interestingly, fatty acid membrane
composition affects the activity of surface membrane receptors, influencing the
activation of downstream intracellular pathways. For example, increased levels of n-3
PUFA can enhance the expression and the signaling activity of glucose receptor
(GLUT-4) on muscle cell membrane, thus potentially ameliorating insulin sensitivity
(Taouis M et al., 2002). Moreover, the fraction of n-3 PUFAs in cell membranes was
directly associated to reduced incidence of cardiovascular diseases: this effect was
associated with the anti-inflammatory role of n-3 PUFAs and to other changes induced
on cardiovascular system physiology by this class of fatty acids (Harris WS & Von
Schacky C 2004). Published evidence proved that in human neutrophil membranes,
increases in n-6 to n-3 PUFA ratios are directly associated with the ability of
synthesizing pro-inflammatory mediators (Zhang L et al., 2009). Such evidence
confirms that cell membrane relative content of total n-6 and n-3 fatty acids can be
considered as a marker of whole body inflammatory condition.
The impact of physical exercise on membrane fatty acid composition was investigated
in several studies. Constant moderate training and acute exercise were shown to
decrease both phosphatidylserine and polyunsaturated fatty acids in erythrocyte
membranes: this effect was hypothesized to be caused by increased lipid peroxidation

37
due to muscle contraction (Sumikawa K et al., 1993). Another study emphasised n-6
PUFA content changes mediated by physical exercise. The authors showed that linoleic
acid and the sum of n-6 fatty acids were decreased in trained skeletal muscle
phospholipids. This reveals that physical exercise can directly exert an anti-
inflammatory role at muscle level, in this way potentially ameliorating insulin
sensitivity (Andersson A et al., 1998). Regular exercise was shown, in rats, to decrease
∆-5 desaturase activity and arachidonic acid content; in addition, docosahesaenoic acid
proportion in cell membrane was decreased while linoleic acid was increased (Helge
JW et al., 1999). Observed effects on membrane composition mediated by regular
exercise where hypothesized to be dependent on energy substrate utilization during
training (Helge JW et al., 1999). Still, a previously published study showed in humans
that fractions of oleic acid and docosahexaenoic acid were significantly higher in
trained muscles when matched to untrained (Helge JW et al., 2001). Similarly, physical
exercise significantly lowered the ratio between n-6 and n-3 PUFA in the trained
muscle vs those untrained (Helge JW et al., 2001). This study confirmed also that
regular physical exercise can reduce whole body and muscle inflammation.

38
 1.3.1.4. Oxidative stress

Oxidative stress is the result of an unbalance between free radicals and the activity of
the antioxidant defense system of the body (Tchou J et al. 1991; Hardmeier R et al.,
1997) (Figure 12).

The anti-oxidant defense includes non enzymatic and enzymatic systems.

• Enzymatic systems. There are several enzyme systems, part of the endogenous
defense mechanisms, which catalyze reactions able to neutralize free radicals
and ROS, thus preventing cell damage. The major systems are: superoxide
dismutase (SOD), catalases, glutathione peroxidase and glutathione reductase,
the last two being part of the glutathione system. Metal ions, including iron,
selenium, copper, zinc and manganese, act as co-factors in these reactions.
• Non-enzymatic systems. This category includes: GSH, ascorbic acid (Vitamin
C), tocopherols (Vitamin E), carotenoids, flavonoids, and ubiquinol.

Figure 12. Oxidants and antioxidants agents in the human body

39
Glutathione is the most important non-enzymic antioxidant in the organism (Pastore A
et al., 2003).
Glutathione synthesis is achieved by the action of two ATP dependent enzymes γ-
glutamil cysteine synthetase (catalyzing the bond between glutamic acid and cysteine)
and glutathione synthetase, leading to the final formation of reduced glutathione
(Majerus PW et al., 1971); the first reaction being the rate-limiting step (Lu, SC 1999)
(figure 13).

Figure 13. Glutathione biosynthetic pathway

Glutathione is further processed (figure 14), within the γ-glutamate cycle, by γ-glutamyl
transpeptidase leading to the formation of a γ-glutamil amino acid and to the dipeptide
cisteinglycine (Pastore A et al., 2003). γ-glutammil amino acid is then transformed to 5-
oxoprolin and glutamic acid by γ-glutamil cyclotransferase and oxoprolinase. Glutamic
acid is re-utilized to synthesize again glutathione (Pastore A et al., 2003).
Cysteinglycine is then catabolized to free cysteine and glycine amino acids. Cysteine is
a limiting substrate for glutathione synthesis, but also glutamate and glycine, as direct
glutathione precursors, can affect its synthesis rate.

40
Figure 14. The metabolic glutathione pathway or the γ-glutamyl cycle

Physical exercise increases oxidative stress; Regular physical exercise enhance total and
reduced glutathione availability (Sen CK & Packer L 2000), thus counteracting the
effect of free radical formation. The ratio between reduced and oxidized glutathione, a
reliable marker of glutathione system activation is increased by exercise (Ji LL 1995).
Thus, increased availability of GSH levels, can be considered, in healthy subjects, as an
active response of the organism to a previous release of free radicals.
Exercise training results in an elevation in the activities of both superoxide dismutase
(SOD) and glutathione peroxidase (GPx), along with increased cellular concentrations
of glutathione (GSH) in skeletal muscles. In contrast, inactivity leads to redox
unbalance both in skeletal muscle (Agostini F et al 2010) and at the systemic level
(Biolo G et al 2008).
Low levels of physical activity also promote oxidative stress, which, through the
activation of specific proteases and of apoptosis (Powers SK et al., 2005; Powers SK et
al., 2007), is involved in the processes leading to muscle atrophy (Laufs U et al., 2005).
Our research group has investigated, for the first time, the association (Brocca L et al
2012) in healthy subjects between immobilization and oxidative stress through human
bed rest. The development of muscle atrophy is associated with impaired anti-oxidant
defense systems (de Boer MD et al 2008), protein oxidation and/or lipid peroxidation
(indices of redox imbalance), suggesting a major role of oxidative stress in disuse
atrophy (Pellegrino MA et al 2011; Dalla Libera L et al 2009).

41
In our previous data, we observed that bed rest caused an early and persistent down-
regulation of myofibrillar protein content, impairment of antioxidant defense systems
and redox imbalance, followed, at later stages, by muscle fiber atrophy (de Boer MD et
al 2008). The experimental inactivity caused an early down-regulation (after 8 days) of
the antioxidant defense system, including: superoxide dismutase (SOD - which converts
the superoxide ion into hydrogen peroxide), peroxiredoxine (such as catalases, which
work downstream of SOD, converting hydrogen peroxide into water), α-β-crystallin and
some heat shock proteins (such as HspB1 and Hsp70 – which remove products induced
by free radicals and have been shown to protect cells against oxidative damage). Only
few components i.e. heme-oxigenase-1 (HO-1) and glucose regulated protein-75
(Grp75), of the antioxidant system showed a transient early, 8 days, post-BR up-
regulation (Mazzucco S et al 2010). After a prolonged period of inactivity (24 days) the
mRNA for NRF2, the major transcription factor for the expression of the antioxidant
defense system, such as heme oxygenase-1 (HO-1), catalase, SOD, peroxiredoxins, and
genes, involved in glutathione synthesis and function, was found to be up-regulated,
possibly in response to the ongoing redox imbalance. In our study we observed an
increased protein carbonylation (an index of oxidative stress) at 35-days post-BR, in
muscle biopsy samples (Mazzucco S et al 2010; Lawler JM et al 2003), which
correlated positively with the decreased muscle fiber, cross sectional areas (CSA)
associated with muscle atrophy and reduced protein synthesis (Mazzucco S et al 2010).
35 days of experimental inactivity determined also an increased muscle glutathione
synthesis. These data correlated with the levels of protein carbonylation, suggesting the
relevance of maintaining adequate GSH levels. Glutathione depletion can influence bed
rest outcomes (Sastre J et al. 1989), while an increased bioavailability of glutathione
precursors, such as cysteine or N-acetyl-cysteine, can improve glutathione system
scavenging action (Khamaisi M et al. 2000) and reduce, in an animal model, muscle
protein catabolism (Ling PR et al. 2007). Thus, dietary glutathione precursor
supplementation may ameliorate immobilization outcomes.
Inactivity leads to insulin resistance and low-grade systemic inflammation (Lawler JM
et al. 2003). Indeed, during bed rest there is an activation of the inflammatory response
(Lawler JM et al. 2003; Biolo G et al 2007) that is related, with reciprocal influences, to
the increased oxidative stress and insulin resistance. We have evaluated in a 14-days
experimental bed rest (Biolo G et al 2007) the effects of immobilization on
inflammatory response by assessing the levels of pro-inflammatory cytokines
(Interleukine-6, IL-6) and anti-inflammatory cytokines (Interleukine-10, IL-10) and of

42
two acute-phase proteins (C-Reactive Protein, CRP or short pentraxine and long
pentraxin-3, PTX3). Bed rest, on eucaloric conditions, significantly increased CRP and
IL-6 concentrations and decreased mRNA transcript levels for IL-10.
Notably oxidative stress, trough the glutathione system, and changes in the reduced
glutathione (GSH)/oxidized glutathione disulfide (GSSG) ratio is related to the
inflammatory response. Indeed ROS modulate the transcription of IL-4, IL-6, IL-8 and
tumor necrosis factor-α (TNF-α), through thiol-dependent mechanisms, while cytokines
can mediate ROS signaling (Laviano,A et al 2007). Our preliminary data suggest that
oxidative stress and changes in glutathione levels are associated with insulin resistance
conditions (such as type II diabetes and obesity) (Badaloo,A et al. 2002; Ikemoto M et
al. 2002). Decreased intracellular glutathione levels have been suggested to play a direct
causative role in the development of impaired insulin action in adipose tissue and
skeletal muscle (Agostini F et al 2010). Insulin resistance and deficiency are often
associated with hyperglycemia in diabetes and critical illness and hyperglycemia seems
to be related with generation of ROS, increased oxidative stress and decreased liver
glutathione concentrations (Bosutti A et al. 2008). Indeed liver is the major glutathione
storage organ and the major source of plasma glutathione; circulating erythrocytes may
reflect the synthetic capacity of the liver in the inter-organ glutathione homeostasis
(Haddada JJ & Harbb HL 2005).
Cross-sectional studies identified strong relationships between insulin resistance,
systemic inflammatory response and alterations in fatty acid (FA) composition of cell
membrane phospholipids (Das UN 2004; Vessby B et al. 2002). The poly-unsaturated
FAs (PUFAs) of the n-6 and n-3 series are involved in up-regulation and down-
regulation of the inflammatory response, respectively.
We have already demonstrated that bed rest prolonged for 35 days causes increased
insulin resistance (Lawler JM et al. 2003). These metabolic alterations were associated
with changes in erythrocyte membrane fatty acid composition. We observed increased
levels of pro-inflammatory n-6 PUFAs and fractional content of pro-inflammatory
arachidonic acid, decreased levels of anti-inflammatory omega 3 PUFAs (alfa-linolenic
and eicosapentaenoic, EPA) and of monounsaturated fatty acids. These changes were
associated with altered activity of the enzymes Δ5 and Δ9 desaturase, affected by
insulin action (Lawler JM et al. 2003). Low Δ5 desaturase activity decreased long-chain
PUFA content. This may cause changes in the cell membrane physical properties,
potentially leading to altered receptor binding capacities and further impairment of
insulin sensitivity (Peter A et al. 2009).

43
 1.3.1.5. Hypoxia

Sarcopenia is caused by multiple factors; recently a role has been attributed to hypoxia
(Di Giulio C et al. 2009). Conditions associated with hypoxia are aging and chronic
respiratory diseases (i.e. COPD), obesity-related obstructive sleep apnea syndrome
(OSAS) and cardiorespiratory disorders. During aging there is a general increased
production of ROS with possible negative effects on proteins, nucleic acids and lipids
and on membrane functions (Di Giulio C et al. 2009; Cataldi A & Di Giulio C. 2009).
This higher oxidative stress can be related, among other factors, to a reduction of the
oxygen flow from the lungs to the tissues, leading to a lower cellular pO2. The reduced
blood flow and oxygenation of skeletal muscle, and the higher ROS production together
with a diminished mitochondrial density (Di Giulio C et al. 2009), can contribute to the
loss of muscle mass of aging (Gunnarsson L et al. 1996; Muller FL et al. 2007; Porter
MM et al. 1995).
Furthermore the normal ventilatory response to hypoxia characterized by increased
volume and ventilatory frequency are attenuated with aging (Fukuda Y et al 1992).
Muscle atrophy has been shown also in subjects with recurrent obstructive sleep apnea
syndrome (OSAS), cardiopulmonary disease and in COPD patient (Di Giulio C et al.
2009). Hypoxia is central to the pathogenesis of both OSAS and COPD, further
sustained by inflammatory and oxidative stress pathways (Figure 15)

Figure 15. Hypoxia, the link among COPD, OSAS, systemic inflammation, and
muscle atrophy.
Adapted from McNicholas WT. Am J Respir Crit Care Med. 2009

44
Patients with chronic heart failure or COPD during the course of the illness lose skeletal
muscle mass leading to exercise limitations and lower quality of life. Epidemiological
studies have shown that between 15-30% of COPD patients were found sarcopenic
(Jones SE et al 2015; Biolo et al 2014; Koo HL et al 2014) with or without sarcopenic
obesity. Sarcopenia and obesity were independent risk factors for respiratory
complications and worsening of COPD, however, the respiratory loss was most severe
in obese sarcopenic subjects (Koo HL et al 2014). Sarcopenic COPD patients had a
greater airflow obstruction, were significantly older and compared to non-sarcopenic
subjects, showed reduced quadriceps strength, functional performance and exercise
capacity (Jones SE et al 2015).

