15
Interactions between Protein
and Energy Metabolism
T.C. Wright
1
, J.A. Maas
2
and L.P. Milligan
1
1
Department of Animal and Poultry Science, University of Guelph, Guelph,
Ontario N1G 2W1, Canada;
2
Centre for Integrative Biology, University of
Nottingham, Sutton Bonnington, Leicestershire LE12 5RD, UK
Introduction
The corresponding chapter in the previous edition of this book concluded by
describing protein and energy metabolism as a unity instead of an interaction of
separate components of metabolism. This edition will examine some of the
recent knowledge generated about this subject with an emphasis on those
metabolites and tissues that serve important roles for biochemical reactions in
which carbon and nitrogen are, in effect, equal partners.
Animals encounter numerous challenges during their lives, and respond to
achieve maximum advantage for their welfare and survival in meeting those
challenges. This does not imply, however, that the response will necessarily be
measured as the most efficient possible in terms of agricultural animal perform-
ance. It is possible to make estimates of the stoichiometry of numerous reactions
for many metabolic pathways involving protein and energy intermediates. The
opportunity for nutritionists is to develop a better understanding of the fate of
nutrients under differing circumstances and of the regulatory system that deter-
mines an end point. The energetic costs associated with disposing of an amino

of the animal through the use of fumarate and malate as feed additives that
would serve as electron acceptors. The importance of AA, peptides and am-
monia as substrates for microbial protein synthesis should be quantitatively
described in terms of both the ruminal environment they contribute to, and as
the major source of protein for the host, as microbes pass from the rumen to
the small intestine. Oldick et al. (1999) and Clark et al. (1992) both reported
that the profile of microbes passing to the small intestine from the rumen
changes depending on the diet, and therefore the AA profile of microbial
protein is not constant, as is commonly assumed in several models. The
availability of AA in the animal can be increased by increasing dry matter
intake, which increases the synthesis of microbial protein, and by providing
dietary proteins that are resistant to ruminal digestion but are digested by the
animal. One of the most important variables associated with abomasal protein
flow is the level of feed intake.
VFA represent the principal form of energy substrate for ruminant animals
(Sutton, 1985). Considerable proportions (30%, 50% and 92% of acetate,
propionate and butyrate, respectively) are subjected to first-pass absorptive
metabolism and never reach the venous blood (Reynolds, 2002). Fermentation
imbalances in the rumen (e.g. resulting from excess supply of degradable
nitrogen) can be minimized by using current feeding recommendations, that
will benefit animal performance as well as reduce the negative impact on the
environment, whether measured locally (e.g. on-farm balance of nitrogen and
phosphorus) or in a more global sense (e.g. greenhouse gases). Further im-
provements to mechanistic models of metabolism will result in more effective
strategies to minimize the potential for negative environmental impact.
Energetics and Protein Metabolism
The synthesis and degradation of protein in the body continues to be the
subject of most research. Energetically costly, the estimate for ATP-equivalent
400 T.C. Wright et al.
cost per peptide bond formed remains at 5 ATP. However, the true cost of

energy expenditures. Wolfe (2002) noted that in burn patients in whom protein
Table 15.1. Energy cost estimates of protein synthesis (revised from Kelly et al., 1993).
Method
Energy cost
(mole ATP per molar peptide
bond synthesized) References
Inhibition (reticulocytes) 3.0 Siems et al. (1984)
Inhibition (chicks) 7.5 Aoyagi et al. (1988)
Inhibition (fish) 4.3–5.6 Storch and Portner (2003)
Stoichiometry 4.0 Buttery and Boorman (1976)
Stoichiometry 5.0 Millward et al. (1976)
Stoichiometry 6.3–7.0 Webster (1981)
Regression (swine) 30.2 Reeds et al. (1980)
Regression (chicks) 18.8 Muramatsu and Okumura (1985)
Interactions between Protein and Energy Metabolism 401
degradation rates were elevated above protein synthesis rates, supplementation
of AA had the effect of reducing protein degradation without an offsetting
effect on protein synthesis rate. The results of this study led the author to ask
the question as to whether or not there is independent regulation of protein
degradation and protein synthesis (Wolfe, 2002). The answer to this question
has important implications for nutritionists who must consider that a variety of
results can be achieved from intake of the same AA. The outcome of a set AA
intake will depend on the dynamics of the governing factors in play in the
metabolic situation being studied. We concur with the conclusion of Wolfe
(2002) that it may be more beneficial in the long run to determine the mech-
anisms by which AA and energy affect muscle protein synthesis and degrad-
ation rather than seeking a particular value for a ‘requirement’. There is
potential for direct regulation of proteolysis by AA (Kadowaki and Kanazawa,
2003). The regulation of protein synthesis by AA in human skeletal muscle (Liu
et al., 2002) has recently been reviewed (Wolfe and Miller, 1999; Yoshizawa,