45
 2. AIM

The aim of the present thesis was to investigate in human healthy volunteers new
biomarkers adequate to define optimal protein intake. We based our research on recent
studies that have determined protein needs by measuring whole-body protein
metabolism using stable labeled isotope-amino acids.

The present work includes two experimental protocols:

1) The PLANHAB study on the effects of hypoxia and inactivity isolated or

combined on muscle mass and function and protein, glucose and lipid
metabolism, oxidative stress and inflammation. Hypoxia and inactivity can be
considered models, in humans, of clinical conditions such as COPD, CVD and
OSAS characterized by loss of muscle mass and function and normal or
expanded fat tissue (sarcopenia and sarcopenic obesity).

2) The INTERREG PANGeA on the effects of aging and immobilization on

muscle mass and function, protein and glucose metabolism in elderly subjects
compared to young controls

46
 3. METHODS

The main goal of this thesis is to find new biomarkers to define optimal protein
requirement in physio-pathological conditions. Specifically we have investigated the
detrimental effects of physical inactivity with or without hypoxia and in aging, on
muscle protein synthesis. Moreover the effects of these conditions on, oxidative stress,
inflammation, lipid metabolism and insulin sensitivity were also evaluated as related
conditions influencing MPS. The bed rest model is known to be a reliable approach to
study the impact of muscle unloading on human metabolism (Biolo G et al., 2005). To
investigate hypoxia, we utilized a special structure with areas at controlled oxygen
levels. The results reported in the present thesis were collected during two different
European funded experimental bed rest study: 1) the FP7 PLANHAB study (Planetary
Habitat simulation). A 7 Framework program 2007-2013 and 2) the INTERREG
PANGeA (Physical Activity and Nutrition for Quality Ageing). Italy-Slovenia
European Program for Cross Border Cooperation 2011-2014.

3.1. Experimental procedures

3.1.1. Whole body protein kinetics

Whole body protein kinetics were determined in plasma by the tracer model of
phenylalanine and tyrosine metabolism, as previously described (Antonione R et al.,
2008).
Briefly, after an overnight fasting, two polyethylene catheters were inserted into a
forearm vein of a subject for isotope infusion and into a wrist vein of the opposite arm,
the latest was heated at 50°C, for arterialized venous blood collection. Before the start
of the infusion, at time 0, a background blood sample was collected to assess the natural
occurring isotopic enrichments of [ring-2H5]-phenylalanine, [ring-2H4]-tyrosine, 2H2-
tyrosine in plasma. After background blood collection, 7-hour primed-continuous
infusions of [ring-2H5]-phenylalanine (3.3 µmol×kg-1×h-1) and 2
H2-tyrosine (1.0
µmol×kg-1×h-1) in parallel with a single bolus of [ring-2H4]-tyrosine (1.1 µmol×kg-1)
(Cambridge Isotope Laboratories, Andover, MA). Blood samples were drawn 180, 300
and 420 minutes after isotope infusion, to assess the plasma enrichments of [ring-2H4]-
tyrosine and [ring-2H5]-phenylalanine. Blood was collected in EDTA tubes,
immediately centrifuged at 3000 g at 4°C for 10 minutes; plasma and erythrocytes were
immediately stored at -80°C for further analysis.

47
Isotopic enrichments of phenylalanine and tyrosine, in plasma, were determined by gas
chromatography–mass spectrometry (GC-MS) (HP 5890; Agilent Technologies, Santa
Clara, CA) as previously described (Biolo G et al., 2008; Antonione R et al., 2008).
Isotopic enrichments were assessed considering the following mass-to-charge ratios
(m×z-1): phenylalanine m×z-1 234-239; tyrosine m×z-1 466-470.

Phenylalanine appearance from protein proteolysis (Phe Ra from protein proteolysis)

was calculated multiplying the infusion rate of [ring-2H5]-phenylalanine for the
enrichment of [ring-2H5]-phenylalanine. The rate of phenylalanine disappearance
through hydroxylation to tyrosine (Phe Rd to hydroxylation), an index of net protein
catabolism, was defined as follows:

Phe Rd to hydroxylation= (IRD2Tyr× ED2Tyr-1) × (ED4Tyr× ED5Phe-1)

where IRD2Tyr is the infusion rate of 2H2-tyrosine; ED2Tyr is the enrichment of 2H2-
tyrosine; ED4Tyr is the enrichment of [ring-2H4]-tyrosine; and ED5Phe is the
enrichment of [ring-2H5]-phenylalanine. Phe Rd to protein synthesis was calculated as
the difference between Phe Ra from protein proteolysis and Phe Rd to hydroxylation
(Figure 16).

Figure 16. Measurement of whole body protein kinetics Humans

In the postabsorptive state there is no entry of amino acids from dietary sources, and the flux of phenylalanine in the body is derived
from entry of phenylalanine released from protein breakdown. That input is matched by phenylalanine removal via protein synthesis

and via metabolic disposal by conversion to tyrosine. Therefore, the measurement of the whole body rate of appearance of

phenylalanine in the postabsorptive state is a measure of the whole body rate of proteolysis (Matthews DE 2007).

48
 3.1.2. Post-prandial anabolic resistance

In the morning a polyethylene catheter was inserted into a forearm vein of subjects,
fasted overnight, for blood collection. After basal blood sampling, at time 0 (t0), an oral
load of 0.3g of 2H5-phenylalanine, dissolved in 15-20mL of water, was administred to
the volunteers to be drank in 5 minutes. Blood samples were drawn at 30, 60, 120, 180,
240, 300 and 360 minutes after the the loading, in EDTA tubes then immediately
centrifuged at 3000g at 4°C for 10 minutes. Plasma was immediately stored at -80°C.
Plasma samples were used to assess the concentration of 2H5-phenylalanine (D5-Phe)
and 2H4-tyrosine (D4-Phe), the product of phenylalanine hydroxylation.

Isotopic enrichments of plasma phenylalanine and tyrosine, derived by phenylalanine

hydroxylation, were determined by gas chromatography–mass spectrometry (GC-MS)
(HP 5890; Agilent Technologies, Santa Clara, CA) as t-butyldimethylsilyl derivatives
(Biolo G et al., 2008). Plasma concentrations of leucine, phenylalanine and tyrosine
were assessed in all samples by GC-MS, using the internal standard technique, as
13 13
previously described (Biolo G et al., 2008). Known amounts of C-leucine, C-
phenylalanine and 2H2-tyrosine (Cambridge Isotope Laboratories) were added as
internal standards. Isotopic enrichments were assessed considering the following mass-
to-charge ratios (m×z-1): phenylalanine m×z-1 234-239; tyrosine m×z-1 466-470. Amino
acids in plasma were monitored using the following m×z-1: leucine m×z-1 302-303,
phenylalanine m×z-1 336-337 and tyrosine m×z-1 466-468.

Amino acid concentrations [aa] was determined by the internal standard technique as
follows:

[aa] = a × TTR

where a is the concentration of the internal standard added to plasma samples and TTR
(Tracer-to-Tracee Ratio) is the isotopic enrichment of the internal standard.

Phe-D5 and Tyr-D4 concentrations, [aa*], was calculated as follows:

[aa*]= [aa] ×TTR*

where [aa] is plasma concentration of the tracee (phenylalanine or tyrosine amino acid)

49
and TTR* is the isotopic enrichment of Phe-D5 or Tyr-D4.
The area-under-the curve (AUC), i.e., the area under the plasma amino acid
concentration versus time curve, was estimated using the linear trapezoidal method.
Blood was collected every 60 minutes for 6 hours. Thus the index of anabolic resistance
was calculated as ratio between AUC Tyr-D4 and AUC Phe-D5, assessed over 6h meal
test.

AUC Tyr-D4/AUC Phe-D5

A new method
After the data analysis, we calculated the same index, using only the blood collections
of the first two 2 hours from meal and [ring-2H5]-phenylalanine load, requiring 2 blood
draws, over the 7 planned, and 2h, over 6 of observation.
A simplify index of anabolic resistance was calculated as ratio between Tyr-D4
concentration and Phe-D5 concentration measured after 2 h from meal and Phe-D5 load.

[Tyr-D4]T120/[Phe-D5]T120

The new, simplify index displayed similar results compared with the (bed-rest effect
p<0.05; bed-rest×group interaction p<0.05) (R=0.75; p<0.001).

3.1.3. Post-prandial insulin resistance

Post-prandial state
In the morning, a polyethylene catheter was inserted into a forearm vein of a subject,
fasted overnight, for blood collection. After basal blood sampling, at time 0 (t0), a
standarized test meal (500mL, 500Kcal, 55% of carbohydrate 15% of protein and 30%
of fat; vanilla flavour, Nutricomp ®, B.Braun), was administred to the volunteers to be
drank in 5 minutes. Blood samples were drawn at 30, 60, 120, 180, 240, 300 and 360
minutes after the the loading, in EDTA tubes then immediately centrifuged at 3000g at
4°C for 10 minutes. Plasma was immediately stored at -80°C. Plasma samples were used
to assess insulinaemia and glycaemia in the fasting and fed state.

Insulin and glucose levels were measured with standard procedures by a certified
external laboratory (Synlab Italia Srl, Italy) and their values were used to calculate:

50
The Area Under the Curve (AUC) of post-load insulinemia and glycemia.

The Matsuda index as parameter of insulin sensitivity in fed state.

Matsuda Index =10000/√(glycT0 × insT0 × insaverage OGTT × glycaverage OGTT)

Where (ins) and (glyc) indicate insulin and glucose plasma levels, respectively.

Fasting state
The HOmeostatic Model Assessment, as index of insulin resistance (HOMA-IR).

HOMA index=(fasting glucose x fasting insulinemia)/405

3.1.4. Amino acids

A known amount of internal standards were added to plasma samples (200 µL) to
determinate the concentration of unlabeled AAs. The plasma samples were
deproteinized adding sulfosalicylic acid (200 µl, 10%). After centrifugation (4000 rpm
per 20 min at 4°C) the supernatants were purified in a cationic resin (AG50W-X8; Bio-
Rad, Hercules, CA) using NH4OH (4N) as eluent. The NH4OH excesses were
evaporated under N2 flux. Samples were then lyophilized and the powders obtained
were derivatized by the addition of 50 µl acetonitrile and 50 µl MTBSTFA and by
heating at 90°C for 45 min. After derivatization, samples were injected into a gas
chromatography-mass spectrometer (GC-MS) (HP 5890, Agilent Technologies, Santa
Clara , CA, USA). Gas chromatographic measurements were performed in single ion
monitoring mode, using the following mass-to-charge ratio (m×z-1): phenylalanine
m×z-1 336; [ring-2H5]-phenylalanine m×z-1 341; tyrosine m×z-1 466; [3,3-2H2]-
tyrosine m×z-1 468; [ring-2H4]-tyrosine m×z-1 470; homocysteine m×z-1 496; [13C]-
homocysteine m×z-1 497; [2H8]-homocysteine m×z-1 500; methionine m×z-1 320; [1-
13C, methyl-2H3]-methionine m×z-1 324; cysteine m×z-1 406; [3,3-2H2]-cysteine
m×z-1 408.

51
 3.1.5. Oxidative stress

Glutathione synthesis rate

In the morning, after an overnight fasting, two polyethylene catheters were inserted: one
into a forearm vein for isotope infusion, and the other into a wrist vein of the opposite
arm, heated at 50°C, for arterialized venous blood collection. Before the beginning of
the infusion (time 0), a background blood sample was collected to assess the natural
isotopic enrichments of 2H2-glycine and 2H2-glutathione in red blood cells (RBC). After
that, a 7-hour primed-continuous infusion of 2H2-glycine (26.5 µmol×kg-1×h-1) was
started (Cambridge Isotope Laboratories, Andover, MA). Blood samples were drawn at
180, 300 and 420 minutes after the start of isotope infusion, to assess enrichments of
2
H2-glycine and [2H2-glycine]-glutathione in RBC. Blood, collected in EDTA tubes,
was immediately centrifuged at 3000 g at 4°C for 10 minutes; plasma and erythrocytes
were immediately stored at -80°C for further analysis.