carbon to the tissues of the gut and the liver. The compromise of incomplete
oxidation leaves the nitrogen in a non-toxic form that can be transported back
to the liver. Because the tissues of the gut almost completely metabolize the
supply of glutamate, aspartate and glutamine during first-pass absorption,
the supply of these AA for protein synthesis in other tissues must be
met almost completely from de novo synthesis (Reeds et al., 1996). These
are likely to be synthesized by transamination from glutamate at a cost of 4
ATP per molecule of non-essential AA. Thus diets balanced for non-essential as
well as essential AA could have an energy sparing effect for the animal.
Lobley et al. (2001) provided an interesting perspective whereby the
metabolism of glutamine was described with respect to its contribution to
whole-body protein and energy metabolism. Glutamine has many metabolic
roles, but responses to glutamine supplementation have been inconsistent and
it is not considered to be limiting for growth or lactation. For example, glutam-
ine is the most abundant free AA in tissues of most animals, which Van
Milgen (2002) noted is energetically favourable compared with protein storage.
Previously, researchers have focused on the extensive use of glutamine and
glutamate as energy substrates by the tissues of the gut.
Alanine
Alanine
TA
BCAA
TA
Glutamine
Glutamine
Glutamine Glutamine
Liver Intestine
Kidney
Muscle
Amino acids

10.0% of AA residues in bovine caseins, therefore uptake and synthesis of
glutamine by the mammary glands must be considerable in a high-producing
dairy cow. In addition, the uptake of many non-essential AA by the mammary
glands is below that required for milk synthesis, and glutamine is likely the
source of both carbon and nitrogen for mammary synthesis of other non-
essential AA. Glutamine also appears to have a role in mediating intracellular
activity through transport-mediated changes in cell volume.
Reeds et al. (2000), using the neonatal pig as a model, suggested mech-
anisms exist that allow pigs to sense an imbalance in the AA supply from milk
so they can make acute metabolic changes to ensure AA are still used with high
efficiency. These mechanisms may also be present in more mature animals.
Data from both the rat and the neonatal pig suggest that the number of
ribosomes decreases but the translational activity of each ribosome increases
as the animal approaches weaning. The reduction in efficiency of protein
utilization in neonatal pigs from birth to 26 days of age is mirrored by changes
in sensitivity and responsiveness of protein deposition to insulin concentration.
Lobley (1992) suggested that in lambs the conversion of dietary nitrogen to
body nitrogen was only 13%. Data from isotopic studies suggest that 50% to
100% of oxidized glucose was synthesized from glutamate, glutamine and
alanine. The incremental efficiency for protein gain of absorbed AA ranges
from 40% to 80% (Lobley, 1992). Tracer approaches suggest that in fasted
sheep, daily protein synthesis amounted to approximately 8% of the whole-
body protein pool. There is some suggestion that gluconeogenesis from AA
occurs even under supramaintenance conditions, which may explain the low
efficiency of incremental AA use as supply increases (Lobley, 1992).
The use of non-essential AA as a fuel source in visceral tissues is, intuitively,
energetically more expensive than the direct use of glucose. Van Milgen (2002)
presented a useful framework to examine the energetics of intermediary
metabolism, wherein this efficiency was re-examined in some detail. The
additional net cost of converting glucose to glutamate and then oxidizing the

drainage is combined and flows into the hepatic portal vein, including the
rumen, reticulum, omasum, abomasum, small intestine, large intestine, spleen,
pancreas, caecum and mesenteric and omental fat tissue. Some small anatom-
ical differences exist between ruminant species but they are generally quite
similar (Seal and Reynolds, 1993). The PDV tissues differ from other tissues of
the body because of their exposure to dual sources of nutrient supply, namely
digesta and arterial blood supply. Ruminant PDV tissues utilize glucose, volatile
or short-chain fatty acids, ketones and AA as oxidative substrates (Reynolds
et al., 1990). The absorption of free AA and peptides across the small intestine
is achieved by specific transporters, some of which require energy. This, and
the high turnover rate of gut tissue, are two significant contributions of the
small intestine to whole-body energy expenditure. Maintenance of Na
þ
,K
þ
,
ATPase activity, substrate cycling, urea synthesis, protein synthesis and deg-
radation in the gastrointestinal tract and liver were estimated together to
account for 22.8% of whole-body oxygen consumption in growing steers
(Huntington and McBride, 1988) and, more recently, Reynolds (2002) esti-
mated that the total splanchnic tissues usually account for 40–50% of total body
oxygen consumption. The energetic cost to the animal for maintenance and
turnover of gut tissues and for nutrient absorption is, therefore, considerable
and a large proportion of this energy expenditure is directly linked to protein
and AA metabolism.
Coordination of nutrient use by the whole animal is an important part
of protein/energy metabolism, particularly in the PDV. Ebner et al. (1994)
conducted an experiment with 2-week-old pigs to examine the effects of a low-
protein diet (15% crude protein (CP)) compared with a control, isocaloric
protein diet (30% CP) on PDV tissue growth and metabolism. In their experi-