Isotopic enrichments of glycine and glutathione, in RBC, were determined by gas

chromatography–mass spectrometry (GC-MS) (HP 5890; Agilent Technologies, Santa
Clara, CA) as previously described (Biolo G et al., 2008; Antonione R et al., 2008).
Isotopic enrichments were assessed considering the following mass-to-charge ratios
(m×z-1): glycine m×z-1 218-220; glutathione m×z-1 363-366. Erythrocyte total
glutathione concentrations were determined in background blood samples using the
internal standard approach, through the addiction of known amount of [13C2-15N-
glycine]-glutathione (Cambridge Isotope Laboratories, Andover, MA).

Glutathione fractional turnover rate (FTR; in % × d-1) was calculated as:

[E(2H2-glutathione)×t-1]×E(2H2-glycine)-1 × 24 × 100

where E(2H2-glutathione) ×t-1 is the slope of the regression line describing the rise in
erythrocyte 2H2-glutathione enrichment as a function of time (hours); E(2H2-glycine) is
the mean glycine enrichment in erythrocytes after steady state achievement.
Coefficients (i.e., 100 and 24) were applied to express glutathione fractional turnover
rate as %×d-1. The absolute turnover rate was calculated as the product of FTR and
glutathione concentrations.

52
The ratio between the concentrations of reduced and oxidized forms of glutathione in
erythrocytes (GSH/GSSG ratio) was assessed in the same background blood sample, by
a commercially available kit (GT40, Oxford Biomedical Research; Oxford, MI).

Glutathione synthetic capacity.

Glutathione peroxidase activity was determined in erythrocytes according to Paglia and
Valentine (Paglia DE & Valentine WN 1967) and expressed as µmol metabolized
NADPH × min-1 × g protein-1 in the presence of an organic hydroperoxide
(cumolhydroperoxide) and of reduced glutathione as enzyme cofactor.
Catalytic and modulator subunit expression of glutamatecysteine ligase (GLC) in
erythrocytes was measured by Western blot analysis (Thompson SA et al., 2000).
Briefly, proteins were extracted from red blood cells by using a lysis buffer (45 mmol/L
Tris-HCl, 0.2% Nlaurylsarcosine; Sigma-Aldrich, St Louis, MO) containing proteinase
and phosphatase inhibitors (0.2 mM phenylmethanesulfonyl fluoride, 1 mM
dithiothreitol, 2 µg aprotinin/mL, 2 µg pepstatin× mL- 1, 0.1 mmol NaF × mL-1, and
0.1 mM Na3VO4; all: Sigma-Aldrich). After centrifugation (10 min, RT, 16 00 × g),
proteins were separated by sodium dodecyl sulfate–polacrylamide gel (12%)
electrophoresis and transferred to a nitrocellulose membrane (Protran; Perkin Elmer,
Boston, MA). Proteins were recognized by using commercial antibodies raised against
the catalytic (GCLc: sc-22755) and modulator (GCLm: sc-22754) (both: Santa Cruz
Biotechnology Inc, Santa Cruz, CA) subunits of the glutamate-cysteine ligase.
Glyceraldehyde-3-phosphate dehydrogenase was recognized by commercial antibody
(sc- 25778; Santa Cruz Biotechnology Inc). A goat anti-rabbit horseradish peroxidase–
conjugated immunoglobulin G (Sigma-Aldrich) was used as secondary antibody.
Protein complexes were detected by enhanced chemiluminescence (Amersham Life
Sciences, Arlington Heights, IL) on photographic film (Kodak Biomax Light Film;
Sigma-Aldrich). Protein concentrations in the catalytic and modulator subunits of
glutamate-cysteine ligase were measured by band densitometry as a ratio with
glyceraldehyde-3-phosphate dehydrogenase protein concentration (Model 45–700
Imaging Densitometer; Bio-Rad).

53
 3.1.6. Lipid pattern

Total cholesterol, HDL cholesterol and triglycerides were measured with standard
methods by a certified external laboratory (Synlab Italia Srl, Italy).
Commercially available kits were used to determine plasma concentrations of:

− Cholesterylester transfer protein (CETP, ALPCO, Salem, NH, USA),

− Lecithin cholesterol acyltransferase (LCAT, MyBioSource, CA, USA),

− Lipoprotein lipase (LPL, MyBioSource, CA, USA),

− Hepatic lipase (HL, MyBioSource, CA, USA),

− Serum amyloid A, (SAA, Abcam, UK),

− TNF-related apoptosis-inducing ligand (TRAIL, R&D Systems,

Minneapolis, MN).

HDL2 and HDL3 concentrations were assessed using the precipitation technique
(chemical reagents were purchased from Gesan Production srl (Italy) and Fitzgerald
Industries International, MA, USA).

Plasma LDL cholesterol was measured by the Friedewald’s formula:

total cholesterol – HDL cholesterol – (triglycerides×5-1).

54
 3.1.7. Systemic inflammation

high-sensitivity C reactive protein, was measured with standard methods by a certified

external laboratory (Synlab Italia Srl, Italy).

3.1.8. Membrane fatty acid composition

Fatty acid membrane composition of red blood cells was analyzed modifying a
previously published method (Burdge GC et al. 2002). Erythrocytes (200 µL) were
washed five times with decreasing concentrations (10 mmol/L, 2.5 mmol/L; 1.25
mmol/L; 0.625 mmol/L; 0.312 mmol/L) of phosphate buffered saline (PBS). Total lipid
extraction was performed in 5 mL of a chloroform–methanol (2:1) solution, containing
50 mg/L of butylhydroxytoluene as antioxidant, and 1mL of 1M NaCl solution. After
centrifugation, the lower lipid phase was collected and dried under nitrogen flux at 40
°C. Pellets were dissolved in toluene (500 µL), and after the addition of 1mL of a
methanol solution containing 2% of H2SO4, were heated at 50 °C for 2 h. A neutralizing
solution (1.0 ml, 0.25 M KHCO3 and 0.5 M K2CO3 in deionized H2O) and hexane (1
mL) was added. After centrifugation, the hexane layer, containing fatty acid methyl
esters (FAMEs), was collected and organic solvents were removed by N2 flux. After the
addition of hexane (150 µL), samples were analyzed by gas-chromatography–flame
ionization detection (GC- FID; GC 6850 Agilent Technologies, Santa Clara, CA, USA).
Specific fatty acid standards were used to identify FAMEs by retention times in
erythrocyte samples. A commercial mixture of purified fish oil fatty acids (Menhaden
oil, Sigma–Aldrich, Inc, MO, US) was used to detect: oleic acid (18:1, n-9), elaidic acid
(trans 18:1, n-9), eicosapentaenoic acid (20:5, n-3), docosapentaenoic acid (22:5, n-3)
and docosahexaenoic acid (22:6, n-3). Retention times of myristic acid (14:00), palmitic
acid (16:00), palmitoleic acid (16:1, n-7), stearic acid (18:00), linoleic acid (18:2, n-6),
a- linolenic acid (18:3, n-3), eicosaenoic acid (20:1, n-9), eicosadienoic acid (20:2, n-6),
dihomo-γ- linolenic acid (20:3, n-6) and arachidonic acid (20:4, n-6) were identified by
commercial standards. Adrenic acid (22:4, n-6) and docosapentaenoic acid (22:5, n-6)
were identified by commercial standards from Nu-Check Prep, Inc, MN, US. Organic
solvents and buffering salts were purchased from Sigma–Aldrich, Inc, MO, US, if not
differently specified.
Area-under-the-curve of each selected peak was determined by highly standardized
hand integration performed using commercial software (HP Chem station; Agilent
Technologies, Santa Clara, CA, USA).
55
Erythrocyte membrane fatty acid (EMFA) composition was assessed by gas-
chromatography-flame ionization detection (GC-FID; GC 6850 Agilent Technologies,
Santa Clara, CA, USA), as previously reported (Mazzucco et al. 2010).
Red blood cell membrane level of each enlisted FA was expressed as percent ratio
between area-under-the-curve of each selected EMFA peak and the sum of all measured
EMFA peaks.
The following indices were calculated:

− Δ-5 desaturase index, as index of insulin sensitivity, was defined as ratio between
arachidonic acid (20:4, n-6) and dihomo-γ-linolenic acid (20:3, n-6) membrane
levels.

− Δ-9 desaturase index, as index of insulin sensitivity, was calculated as ratio

between oleic (18:1, n-9) and stearic (18:0) acid contents.

− Arachidonic-to-eicosapentaenoic acid ratio, as index of inflammation, was

calculated as ratio between arachidonic acid (20:4, n-6) and eicosapentaenoic acid
(20:5, n-3) membrane levels.

56
 3.2. Human study protocols

3.2.1. The PLANHAB study (experimental hypoxia and bed rest)

This study is was conducted at the Olympic Sport Center Planica, in Rateče, Slovenia.
Two floors of the Olympic center are structured to simulate any altitude conditions in
the all areas and in each room individually. Eleven male young subjects (mean ± sem;
age 24 ± 4 yr; BMI 22 ± 2 kg/m²) were selected to participate to the experimental study.
The experimental protocol was approved by the ethical committee of the University of
Ljubljana (Slovenia) and was in accordance with the Declaration of Helsinki and
following amendments. A written informed consent was obtained from each
individuals. Subjects were physically active before the study and none of them was
under medication.
Subjects have been randomly assigned to three groups. Each group participated to three
experimental campaigns: i) 10-day normoxic normoxia (21 kPa oxygen) bed rest, NBR
ii) 10-day hypoxic (12.5 kPa oxygen, corresponding to 4000 m above sea level) bed
rest, HBR iii) 10-day hypoxic ambulation HAMB. Each campaign was preceded by 5
days of dietary and environmental adaptation and was followed by 5 days of recovery
(Figure 17). A 2-month wash-out period was allowed between each campaign. Dietary
intake was controlled to maintain subjects in eucaloric condition; before the study,
individual resting energy expenditure (REE) was calculated according to the
FAO/WHO equations (Muller MJ et al. 2004).
Each subject received a dietary energy intake equals to 1.4 and 1.1 times his REE in the
ambulatory (adaptation and recovery periods as well as hypoxic ambulation) and bed
rest (normoxic and hypoxic bed rests) periods, respectively. Menu composition was
adapted to individual dietary habits. Subjects received six meals daily (i.e., 3 main
meals and 3 snacks). Each individual was tested at the baseline (normoxic ambulation)
and at the end (day 10) of each experimental condition. All foods were weighed for
each participant, and volunteers were asked to consume the complete meal. At the
beginning and at the end of each experimental condition, body composition was
assessed by DXA (Discovery W—QDr series, Hologic, Bedford USA). Whole body
protein turnover as well as glutathione kinetics were determined by stable isotope
infusion technique, as previously described (see experimental design section 2.1.1,
2.2.5).

57
Statistics
All data were expressed as mean ± S.E.M. Hypoxia and bed rest effects were analyzed
by repeated measure ANOVA with two levels of interaction, physical activity and
oxygen partial pressure. If significant interaction was evidenced, post hoc analysis was
performed using Student t-test with Bonferroni correction. Statistical significance was
achieved with p-value <0.05. Statistical analysis was performed using SPSS software
(version 12; SPSS, Inc., Chicago, IL).
METHODS

10-d BED REST IN NORMOXIC CONDITION (n=11)

21 kPa oxygen

10-d BED REST IN HYPOXIC CONDITION (n=11)

12.5 kPa oxygen

10-d NORMAL PHYSICAL ACTIVITY IN HYPOXIC CONDITION (n=11)

12.5 kPa oxygen

• BODY COMPOSITION: DXA

• WHOLE-BODY PROTEIN KINETICS
Phe/Tyr stable isotope infusion in the
postabsorptive state

Figure 17. The PLANHHAB Study experimental design

Subjects were randomly assigned to three groups. Each group participated to three experimental campaigns: i) 10-day normoxic
normoxia bed rest (yellow) ii) 10-day hypoxic bed rest (red) iii) 10-day hypoxic ambulation (blue). A 2-month wash-out period was

allowed between each campaign.

58
 3.2.2. The PANGeA study (bed rest in the elderly)

PANGeA is an European program (INTERREG) aiming to answer to some scientific

questions that refer to the impact of motor inactivity on the elderly and, consequently,
on their health.
The bed rest study was conducted at the Valdoltra Hospital (University of Primorska,
Ankaran-Capodistria, Slovenia). Fifteen healthy male subjects were enrolled: 7 young
males (mean ± S.E.M.; age 23±1 years; BMI 24.0±0.9 kg/m2) and 8 elderly subjects
(mean ± S.E.M.; age 59±1 years; BMI 26.8±1.5 kg/m2). Body weight of all subjects had
been stable from the previous 3 months. Preliminary standard anthropometric measures
and routine medical screening were performed. Volunteers were admitted at the hospital
one week before the bed rest period for dietary and environmental adaptation phase
(Ambulatory period). At the end of this period, each subject underwent 14 days of bed
rest in which all daily activities were performed in horizontal conditions.
The experimental protocol was approved by the ethical committee of the University of
Ljubljana (Slovenia) and was in accordance with the Declaration of Helsinki and
following amendments. A written informed consent was obtained from each persons.
Subjects were physically active before the study and none of them was under
medication, or had any acute or chronic illness at least from the three months preceding
the protocol.
Dietary intake was controlled to maintain subjects in eucaloric condition; before the
study, individual resting energy expenditure (REE) was calculated according to the
FAO/WHO equations (Muller MJ et al. 2004). Participants received a diet containing
1.4 and 1.1 times their calculated REE during the ambulatory and the bed rest periods,
respectively; All foods were weighed for each participant, and volunteers were asked to
consume the complete meal.
Menu composition was adapted to individual dietary habits. Subjects received three
meals daily (i.e., breakfast, lunch and dinner). Each individual was tested at the baseline
(BR0) and at the end (BR+14) of the experimental condition. In these days all subjects
underwent to a metabolic test in order to estimate both insulin sensitivity and anabolic
sensitivity in fed state through a new, simple, safe and quick method, as previously
described (see experimental design section - 2.2.2, 2.2.3)
At the beginning and at the end of each experimental condition, body composition was
assessed by bioimpedentiometry (BIA 101, Akern Srl, Italy, following manufacture
instructions) (Figure 18).

59
Statistics
Data are expressed as mean ± SEM. In order to evaluate the effect of the bed rest and/or
the effects of aging on the insulin resistance and on the anabolic resistance, multivariate
ANCOVA statistical analysis was applied. Basal values were used as covariate. the
changes induced by bed rest and/or aging, were assessed through a Student T-test.
Linear regression analyses were performed using Pearson’s correlation. Data were
logarithmic transformed when appropriate. P-values <0.05 were considered statistically
significant. Statistical analysis was performed using SPSS software (version 12; SPSS,
Inc., Chicago, IL).

Figure 18. The PANGeA Study experimental design.

During each experimental day (BR0 and BR+14), after basal blood sampling, at time 0 (t0), an oral load of 0.3g of 2H5-

phenylalanine, dissolved in 15-20mL of water, was administred to the volunteers to be drank in 5 minutes. Blood samples were

drawn at 30, 60, 120, 180, 240, 300 and 360 minutes after the the loading.

60
 4. RESULTS

4.1. The PLANHAB study (experimental hypoxia and bed rest).

4.1.1. Body composition

As reported elsewhere (Debevec T. et al. 2014) there was a significant reduction in

body weight in all 3 conditions (–2.1%, –2.8%, and –2.0% for HAMB, HBR, and NBR,
respectively; p<0.05), due to a significant decrease in lean body mass (–3.8%, –3.8%, –
4.3% for HAMB, HBR, and NBR, respectively; figure 19A) with a slight but not
significant increase in fat mass (Figure 19B).

Figure 19. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on body composition.
N=11. Body composition changes before (basal, white columns) and after (intervention, black columns) ambulatory hypoxia
(HAMB), bed rest Hypoxia (HBR) and bed rest normoxia (NBR) conditions * for p<0.05.

4.1.2. Whole body protein kinetics

As showed on table 3 Hypoxia significantly decreased whole body protein turnover

(protein synthesis, -8±3%; protein degradation, -9±3%) in ambulatory conditions, while
hypoxia tended to increase the rates of synthesis (+4±4%) and degradation (+2±4%) in
bed rest. There were not significant effects of hypoxia or bed rest on the rates of
phenylalanine hydroxylation to tyrosine (Figure 18), an index of net protein loss in the
postabsorptive state (Basal 0.12±0.01, HAMB 0.11±0.01, NBR 0.12±0.01, HBR

61
 0.11±0.01 mmol/min/kg LBM; bed rest effect p=0.98, hypoxia effect p=0.08,
interaction 0.66).

Table 3 Effects of 10-d bed rest and hypoxia, per se or in combination, on protein
metabolism.
Ambulatory Bed Rest p#
Bed rest Hypoxia
Control Hypoxia Control Hypoxia Interaction
effect effect
Rd to protein synthesis
0.96±0.03 0.88±0.03* 0.92±0.02 0.95±0.03 0.86 0.57 0.001
(mmol/min/kg LBM)
Ra from proteolysis
1.08±0.03 0.99±0.04* 1.04±0.02 1.06±0.03 0.91 0.36 0.001
(mmol/min/kg LBM)
Rd to hydroxylation
0.12±0.01 0.11±0.01 0.12±0.01 0.11±0.01 0.98 0.08 0.66
(mmol/min/kg LBM)
N=11. Data were expressed as mean±S.E.M. # Data were analyzed with the use of a 2-factor repeated measure ANOVA. Rd, rate of

disappearance. Ra, rate of appearance. * p<0.05

On figure 20 are shown the effects of hypoxia and bed rest, alone or in combination, on
the rates of whole body protein synthesis and degradation. There was significant
hypoxia×bed rest interaction for the rates of protein synthesis and degradation.
PROTEIN KINETICS
Normoxia Hypoxia

PROTEIN SYNTHESIS PROTEIN DEGRADATION

µmol/kg/min

µmol/kg/min

*
*

Ambulatory Bed rest Ambulatory Bed rest

OXYGEN EFFECT p=0.86 OXYGEN EFFECT p=0.91

PHYSICAL ACTIVITY EFFECT p=0.57 PHYSICAL ACTIVITY EFFECT p=0.36
INTERACTION p=0.001 INTERACTION p=0.001

FigureRepeated
20. Effect of hypoxia in with
measurement-ANOVA ambulatory conditions
two-factor interactions and during
(physical 10-d
activity and of bed
oxygen level)rest

on whole body protein kinetics.

N=11. Whole body protein kinetics changes in ambulatory and bed rest states during hypoxia (white columns), or normoxia (black

columns) conditions * for p<0.05.

62
No changes were observed on phenylalanine hydroxylation an index of net protein loss
(Figure 21).

0.2
PHENYLALANINE HYDROXYLATION

0.15 ns ns
µmol/kg/min

0.1

0.05

0
Ambulatory Bed rest

Normoxia Hypoxia

Figure 21. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on phenylalanine hydroxylation.
N=11. phenylalanine hydroxylation changes in ambulatory and bed rest states during hypoxia (white columns), or normoxia (black

columns) conditions * for p<0.05. ns = non significant

63
 4.1.3. Plasma lipid profile

Effects of hypoxia per se and/or combined to bed rest lipid profile are reported on table
4.

Table 4. Effects of 10-d bed rest and hypoxia, per se or in combination on plasma
lipid profile.
Ambulatory Bed Rest p#
Bed rest Hypoxia
Normoxia Hypoxia Normoxia Hypoxia Interaction
effect effect

PLASMA LIPIDS
Total cholesterol (mg·dL-1) 184±13 173±8 174±10 180±11 0.66 0.67 0.12
-1
Tryglicerides (mg·dL ) 94±14 113±14* 109±20 102±13 0.65 0.38 0.02
HDL cholesterol (mg·dL-1) 49±3 41±2* 42±2 39±3 <0.01 <0.001 0.02
-1
LDL cholesterol (mg·dL ) 117±12 110±7 110±7 121±9 0.57 0.70 0.14
HDL2-to-HDL3 ratio 0.48±0.08 0.43±0.07 0.55±0.09 0.38±0.05 0.93 0.26 0.55
ENZYMES INVOLVED IN LIPID METABOLISM
Cholesteryl ester transfer
2.91±0.23 3.00±0.16 2.86±0.20 3.01±0.19 0.87 0.31 0.85
protein (µg·mL-1)
Lecithin-cholesterol
21.8±4.2 20.3±4.3 21.7±4.7 23.8±6.3 0.66 0.99 0.45
acyltransferase (ng·L-1)
Lipoprotein lipase (ng·L-1) 28.4±3.3 23.9±3.6 27.5±4.0 26.8±3.7 0.48 0.28 0.43
Hepatic lipase (U·mL-1) 0.72±0.10 0.79±0.09 0.80±0.09 0.90±0.08 0.15 0.04 0.81
N=11. Data were expressed as mean±S.E.M. # Data were analyzed with the use of a 2-factor repeated measure ANOVA. *p<0.05

HDL concentrations were significantly decreased both by bed rest and hypoxia with a
significant negative bed rest×hypoxia interaction (Figure 20).
Furthermore the ratio between HDL and total cholesterol (Figure 20) was significantly
decreased by physical inactivity (-8±4%, p=0.001) and hypoxia (-11±4%, p=0.003).
The ratio between HDL2 and HDL3 were not significantly changed. Plasma levels of
total cholesterol, LDL and triglycerides were not significantly affected by 10-d of bed
rest with or without hypoxia. On the other hand, the exposure to hypoxia in ambulatory
conditions increased the triglycerides concentrations without changes in the other
plasma lipid fractions (Figure 22).

64
Figure 22. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on total and HDL cholesterol.
N=11. HDL cholesterol (A), Total Cholesterol (B) and HDL-to-total cholesterol ratio (C) modifications in ambulatory and bed rest

states during hypoxia (black columns), or normoxia (white columns) conditions. Data were analyzed with the use of a 2-factor
repeated measure ANOVA. Significance for p<0.05.

65
 Among the enzymes involved in lipid metabolism only HL was modified, with a
significant increase in hypoxic conditions, together with a decreased LPL-to-HL ratio
(p=0.05), independently from the physical activity levels (Figure 23).

Figure 23. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on enzymes involved in lipid metabolism.
N=11. LPL-to-HL ratio modifications in ambulatory and bed rest states during hypoxia (black columns), or normoxia (white

columns) conditions. Data were analyzed with the use of a 2-factor repeated measure ANOVA. Significance for p<0.05. LPL,
Lipoprotein lipase. HL, Hepatic Lipase.

4.1.4. Insulin sensitivity.

There were no significant effects of hypoxia or bed rest on plasma glucose and insulin
concentrations or on HOMA-IR index (Table 5). However the Δ-5 desaturase index was
significantly reduced by both exposure to bed rest (p=0.03) and hypoxia (p=0.02). The
index decreased by 5±2% after 10 days of physical inactivity and by 6±2% after 10 days
of hypoxia, as compared to baseline conditions (Figure 24). A bed rest×hypoxia
interaction was not observed (p=0.15).

Table 5. Effects of 10-d bed rest and hypoxia, per se or in combination, on glucose
metabolism.
Ambulatory Bed Rest p#

Bed rest Hypoxia

INSULIN SENSITIVITY Normoxia Hypoxia Normoxia Hypoxia Interaction
effect effect

Fasting insulin (µU·mL-1) 7±1 7±1 7±1 6±1 0.85 0.93 0.27
-1
Fasting glucose (mg·dL ) 94±2 93±1 93±1 92±1 0.35 0.57 1.00
HOMA index 1.5±0.2 1.7±0.2 1.6±0.2 1.5±0.1 0.74 0.98 0.29
N=11. Data were expressed as mean±S.E.M. # Data were analyzed with the use of a 2-factor repeated measure ANOVA. *p<0.05

66
Figure 24. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on the activity of Δ-5 desaturase enzyme.
N=11. Δ-5 desaturase activity modifications in ambulatory and bed rest states during hypoxia (black columns), or normoxia (white

columns) conditions. Data were analyzed with the use of a 2-factor repeated measure ANOVA. Significance for p<0.05.

Tumor necrosis factor Related Apoptosis Induce Ligand (TRAIL)

Plasma TRAIL concentration was significantly increased by hypoxia (+36±6%,
p=0.001) and a significant bed rest×hypoxia interaction was reported (p=0.04). A
significant direct correlation between TRAIL and GSH-to-GSSG ratio was observed, as
reported in figure 25.

70%
60%
50%
40%
30%
TRAIL

20%
10%
Pearson's correlation
0%
R = 0.43
"10% p<0.05
"20%
"30%
"300% "200% "100% 0% 100% 200% 300% 400% 500%

GSH/GSSG!ratio!

Figure 25. Correlation between TRAIL and GSH-to-GSSG ratio.

N=11. TRAIL and GSH-to-GSSG ratio positive relationship in hypoxic bed rest conditions. Significance for p<0.05.

67
 4.1.5. Inflammatory responses.

Hypoxia, but not bed rest, significantly increased the levels of C reactive protein (CRP)
and serum amyloid A (SAA) indices of systemic inflammation, without a significant
bed rest×hypoxia interaction (Figure 26).

Figure 26. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on the activity of inflammatory status.
N=11. SAA (A) and PCR (B) modifications in ambulatory and bed rest states during hypoxia (black columns), or normoxia (white
columns) conditions. Data were analyzed with the use of a 2-factor repeated measure ANOVA. Significance for p<0.05. SAA,
serum amyloid A. CRP, C reactive protein.

68
 4.1.6. Oxidative stress

Glutathione redox capacity

Data are reported on table 6. Hypoxia conditions significantly increased the hematocrit
values (Figure 27). Thus, the results of glutathione concentrations and glutathione
absolute turnover rate (ATR) were normalized by whole blood volume, taking into
account the hypoxia-induced hematocrit changes, and also by erythrocyte volume.

Table 6. Effects of 10-d bed rest and hypoxia, per se or in combination, on

glutathione redox capacity in erytrocytes (RBC) and in whole blood (WB).
Ambulatory Bed Rest p#
Bed rest Hypoxia
Normoxia Hypoxia Normoxia Hypoxia Interaction
effect effect
Hematocrit (%) 46±1 51±1 47±1 51±1 0.07 0.03 0.10
Total glutathione
2692±84 2620±79 2564±85 2547±86 0.03 0.22 0.42
(mmol/L RBC)
Glutathione ATR
656±135 858±88 869±82 1250±263 0.05 0.06 0.53
(mmol/day/L RBC)
Total glutathione
1245±49 1332±51 1207±37 1302±45 0.21 <0.001 0.82
(mmol/L WB)
Glutathione ATR
299±61 430±44 409±38 637±134 0.04 0.03 0.49
(mmol/day/L WB)
Glutathione FTR
25±5 33±3 35±4 50±10 0.04 0.05 0.58
(percent/day)
GSH/GSSG (ratio) 137±26 237±43* 221±37 176±36 0.81 0.40 0.009
N=11. Data were expressed as mean±S.E.M. # Data were analyzed with the use of a 2-factor repeated measure ANOVA. ATR,

absolute turnover rate. FTR, fractional turnover rate. GSH/GSSG, ratio between reduced and oxidised glutathione. RBC, red blood

cell. WB, whole blood. Significance for p<0.05.

69
Figure 27. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on Hematocrit.
N=11. Hematocrit modifications in ambulatory and bed rest states during hypoxia (black columns), or normoxia (white columns)

conditions. Data were analyzed with the use of a 2-factor repeated measure ANOVA. Significance for p<0.05.

Total glutathione concentrations normalized by erythrocytes volume significantly

decreased following bed rest in both normoxic or hypoxic conditions, with no
significant bed rest×hypoxia interaction (Figure 28A).

Figure 28. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on glutathione metabolism in erythrocytes.
N=11. Total glutathione concentration (A) and Glutathione Absolute synthesis rate (B) modifications in red blood cells during

ambulatory and bed rest states in hypoxia (black columns), or normoxia (white columns) conditions. Data were analyzed with the
use of a 2-factor repeated measure ANOVA. Significance for p<0.05. GSH, glutathione. ATR, absolute synthesis rate. RBC, red

blood cell.

70
The glutathione ATR normalized by RBC volume, shows a significant increase
following 10-d of immobilization in both normoxic and hypoxic states (Figure 28B).
The total glutathione concentration normalized by whole blood volume shows a
significant positive effect of hypoxia in both ambulatory and BR conditions (Figure
29A). The glutathione ATR normalized by whole blood volume shows an increased
turnover rate influenced both by hypoxia and physical inactivity (Figure 29B).
The GSH/GSSG ratio, an index of the redox balance, indicates a significant
hypoxia×bed rest interaction on the ratio. Hypoxia and BR conditions significantly
increased the glutathione FTR (Figure 29C).

Figure 29. Effect of hypoxia in ambulatory conditions and during 10-d of bed rest
on glutathione metabolism in whole blood.
N=11. Total glutathione concentration (A) and Glutathione Absolute synthesis rate (B) modifications in whole blood and

glutathione fractional synthesis rate (C) changes, measured during ambulatory and bed rest states in hypoxia (black columns), or

normoxia (white columns) conditions. Data were analyzed with the use of a 2-factor repeated measure ANOVA. Significance for

p<0.05. GSH, glutathione. ATR, absolute synthesis rate. FTR fractional synthesis rate. WB, whole blood.

71
 4.1.7. Plasma and erythrocyte GSH amino acid precursor

Plasma concentrations of glutamate and cysteine were increased due to hypoxia, for
glycine plasma levels there is a bed rest×hypoxia interaction. In erythrocytes, hypoxia
induced a statistically significant increase in glycine concentrations while no effects
were observed on the concentrations of the other glutathione amino acid precursor
(Table 7). Hypoxia inversely affected plasma concentrations of glutamate and
glutamine. Glutamine decreased by about 4%, while glutamate increased by about 12%,
both in ambulatory and bed rest conditions. There was a significant hypoxia-induced
increase (+19±6%, p<0.01) on the glutamate-to-glutamine ratio. The 5-oxoproline
plasma concentrations tended to decrease in hypoxic condition both in ambulatory and
bed rest states

Table 7. Effects of 10-d bed rest and hypoxia, per se or in combination, on amino
acid concentrations in plasma and red blood cells.
Ambulatory Bed Rest p#
Bed rest Hypoxia
Normoxia Hypoxia Normoxia Hypoxia Interaction
effect effect
PLASMA AMINO ACID CONCENTRATIONS
Cysteine (µmol×L-1) 294±12 299±11 277±10 304±11 0.45 0.005 0.27
-1
Glycine (µmol×L ) 172±9 185±10* 196±12 185±12 0.04 0.78 0.01
Glutamate (µmol×L-1) 61±4 69±5 62±3 67±3 0.81 0.03 0.59
Glutamine (µmol×L-1) 541±10 515±9 552±21 522±7 0.55 0.01 0.86
-1
Homocysteine (µmol×L ) 15.3±1.7 17.0±1.4 14.7±1.4 16.2±1.4 0.38 0.003 0.93
a -1 a
5-oxoproline (µmol×L ) 1.01±0.08 0.91±0.06 1.00±0.07 0.91±0.06 0.92 0.08 0.90
ERYTHROCYTE AMINO ACID CONCENTRATIONS
Cysteine (µmol×L-1) 70±3 79±3 72±4 70±3 0.20 0.33 0.07
Glycine (µmol×L-1) 368±11 389±13 372±12 393±12 0.49 <0.001 0.98
-1
Glutamate (µmol×L ) 477±24 481±18 448±16 478±17 0.33 0.16 0.37
-1
Glutamine (µmol×L ) 1053±147 1015±209 1042±141 1000±215 0.79 0.73 0.97
N=11. Data were expressed as mean±S.E.M. # Data were analyzed with the use of a 2-factor repeated measure ANOVA. a, 5-

oxoproline concentrations have been estimated as ratio between 5-oxoproline and glutamate. Significance for p<0.05.

72
 4.1.8. Red blood cell membrane lipid composition.

Data on lipid composition in RBC membranes are reported in Table 8. There are no
changes in FAs concentrations in any of the study protocols.

Table 8. Effects of 10-d bed rest and hypoxia, per se or in combination on fatty
acids composition (%) of erythrocyte membranes.
Ambulatory Bed Rest p#
Bed
Hypoxia
Normoxia Hypoxia Normoxia Hypoxia rest Interaction
effect effect
SATURATED FATTY ACIDS
Myristic 14:00 0.20 ± 0.024 0.31 ± 0.32 0.3 ± 0.025 0.25 ± 0.020 0.45 0.08 0.01
Palmitic 16:00 20.61 ± 0.36 20.9 ± 0.31 20.62 ± 0.26 20.81 ± 0.29 0.71 0.15 0.76
Stearic 18:00 18.54 ± 0.20 18.42 ± 0.08 18.64 ± 0.17 18.45 ± 0.14 0.58 0.27 9.64
SUM 39.35 ± 0.45 39.63 ± 0.37 39.55 ± 0.32 39.52 ± 0.34
MONOUNSATURATED FATTY ACIDS
Palmitoleic 16:1 n7 0.25 ± 0.04 0.27 ± 0.04 0.28 ± 0.04 0.26 ± 0.03 0.72 1.0 0.26
Oleic 18:1 n9 13.34 ± 0.33 12.87 ± 0.33 12.81 ± 0.28 12.94 ± 0.34 0.08 0.1 0.06
Elaidic trans 18:1n-9 1.03 ± 0.04 1.02 ± 0.03 1.01 ± 0.05 1.05 ± 0.05 0.7 0.45 0.14
Eicosenoic 20:1n-9 0.31 ± 0.01 0.3 ± 0.01 0.3 ± 0.01 0.31 ± 0.01 0.86 0.75 0.1
SUM 14.92 ± 0.37 14.46 ± 0.34 14.39 ± 0.23 14.56 ± 0.37
n-3 POLYUNSATURATED FATTY ACIDS
α-Linolenic acid 18:3
0.04 ± 0.02 0.04 ± 0.02 0.05 ± 0.02 0.04 ± 0.02 ns ns ns
n3
Eicosapentaenoic acid
0.53 ± 0.04 0.58 ± 0.04 0.56 ± 0.03 0.54 ± 0.04 0.94 0.5 0.1
20:5n-3
Docosapentaenoic
2.50 ± 0.08 2.57 ± 0.10 2.55 ± 0.09 2.57 ± 0.09 0.43 0.02 0.41
22:5n-3
Docosahexaenoic acid
4.48 ± 0.23 4.69 ± 0.26 4.63 ± 0.25 4.80 ± 0.2 0.02 0.01 0.83
22:6n-3
SUM 7.50 ± 0.25 7.84 ± 0.30 7.74 ± 0.26 7.91 ± 0.19
n-6 POLYUNSATURATED FATTY ACIDS
Linoleic acid 18:2 n6 11.87 ± 0.35 11.72 ± 0.32 11.6 ± 0.35 11.39 ± 0.29 0.08 0.21 0.83
Eicosadienoic acid
1.09 ± 0.51 1.47 ± 0.56 1.13 ± 0.19 0.89 ± 0.24 0.6 0.63 0.5
20:2n-6
Dihomo-γ-linolenic
1.97 ± 0.13 2.04 ± 0.13 2.08 ± 0.12 2.16 ± 0.13 0.001 0.02 0.98
20:3n-6
Arachidonic acid
18.11 ± 0.28 17.7 ± 0.27 18.16 ± 0.21 18.39 ± 0.32 0.04 0.49 0.02
20:4n-6
Adrenic 22:4n-6 4.15 ± 0.20 3.99 ± 0.18 4.12 ± 0.17 4.17 ± 0.18 0.07 0.02 0.02
Docosapentaenoic
0.99 ± 0.08 1.10 ± 0.13 1.17 ± 0.17 0.98 ± 0.08 0.71 0.47 0.17
22:5n-6
SUM 38.22 ± 0.79 38.08 ± 0.72 38.31 ± 0.65 38.01 ± 0.64
N=11. Values are percent of total Fatty Acids. Data were expressed as mean±S.E.M. # Data were analyzed with the use of a 2-factor
repeated measure ANOVA Significance for p<0.05.

73
 4.2. The PANGEA STUDY (bed rest in the elderly)

4.2.1. Body composition

Body composition was significantly modified after 14 days of BR both in young and
elderly subjects. As showed in table 9, body weight and BMI significantly decrease
after 14-d of resting with a reduction in FFM (Figure 30A) and a slight but significant
increase in FM (Figure 30B).

Table 9. Anthropometrical data before (BDC-1) and after (BR+14) bed rest in
young and elderly subjects.
p#
bed rest ×
Bed rest
subjects BDC-1 BR+14 group
effect
interaction
Body weight Young 74.84 ± 3.32 71.59 ± 3.14
<0.001 0.08
(kg) Elderly 79.64 ± 3.71 77.58 ± 3.67
Young 23.96 ± 0.89 22.91 ± 0.81
BMI (kg/m2) <0.001 0.12
Elderly 26.85 ± 1.47 26.15 ± 1.45
Young 60.86 ± 1.46 56.36 ± 1.43
FFM (kg) <0.001 0.18
Elderly 59.79 ± 2.43 56.66 ± 2.41
Young 13.99 ± 2.34 15.33 ± 2.66
FM (kg) 0.01 0.74
Elderly 19.85 ± 1.75 20.91 ± 1.69
N=7 young group; N=8 elderly gourp. Data were expressed as mean±S.E.M. #Data were analyzed with the use of a 2-factor

repeated measure ANCOVA. BMI: Body Mass Index, FFM: Fat-Free Mass, FM: Fat Mass.

Figure 30. Body composition modifications after 14-d of bed rest in young and
elderly subjects.
N=7 young group; N=8 elderly group. Body composition modifications in ambulatory conditions (black column) and after 14-d of
bed rest (white column). in young and elderly subjects. Data were expressed as mean±S.E.M. Data were analyzed with the use of a

2-factor repeated measure ANCOVA. FFM, Fat-free Mass. FM Fat Mass.

74
 4.2.2. Insulin sensitivity.

Glucose and insulin metabolism data, assessed in basal conditions and during load,
before and after 14-d of experimental inactivity, are reported in table 10. The metabolic
basal values were similar between the two groups. Plasma glucose concentration in
fasting state were not significantly modified after 14-d of BR while the fasting insulin
plasma concentration were augmented especially in young subjects (+74±21% and
+16±12% in young and elderly individuals respectively).
The HOMA-IR, as index of insulin resistance in fasting state, significantly increase
after BR in both young and elderly subjects, however, young individuals shown
significantly (p=0.04) higher values (+80±29%) than eldelry (+12±13%).

Table 10. Glucose metabolism data before (BDC-1) and after (BR+14) bed rest in
young and elderly subjects.
p#
Delta %
Bed bed rest ×
subjects BDC-1 BR+14 (BR+14 – rest group
BDC-1) effect interaction
Fasting glycaemia Young 87 ± 4.30 86.57 ± 1.90 1.09 ± 5.74
0.45 0.54
(mg/dL) Elderly 96.38 ± 4.00 92.38 ± 2.39 3.29 ± 3.75
Fasting insulinaemia Young 5.14 ± 1.10 8.43 ± 1.54 74.32 ± 21.06
<0.01 <0.05
(UI/L) Elderly 5.25 ± 0.77 5.88 ± 0.90 15.63 ± 11.52
Young 1.11 ± 0.23 1.82 ± 0.35 79.67 ± 28.86
HOMA index 0.02 <0.05
Elderly 1.27 ±0.20 1.34 ± 0.21 12.44 ± 12.72
Young 26.93 ± 4.24 14.21 ± 2.38 -45.95 ± 4.51
Matsuda index <0.001 0.01
Elderly 18.79 ± 1.50 14.95 ± 2.09 -20.34 ± 9.42
Young 99.86 ± 17.47 207.68 ± 37.96 113.29 ± 19.12
AUCIns (UI h/L) <0.01 0.02
Elderly 133.84 ± 12.97 198.38 ± 26.82 48.78 ± 14.24
Young 311.61 ± 13.76 334.50 ± 13.82 7.84 ± 3.64
AUCGluc (mg h/dL) <0.05 0.94
Elderly 372.13 ± 23.56 396.81 ± 24.47 7.58 ± 5.66
Young 0.33 ± 0.06 0.62 ± 0.11 98.31 ± 17.22
AUCIns/AUCGluc 0.001 0.02
Elderly 0.37 ± 0.04 0.51 ± 0.07 38.93 ± 12.73
N=7 young group; N=8 elderly gourp. Data were expressed as mean±S.E.M. #Data were analyzed with 2-factor repeated measure
ANCOVA. data were logarithmic transformed when appropriate

75
Matsuda index, as parameter of insulin sensitivity in fed state (loading), showed a
reduction due to the 14-d of BR (Figure 31) in both groups but it was doubled in young
than in elderly (-46±5% vs -20±9%, respectively, p=0.04).

Figure 31. Matsuda index modifications after 14-d of bed rest in young and elderly
subjects.
Interaction = bed rest × group interaction. Data analyzed with the 2-factor repeated measure ANCOVA

76
The area-under-the curve (AUC), i.e., the area under of both plasma glucose (AUCGly)
and plasma insulin (AUCIns) concentrations during the test meal versus time, was
estimated using the linear trapezoidal method. In basal condition the AUCIns values
were comparable between the two groups while the AUCGly values were greater in
elderly than in young people about of 19% (Figure 32B and D). After BR the AUCIns
was augmented in both groups with a significant (p=0.02) higher increase in the young
population (+113±19%) if compared to the elderly group (+49±14%). (Figure 32A and
C).
The AUCIns/AUCGly ratio, as index of insulin resistance after load, was augmented in
both young and elderly subjects after bed rest however the young individuals showed a
significant (p=0.01) higher increase of this value (+98±17%) than elderly population
(+39±13%) (Table 9).

Figure 32. Glycaemia and insulinaemia modifications before (◼︎) and after 14-d of
bed rest ( ) in young (A and B) and elderly (C and D) subjects.
Interaction = bed rest × group interaction. Data analyzed with the 2-factor repeated measure ANCOVA

77
 4.2.3. Anabolic resistance

The plasma AA concentrations as well as the isotopic enrichment of D5-Phe and D4-
Tyr, before and after the bed rest period, were assessed by GCMS as previously
reported (see methods section).
To evaluate the effect of ageing and bed rest on anabolic resistance the AUC i.e., the
area under of both plasma D5-Phe (AUCD5-Phe) and plasma D4-Tyr (AUCD4-Tyr)
concentrations during the test meal versus time, were calculated. Moreover the AUCs of
unlabeled phenylalanine (AUCPhe), tyrosine (AUCTyr) and leucine (AUCLeu) were also
assessed (Table 11).
As showed in table 10 AUCPhe significantly increased after 14-d of bed rest (4%) with
no time×group interaction. No significant changes were observed after 14-d of
experimental inactivity on AUCTyr and AUCLeu values. Leucine plasma concentrations
are considered an index of the absorption of the amino acids from the protein
administered with the teat meal. We observed that the leucine peak at 60 minutes was
comparable between the ambulatory and bed rest conditions both in young and elderly
subjects, showing no interference from the amino acid absorption rate.

Table 11. Bed rest effect on labeled and unlabeled plasma amino acids
concentration in young and elderly subjects
p#
Delta %
Bed rest bed rest ×
(BR+14 – effect group
BDC-1 BR+14 BDC-1) interaction
Young 413.14 ± 27.58 430.49 ± 19.37 6.10 ± 5.93
AUC Phenylalanine 0.03 0.60
Elderly 401.27 ± 11.51 415.51 ± 16.13 3.61 ± 3.19
Young 516.02 ± 58.58 502.44 ± 44.51 0.47 ± 6.21
AUC Tyrosine 0.37 0.23
Elderly 599.72 ± 44.09 614.95 ± 52.29 2.12 ± 2.92
Young 818.32 ± 41.91 815.92 ± 58.18 -0.31 ± 4.77
AUC Leucine 0.55 0.93
Elderly 793.23 ± 43.15 798.06 ± 37.71 1.06 ± 2.64
Young 16.33 ± 1.67 16.44 ± 1.39 4.19 ± 9.41
AUC D5-Phe 0.14 0.54
Elderly 16.13 ± 1.55 17.99 ± 2.29 13.24 ± 12.13
Young 3.38 ± 0.60 3.30 ± 0.36 9.06 ± 14.06
AUC D4-Tyr <0.01 0.05
Elderly 3.84 ± 0.43 4.93 ± 0.66 33.32 ± 15.78
AUC D4-Tyr/ Young 0.20 ± 0.03 0.20 ± 0.01 5.20 ± 9.36
AUC D5-Phe Elderly <0.01 0.01
0.24 ± 0.02 0.28 ± 0.02 18.5 ± 7.27
(T0-T360)
AUC D4-Tyr/ Young 0.19 ± 0.02 0.19 ± 0.02 -0.53 ± 1.85
<0.05 <0.05
AUC D5-Phe (T120) Elderly 0.21 ± 0.02 0.27 ± 0.03 6.79 ± 3.85
N=7 young group; N=8 elderly gourp. Data were expressed as mean±S.E.M. #Data were analyzed with 2-factor repeated measure

ANCOVA. data were logarithmic transformed when appropriate. AUC, area under the curve.

78
On figure 33 were reported the D5-Phe and D4-Tyr plasma concentrations during the
test meal before and after the BR period on both young and elderly subjects.

Figure 33. D5-Phe and D4-Tyr plasma concentration before (◼︎) and after 14-d of
bed rest ( ) in young (A and B) and elderly (C and D) subjects.
Interaction = bed rest × group interaction. Data analyzed with the 2-factor repeated measure ANCOVA

No significant changes were observed on AUCD5-Phe after 14-d of experimental BR

while the AUCD4-Tyr vales were significantly modified.
In basal condition, the AUCD4-Tyr/AUCD5-Phe - T360 ratio, as index of anabolic resistance,
was comparable between the two groups, after 14-d of BR. It appear increased in both
young and elderly subjects, but with higher values in the elderly (+19±7%) than in the
young (+5±9%) population, as confirmed by the significant time×group interaction
(Table 10).

79
Figure 34. Bed rest effect on anabolic resistance expressed as AUCD4-Tyr/AUCD5-Phe
ratio in young and elderly subjects
Interaction = bed rest × group interaction. Data analyzed with the 2-factor repeated measure ANCOVA

The AUCD4-Tyr/AUCD5-Phe - T360 ratio was evaluated considering all the blood draw
points as reported in the experimental design (Figure 18). The same index was also
calculated using the blood draw points of the first two hours of the experimental
protocol (AUCD4-Tyr/AUCD5-Phe - T120). As reported on figure 34 (A and B) the AUCD4-
Tyr/AUCD5-Phe - T120 ratio showed the same trend observed with the AUCD4-Tyr/AUCD5-Phe
- T360 ratio, moreover, a significantly direct correlation (R=0.75; p<0.001) was find
comparing the two indices (Figure 35).

80
 0,4
Pearson’s
Test di Pearson
0,35 R=0,5
AUC D4-Tyr/AUC D5-Phe (T120) p<0,001
0,3

0,25

0,2

0,15

0,1

0,05

0
0 0,1 0,2 0,3 0,4

AUC D4-Tyr/AUC D5-Phe (T0-T360)

Figure 35. Correlation between the AUCD4-Tyr/AUCD5-Phe - T360 ratio and the
AUCD4-Tyr/AUCD5-Phe - T120 ratio

81
 5. DISCUSSION

The primary aim of the present work was to develop biomarkers useful to define, in
vivo, in humans, the optimal protein intake in different physiological and pathological
conditions. Within the frame of two studies sponsored by the European Community
(i.e., the FP7 PLANHAB Study and the INTERREG ITA-SLO PANGEA bed rest
study), we applied such methodology to investigate protein requirement in conditions of
inactivity, with or without hypoxia, as well as in a population of healthy elderly subjects
assessed at different levels of physical activity, from ambulatory to bed rest.
The assessment of whole body protein kinetics is a standard methodology to detect
conditions of impaired protein synthesis and anabolic resistance, in the fasting state and
stimulation by protein nutrition. We applied this method in the PLANHAB Study to
investigate protein requirement in hypoxic condition both in ambulatory and bed rested
individuals. The PANGeA study was the second study in the world where aged healthy
people were bed ridden and the first one in which healthy elderly subjects were
compared to healthy young individuals (as control group), during a period of
experimental inactivity. Moreover it was the first time that both glucose and protein
metabolism were assessed in healthy elderly volunteers undergoing bed rest.
In the PANGEA study we developed a new method to assess the ability to utilize
dietary protein during and after a mixed meal. Anabolic resistance is a condition
characterized by reduced ability of meal protein to stimulate protein synthesis, thus
increasing protein requirements. Our new method was used to compare anabolic
resistance and protein requirement in elderly and young subjects either in ambulatory or
in bed rest conditions.
Moreover this new method allowed evaluate at the same time anabolic and insulin
resistance in the post-prandial state. We had therefore the opportunity to explore the
relationships between changes in insulin and anabolic resistance in elderly and young
subjects at different levels of physical activity. The quantity of protein intake, not only
influences the rate of protein synthesis but has an impact also on other metabolic
targets, such as the redox balance. In particular the glutathione system is influenced by
the availability of the precursor amino acids. We have therefore investigated different
aspects of the glutathione system in the PLANHAB study with the aim to define the
effects of hypoxia, with or without bed rest.

82
The relative proportion of cell membrane fatty acids was determined in the PLANHAB
study in order to detect changes induced by insulin resistance or systemic inflammation
and oxidative stress.

The main results of our work can be summarized as follows:

5.1. Hypoxia decreases protein synthesis.

5.2. Aging is characterized by anabolic resistance.

5.3. Aging is a protective factor against inactivity mediated insulin resistances?

5.4. Hypoxia per se improves oxidative stress.

5.5. Hypoxia-bed rest interaction induces multiple metabolic changes.

5.6. Identification of a new biomarker to evaluate anabolic (and insulin) resistance

to define optimal protein requirements.

83
 5.1. Hypoxia decreases protein synthesis

Chronic hypoxia and unloading are well-known protein catabolic factors for skeletal
muscle (Biolo et al 2014; Di Giulio C et al. 2009). Ten days of normobaric hypoxia in
ambulatory conditions or in horizontal bed rest in healthy volunteers led to similar
losses of lean body mass. Bed rest in normoxic conditions led to a similar muscle
catabolic response. Our study shows that when the two factors are applied in
combination for 10 days in healthy volunteers they do not exhibit additive effects. The
hypertrophic response of skeletal muscle trained in chronic hypoxia conditions was
significantly lower than that produced in normoxia (Narici MV et al. 1995). Muscle loss
associated with unloading is characterized by decreased protein turnover with a
resistance to amino acid-mediated stimulation of protein synthesis as key catabolic
mechanism. Protein synthesis is an energy consuming process. Hypoxia, which limits
energy production, has been shown to decrease muscle protein synthesis (Preedy VR et
al. 1985) in animal models, in vitro and in vivo. Decreased muscle protein synthesis has
been observed also in emphysema patients with chronic respiratory failure (Morrison
WL et al. 1988). In parallel with the rate of protein synthesis, hypoxia in ambulatory
conditions also decreased by about 8% the rate of whole body protein degradation,
leading to a decreased protein turnover.
While normoxic subjects are characterized by accelerated post-exercise muscle protein
turnover, in COPD patients with emphysema the rates of protein synthesis and
degradation were suppressed after low-intensity exercise (Engelen MP et al. 2003). A
blunted post-exercise suppression of muscle protein synthesis was also observed in
experimental hypoxia (Etheridge T et al. 2011). Our results are in perfect agreement
with these previous observations. Hypoxia in ambulatory conditions decreased by about
8% the rates of whole body protein synthesis and degradation. In contrast, hypoxia in
bed rest conditions did not significantly affect whole body protein turnover. As
expected the net protein loss in the post-absorptive state was not accelerated by bed rest.
In fact, post-prandial resistance to amino acid-mediated stimulation of protein synthesis
is the key catabolic mechanism during muscle unloading. Net protein loss in the post-
absorptive state was not accelerated during hypoxia suggesting postprandial anabolic
resistance also in this condition.

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 5.2. Aging is characterized by anabolic resistance

During the PANGeA study we tested a new, simple, safe and quick method to evaluate
the anabolic resistance to stimuli inducing protein synthesis (e.g. exercise, amino acids,
etc.), associated with ageing.
Our results confirm that both aging and physical inactivity impairs the sensitivity to
anabolic stimuli (Kim IY et al. 2015; Biolo G et al. 2004).
The development of anabolic resistance following physical inactivity was higher in the
elderly than in the young subjects, evidencing that aging is a detrimental factor in the
risk of an impaired inactivity-mediated protein metabolism. There is a potential decline
in the efficiency of muscle protein synthesis with advancing age when a small amount
of EEAs are ingested (Katsanos CS et al. 2005). Moreover, it was previously
demonstrated (Biolo G et al. 2004) that decreased levels of physical activity in healthy
young subjects were associated to compromise anabolic sensitivity to an amino acidic
stimulus. After 14 days of experimental bed rest, in fact, a reduction in protein synthesis
after AA administration was observed (Biolo G et al. 2004). Consequently, after a
period of inactivity, it seems necessary to increase the protein intake in order to improve
the protein anabolism in the fed state and overcome the anabolic resistance. The
reduction of the sensibility of the muscle protein synthesis to an anabolic stimulus (i.e.
protein intake and/or physical activity) associated with aging (Kim IY et al. 2015), leads
to a reduction in muscle mass and strength, to an increased frailty with a higher risk of
falls and generally to a higher morbidity and mortality (Breen L & Phillips SM. 2011).
Moreover, this resulting loss of skeletal muscle protein could be a key factor in the
development and the progression of sarcopenia and loss of function (Volpi E et al
2000).
Thus anabolic resistance is the principal catabolic mechanism responsible of muscle
atrophy after a period of physical inactivity or during aging (Biolo G et al. 2004). The
decreased ability to use AA for the MPS observed either with aging or in bed ridden
young subject, seems to require an increased protein intake with the diet, especially
when the two condition are associated. Furthermore, a larger intake of EAAs (i.e., 15 g
in the form of beef) resulted in a similar stimulation of muscle protein synthesis (MPS),
in young and old adults (Symons TB et al. 2007).
However, a diet with high protein content could determine harmful effects in elderly
subjects such as impairment of kidney function (Deutz NE et al. 2014). Nonetheless, a
regular physical activity stimulates the MPS and contributes to maintain a positive
protein balance, favoring the muscle mass gain both in young and elderly subjects

85
(Fiatarone MA et al.1990; Fiatarone MA et al. 1994). In healthy adults, both young and
elderly, resistance and endurance exercise acutely stimulates muscle protein synthesis
(Cuthbertson D et al. 2005; Short KR et al. 2004) and this effect is increased when
physical activity is associated to protein administration (Breen L & Phillips SM. 2011).
Our evidences suggest that during inactivity or aging there is an augmented protein
need, and both exercise and proper nutrition could have intense effects on muscle mass
and function. Therefore a correct lifestyle could counteract the consequences of aging.
Specific nutritional programs can attenuate or even overcome the muscle loss associated
to these conditions should be associated to specific training protocol. The hypothesis
that an association of resistance training with higher protein intake from the diet or
dietary supplements may have synergic effects has been confirmed in acute exercise
conditions both in young and elderly subjects, (Biolo G et al. 1997; Rasmussen BB et
al. 2006)

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 5.3. Aging is a protective factor against inactivity mediated insulin resistances?

Aging can determine a progressive reduction of the tissues sensitivity to the insulin
stimulation and therefore an impaired capability of the tissues to metabolize glucose
(DeFronzo RA. 1981). The same detrimental effect is observed after a period of
physical inactivity. Bed rest studies (Stuart CA et al. 1988) conducted on healthy young
subjects showed that reduced insulin sensitivity develops soon after few days.
Trained subjects show greater insulin sensitivity than individuals with a sedentary
lifestyle. Both strength and resistance training improve insulin sensitivity and therefore
glucose metabolism either in young or elderly subjects (Henriksen EJ. 2002), however
if young individuals have an improved insulin sensitivity even after a single bout of
exercise, in aged subjects more bout are required to observe the same effect (Henriksen
EJ. 2002). These data seems to show that in healthy young trained persons the glucose
level control is more efficient. Smorawiński J and his group have shown how, after
three days of bed rest, both trained and untrained subjects exhibit a higher insulin
resistance. Surprisingly the values of insulin resistance were grater in active than in the
sedentary lifestyle subjects (Smorawiński J et al. 2000). These findings suggest that in
trained subjects there is a faster compensatory response than in sedentary lifestyle
individuals.
The data observed in the PANGeA study confirm the findings of Smorawiński J and his
group. The healthy young subjects, apparently more active than the elderly individuals,
showed higher levels of insulin in fed state, after a period of 14-d of bed rest. The
mechanisms of this peculiar insulin response require indeed further investigations.
Probably aging determines a sort of adaptation to physical inactivity and consequently a
higher resistance to alteration of the glucose metabolism.
In ambulatory condition no insulin sensitivity changes were observed in both young and
elderly groups, neither in fasting or fed state.

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 5.4. Hypoxia per se improves oxidative stress

Glutathione is a critical factor in protecting cells against oxidative stress and in

erythrocytes is critical for hemoglobin function (Dumaswala, UJ et al. 2001). In
addition, erythrocyte glutathione contributes to the whole body redox equilibrium and is
a marker of the glutathione status in other tissues, in particular in the liver (Biolo, G., et
al. 2007). Glutathione redox capacity depends on the availability of total glutathione
and by the ratio between reduced and oxidized glutathione (i.e., the GSH/GSSG ratio)
(Biolo, G., et al. 2007).
We have observed that hypoxia directly increased the hematocrit and total whole blood
erythrocyte availability, while glutathione concentration per erythrocyte volume was not
significantly changed. Such increase in total glutathione availability was associated with
a parallel acceleration in the rate of glutathione turnover, expressed either as Fractional
and Absolute Turnover Rate.
The amino acid 5-oxoproline is an intermediate of the γ -glutamyl cycle and has been
proposed as marker of glutathione status and turnover in cell systems and in animal
models (Geenen S et al., 2013). In preliminary work we have tested the hypothesis that
plasma 5-oxoproline level is a marker of in vivo glutathione synthesis and turnover in
humans. We have developed a method to determine plasma 5-oxoproline levels in
humans by stable isotopes, used as internal standard, and gas chromatography - mass
spectrometry. The method has been validated in humans and first results have been
published (Qi L et al. 2013). We have used the data obtained in the PLANHAB study to
test the hypothesis that plasma 5-oxoproline levels could be suitable markers of
glutathione turnover in red blood cells as determined by stable isotope infusions. The
relationship between red blood cell glutathione synthesis rate and plasma 5-oxoproline
levels, relative to the basal condition in healthy volunteers, are shown in figure 36.
According to these results we are using plasma 5-oxoproline in conjunction with
expression of the key enzyme for the glutathione synthesis glutamate cysteine ligase to
assess glutathione synthesis capacity at the whole body level in the PLANHAB study.
The 5-oxoproline in plasma tended to decrease suggesting an increased activity of the γ-
glutamyl cycle and glutathione turnover, not only in erythrocytes but also at the whole
body level. Moreover the glutamate-to-glutamine ratio in plasma was increased in
hypoxic conditions both in ambulatory and bed rest state, suggesting a higher disposal
of this glutathione precursor.

88
Figure 36. Relationship between glutathione fractional synthesis rate in red blood
cells and plasm 5-Oxoproline concentration, in healthy volunteers in the
postprandial state.

The hypoxia-mediated increase in glutathione turnover was accompanied by a rise in

the plasma concentrations of precursor amino acids involved in the glutathione
synthesis, i.e. glutamate and cysteine. Glycine plasma concentration is also increased by
hypoxia but only in ambulatory conditions, however the erythrocyte concentration of
this amino acid is increased by hypoxia in both ambulatory and bed rest condition.
Hypoxia in ambulatory conditions also improved the GSH/GSSG ratio, suggesting a
decreased production of reactive oxygen species (ROS). Moderate hypoxia is known to
limit the excess production of ROS as well as reduce the oxidative damage to cells
(Lopez-Barneo J et al. 2001). In contrast to the ambulatory condition, hypoxia in bed
rest did not change significantly the GSH/GSSG ratio. Erythrocyte GSSG levels
increased by about 100% following hypoxia in bed rest conditions, while it tended to
decrease in ambulatory conditions. Evidence in vivo indicates that muscle unloading is
characterized by oxidative stress and increased oxidized glutathione in skeletal muscle.
We have recently shown that bed rest combined with overfeeding was associated with
activation of erythrocyte glutathione system and markers of oxidative stress in plasma
(Biolo, G. et al. 2008). In the present study, hypoxia enhanced glutathione redox
capacity in either bed rest or ambulatory conditions. An increased hypoxia-mediated
oxidative stress was demonstrated only in bed rest condition.

89
Plasma glutamine concentration.
In our study, plasma glutamine concentrations decreased by about 4% following
hypoxia. Glutamine depletion is often associated with stress conditions and systemic
inflammatory response due to decreased muscle production and increased utilization by
the immune system. Plasma glutamine concentrations have been found either normal or
decreased in patients with COPD (Engelen MP et al. 2003). Pulmonary emphysema was
associated with depletion of muscle glutamine (Engelen MP et al. 2000). Moreover,
glutamine has been shown to upregulate glutathione recycling enzymes including 5-
oxoprolinase (Kaufmann Y et al. 2008).

Tumor necrosis factor Related Apoptosis Induce Ligand (TRAIL)

TRAIL is a member of the TNF-ligand family emerging as protecting factor against
atherosclerosis (Volpato S. et al. 2011). As previously reported, TRAIL is inversely
related to the risk of mortality in patients affected by cardiovascular disease (Biolo G. et
al. 2012; Secchiero P. et al. 2009; Volpato S. et al. 2011). In moderately active subjects,
in hypoxic conditions, we observed an increased TRAIL concentration that is directly
correlated to changes in the ratio between GSH and GSSG. These data suggest that, in
hypoxic condition, the anti-atherosclerosis properties of TRAIL are strictly related to
hypoxia-induced increase in glutathione antioxidant defenses. The hypoxia mediated
protective effects of TRAIL are completely blunted by exposure to physical inactivity.

90
 5.5. Hypoxia-bed rest interaction induces multiple metabolic changes.

Hypoxic diseases such as COPD and OSAS are associated with an accelerated
atherogenesis and an increased cardiovascular morbidity and mortality (Schneider C et
al., 2010; Ozkan Y et al., 2008). Hypoxic diseases are often associated with decreased
physical activity till immobilization. Physical inactivity per se causes insulin resistance,
dyslipidemia, hyperhomocysteinemia, flogosis and altered redox balance (Mazzucco S.
et al., 2010; Biolo G. et al., 2005; Biolo G. et al., 2008). However, the separated and
combined effects of hypoxia and/or inactivity on cardiovascular risk are not well
defined (Drager L. F. et al., 2010; Taylor T. C. 2009). In the PLANHAB study we have
investigated the net effects of normobaric hypoxia and/or physical inactivity, using the
experimental model of bed rest, in healthy young volunteers.
Considering the relevance of the protective effect of HDLs against atherosclerosis, the
significant reduction in the plasma levels of these lipoproteins, observed after 10 days
of normobaric hypoxia in all conditions (HBR, HAMB), can contribute to the increased
cardio vascular risk of hypoxia, which worsens the negative effects of physical
inactivity. This relation between hypoxia and decreased levels of HDLs was recently
confirmed by extensive a meta-analysis (Nadeem R. et al. 2014). Other studies,
however, have shown contrasting results (Wee J et al. 2013) possibly from
inhomogeneous research protocols.
In our study, hypoxia condition per se caused an increment of HL, a lipolytic enzyme
synthesized and secreted by the liver, localized mostly at the surface of hepatic
sinusoidal capillaries. HL is responsible for a faster hepatic HDL clearance, through
various mechanisms, including the insulin resistance induced by both hypoxia and/or
inactivity. The higher HL levels with a lower LPL/HL ratio correlated with the HDL
plasmatic levels; HL and LPL being enzymes both involved in the clearance of these
lipoproteins (Chatterjee C.& Sparks D. L. 2011; Annema W. & Tietge UJ. 2011; Blades
B. et al., 1993). HL transforms HDL2 to smaller and denser HDL3 particles, which are
probably cleared faster by the liver, causing a reduction of total HDL levels. In our
study, the HDL2-to-HDL3 ratio did not significantly change suggesting an increased
clearance of HDL3 possibly combined with a higher HDL3 formation from HDL2,
from an enhanced HL activity. We may speculate that a higher HL concentration
associated with reduced HDL plasma levels, may result in an increased atherosclerosis
risk, through the of highly atherogenic, small, low-density-lipoproteins (LDL). Other
enzymes, including LPL, LCAT and CETP can be involved in such alteration of lipid

91
metabolism (Mazzucco S. et al., 2010), nevertheless we did not observe changes in
these enzyme levels, possibly because of the shorter protocol duration.
Our data showed that triglyceride (TG) levels were increased in hypoxic ambulatory
conditions, but not after bed rest. Different studies have shown an association between
hypoxia, from high altitude exposure (Barnholt KE et al. 2006; Farias JG et al. 2006;
Young PM et al. 1989) or respiratory clinical disorders (Sharma SK et al. 2011; Drager
LF et al. 2010), and increased TG plasma concentrations. The mechanisms underling
these changes, however, have not been completely defined (Adedayo AM et al. 2014;
Jun JC et al. 2012).
Inflammation plays a key role in the pathogenesis of many clinical conditions. Biolo G.
et al. have previously reported an activation of systemic inflammation following 5
weeks of physical inactivity, during bed rest (Biolo G. et al. 2008). Other studies
(Siervo M et al. 2014; Regazzetti C. et al., 2008) have shown the role of hypoxic
conditions on local and/or systemic inflammation. In our study short-term BR did not
modify CRP and SAA levels but they were augmented in all hypoxic conditions. Our
findings are consistent with reports showing that high altitude hypoxia stimulates the
expression of pro-inflammatory markers, including CRP (Bailey DM et al. 2004;
Hartmann G et al. 2000) and with studies on patients with severe OSAS, characterized
by elevated SAA level.
SAA is a HDL-associated apolipoprotein, which is considered an acute-phase factor
during inflammation (Kotani K et al. 2013) and a biomarker of CVD and COPD (Korita
I et al. 2013; Bozinovski S et al. 2008). SAA may act as pro-atherogenic agent inducing
HDL dysfunction by apoA-1 displacement (Kotani K et al. 2013; Van Lenten B et al.
2006; Yokoyama S et al 2006; Jahangiri A. et al. 2009; Tölle M et al. 2012; Alwaili K
et al. 2012). Furthermore, SAA has been showed to affect cholesterol efflux from cells
to HDL (Wroblewski J.M. et al. 2011). This could have contributed to the increased
plasma HDL clearance leading to the decreased HDL plasma levels that we have
observed. In our study the ratio between HDL and SAA was significantly reduced, more
by hypoxia (-37±7%, p<0.01) than bed rest (-28±9%, p<0.01) suggesting that different
mechanisms are involved in the combined effect of inactivity and hypoxia on HDL
metabolism.
Homocysteine plasma level is a well-recognized marker of atherosclerosis development
(Glueck CJ et al. 1995; Baszczuk A et al. 2014), it might be responsible, alone, for 10%
of heart failures, with a rising of this percentage to 90% when this marker is associated
with other classic risk factors, including reduced HDL plasma levels (Baszczuk A et al.

92
2014). Lately, evidence (Nunomiya K et al. 2013; Kai S et al. 2006; Seemungal TA et
al. 2007) indicates an involvement of homocysteinemia in the pathogenesis of COPD
(Seemungal TA et al. 2007). Furthermore an association between CRP and
homocysteine plasma levels was reported in these patients (Seemungal TA et al. 2007),
suggesting a possible contribution of hyperhomocysteinemia to the development of the
systemic inflammation. We reported a significant increase in homocysteine plasma
levels due to hypoxia per se in all conditions.
Activation of systemic inflammation is often observed during the development of
insulin resistance. Ten days of bed rest and/or hypoxia did not significantly affected
HOMA-IR index of insulin resistance (Blanc S. et al., 2000; Alibegovic A. C. et al.,
2010; Bergouignan A. et al., 2011; Bienso R. S. et al., 2012), however, we found a
significant negative effect of both hypoxia and bed rest on the Δ5-desaturase activity
index in erythrocyte membranes. As previously reported Δ-5 desaturase activity, can be
considered a reliable marker of insulin sensitivity (Mazzucco S. et al., 2010).

93
 5.6. Identification of a new biomarker to evaluate anabolic (and insulin)
resistance to define optimal protein requirement.

It is well documented that life expectancy in Europe is increasing. In the last years there
has been a higher awareness of the metabolic alteration, associated to aging. The goal is
to ensure a longer lifespan, together with an improved quality of life. Several metabolic
pathologies are related to aging including: loss in muscle mass and strength (i.e.
sarcopenia), T2DM, atherosclerosis and cardiovascular disease. These disorders are
frequently associated to a reduced physical activity level, exercise being a key factor in
the maintenance of the homeostasis of glucose and protein metabolism. Indeed,
inactivity promotes the development of insulin resistance, T2DM and CVD, and can
impair the muscle sensitivity to anabolic stimuli (e.g. amino acids, protein, etc.). On the
contrary, higher levels of physical activity are associated with improved insulin
sensitivity and enhanced protein synthesis in the fed state. Thus, both aging and
physical inactivity have a negative impact on insulin and anabolic sensitivity.
Moreover, because of the few available scientific evidences, one of the goals of the
present work was to investigate the possible synergic effect of these two factors.
The evaluation of “insulin resistance” and “anabolic resistance” requires complex and
invasive techniques. The “gold standard” procedure for insulin sensitivity in human is
the euglycemic-hyperinsulinemic clamp. This measurement consists in an infusion of
insulin and glucose in the blood stream in order to evaluate the glucose uptake capacity
of the tissues. Despite the extreme precision of this technique, the procedure is very
invasive, time consuming and expansive, so it is frequently replaced by the oral glucose
tolerance test (OGTT). This method, commonly used in clinical diagnosis, allows an
evaluation of the glycaemia levels during and after a glucose load. The procedure
however still remain invasive requiring at least 3 blood draws. Other methods utilize
mathematical models that calculate insulin sensitivity in fasting or fed states (e.g.
HOMA-IR index and Matsuda index, respectively). These models require few blood
samplings (one for the fasting state evaluation and at least two for the after load
measurements), are the easiest to aplly.
To assess anabolic resistance and protein metabolism the most effective technique is the
infusion in the blood stream of stable-labeled isotopes (tracers) and the measurement of
the isotopic enrichment at the steady state. One the most used tracer in the assessment
of the protein metabolism is the L-[ring-2H5]-phenylalanine (D5-Phe). After
administration, D5-Phe is hydroxylated in [ring-2H4]-tyrosine (D4-Tyr), that represents

94
an index of net protein loss. For gold standard procedures this protocol has to be assess
during an anabolic stimulus, such as amino acids infusion.
Stable-labeled isotopes can be administered either orally or intravenously, but the latter
way is the most used. The intravenous infusion protocol, however, is complex (it
requires asepsis of the tracer solutions, two vein catheters etc.) and expansive and
requires several blood samples, decreasing the compliance of the evaluated subjects.
The oral administration protocol, on the other hand, consists in repeated intake of the
tracer solution, at fixed period intervals. This is safer (just one vein catheter for the
blood draws) and requires a simpler protocol (there is no need of an aseptic tracer
solution). However, it still remains expansive, because of the quantity of stable-isotopes
needed, and time consuming.
With the above-mentioned techniques is not possible to assess “insulin resistance” and
“anabolic resistance” at the same time or, at least, in the same experimental protocol.
The present work proposes a new, simple, safe and quick method to evaluate
simultaneously the “insulin resistance” and the “anabolic resistance” in fed state i.e.
after a total meal (500 ml, 500 kcal, 55% carbohydrate, 15% protein, 30% fat) load with
the administration of a single dose of tracer solution. This method was used to assess
insulin and anabolic resistance after 14-d of bed rest in young and elderly subjects at
different level of physical activity. The anabolic resistance was evaluated through a new
index (i.e. AUCD4-Tyr/AUCD5-Phe -t120 ratio), proposed in this work for the first time,
which needs a two hours duration procedure (instead of the 6h often required) and just 2
blood draws versus the 7 samplings required in other protocols. This method does not
allow to determine the protein kinetics (i.e. rate of synthesis and degradation) but it
permits to calculate an important metabolic parameter, the anabolic sensitivity. The
anabolic sensitivity, more than protein kinetics is an important index of the detrimental
effect of aging or sedentary life style on muscle mass and protein metabolism. A safe,
simple and quick method with limited cost is proposed. Moreover, as already said, this
new method allows to evaluate simultaneously, and with the same procedure, anabolic
and insulin resistance in post-prandial conditions, with an additional reduction in time
and cost and with an improved compliance of the evaluated individuals.

95
 6. CONCLUSIONS

− Aging and bed rest are characterized by a higher protein requirement,

confirming data from other studies. Anabolic resistance and insulin resistance are
shared biomarkers of these conditions associated with skeletal muscle mass and
function loss. However, the effect of inactivity on insulin resistance is blunted in aging.
− We developed a new method to assess at the same time the two most relevant
metabolic biomarkers (anabolic resistance and insulin resistance) of protein requirement
in humans.
− Other biomarkers related to these sarcopenic conditions are inflammation and
redox balance.
− Chronic exposure to experimental hypoxia decreases whole body protein
synthesis in the post-absorptive state, suggesting an increased protein requirement also
in this condition.
− In addition, chronic hypoxia has a strong impact on cardiovascular risk markers
(insulin resistance, homocysteine, systemic inflammation), often additive to that of
inactivity (HDL-cholesterol).
− Furthermore, hypoxia, in either ambulatory or bed rest conditions, significantly
increased total glutathione concentrations and synthesis rate in erythrocytes. Hypoxia
significantly increased redox glutathione capacity (i.e., GSH/GSSG ratio) in ambulatory
conditions while this effect was blunted in bed rest.

96
7. ACKNOWLEDGMENT

97
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PUBLICATIONS

• Cucca A, Mazzucco S, Bursomanno A, Antonutti L, Di Girolamo FG, Pizzolato

G, Koscica N, Gigli GL, Catalan M, Biolo G. Amino acid supplementation in L-
dopa treated Parkinson’s disease patients. Clin Nutr. 2014 23 Dec (online
version) doi:10.1016/j.clnu.2014.12.007

• Biolo G, Di Girolamo FG, Breglia A, Chiuc M, Baglio V, Vinci P, Toigo G, Lucchin L,

Jurdana M, Pražnikar ZJ, Petelin A, Mazzucco S, Situlin R. Inverse relationship
between "a body shape index" (ABSI) and fat-free mass in women and men: Insights
into mechanisms of sarcopenic obesity. Clin Nutr. 2014 Apr 13. Pii: S0261-
5614(14)00092-2.

• Di Girolamo FG, Situlin R, Mazzucco S, Valentini R, Toigo G, Biolo G. Omega-3 fatty

acids and protein metabolism: enhancement of anabolic interventions for sarcopenia.
Curr Opin Clin Nutr Metab Care. 2014 Mar;17(2):145-50.

• Effects of experimental chronic hypoxia with inactivity on cardiovascular risk markers,

lipid metabolism and redox capacity in humans - Manuscript in preparation

• Effects of experimental chronic hypoxia with inactivity on protein kinetics and muscle
metabolism in humans - Manuscript in preparation

• Detecting anabolic resistance in a clinical setting: a fast method based on bolus

ingestion of stable-labeled isotopes - Manuscript in preparation

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