exported proteins is noteworthy because the opportunity for energetically
efficient reuse of their carbon and nitrogen metabolites is reduced.
The important role of the PDV and the liver to modulate the quantity and
concentration of nutrients supplied to peripheral tissues was reported by
Lapierre et al. (2000) using multi-catheterized animals. These authors used
growing steers and achieved three different levels of intake of a single diet,
calculated to provide 0.6, 1.0 and 1.6 times the estimated requirements for ME
and CP. Their experiment examined in detail the uptake and release of AA,
hormones and key metabolites across tissues and provided a better understand-
ing of nutrient fluxes in total splanchnic metabolism. The information gained
from this intricate type of research provides important data on nutrient use and
systemic regulation that will ultimately permit the development of diets that
improve efficiency of the conversion of dietary nitrogen to animal protein.
Further improvements in our understanding of PDV metabolism might be
achieved if the luminal nutrients that can directly signal protein synthesis or
degradation were determined. Identification of these nutrients through the use
of normal feeding trials is difficult because as the luminal nutrient supply
changes, both basolateral nutrient concentrations and hormonal changes
will result.
The kinetics of AA use by the PDV are complex, in part because the use of
AA of arterial origin appears to increase concomitantly with increases in
luminal AA supply (Reynolds, 2002). The sensitivity of intestinal protein
synthesis to the avenue of nutrient supply is unique. Discerning systemic effects
from the direct effects of increased luminal nutrient concentration is difficult
because techniques to distinguish these two events are a challenge to
develop, and, invariably, increased luminal nutrient concentrations lead to
systemic responses for growth factors and hormones that can stimulate protein
synthesis.
406 T.C. Wright et al.
Recently, a technique has been validated in piglets to determine the acute

tested by perfusing intestinal segments with buffer, 30 mmol/l mixture of AA
or two concentrations of ammonium chloride. Their results (Table 15.2) indi-
cated that there was a 26% reduction in K
s
when the AA mixture was perfused,
while ammonium chloride perfusion had the effect of raising tissue ammonia
levels to those that resulted with AA perfusion, but without an equivalent effect
on K
s
. Thus, the signal for protein synthesis is mediated by AA. Adegoke et al.
(1999b) noted the rapid (90 min) time frame for the changes detected in
Table 15.2. Effect of buffer, an AA mixture or ammonium chloride on mucosal protein
fractional synthesis (K
s
) in piglets (from Adegoke et al., 1999b).
Treatment Buffer (PBS)
Amino acids
30 mmol/l
Ammonium chloride
0.5 mmol/l 1.0 mmol/l
Tissue ammonia, mg/g
wet weight
6.30 + 0.17
a
8.42 + 0.29
b
7.46 + 0.28
ab
8.39 + 0.28
b

sis and degradation in the PDV. The relative importance of intracellular protein
degradation routes (e.g. ATP–ubiquitin system, calcium-dependent or lysoso-
mal pathways) in the gut and their energetic costs are unknown in ruminant
animals, which also needs to be resolved.
Hepatic Metabolism
Seal and Reynolds (1993) suggested that, excluding acetate, 85–100% of VFA
arriving at the liver via the portal vein is removed from the blood. Acetate is the
only VFA that is not almost completely removed and thus is found in peripheral
blood in substantial concentrations. Propionate is a principal carbon source for
hepatic glucose synthesis. Most AA are removed to some degree by the liver,
the exceptions being branched chain AA and glutamate which appear to be
produced by hepatic metabolism. Alanine, glycine and glutamine from periph-
eral tissues are carried to the liver where they serve as amino donors, are used
in gluconeogenesis or protein synthesis or are degraded to yield urea
(Fig. 15.1). Alanine and glycine also serve as amino group transporters for
tissues of the PDV and thereby avoid potentially toxic ammonia concentra-
tions. The kinetics of AA use by hepatic tissue is far from clear. Blouin et al.
(2002) fed lactating dairy cows isonitrogenous diets that differed in rumen
protein degradability and, hence, metabolizable protein (MP), and measured
the effects on splanchnic (PDV and liver) fluxes of nutrients. Portal absorption
of AA was increased on the high (1930 g/day) MP diet compared with the low
(1654 g/day) MP diet; however, there was no difference in liver removal of AA
between the diets. The similar AA removal from blood by the liver permitted
more AA to be delivered to peripheral tissues, including the mammary glands
with the higher MP diet. Milk and milk protein yield increased 1.8 kg/day and
64 g/day, respectively, as a result. In their experiment, the ratio of ammo-
nia:AA-nitrogen in portal venous blood was affected by diet (0.91 and 1.3 for
408 T.C. Wright et al.
the higher and lower MP diets, respectively), which reflects the importance of
ruminal energy and nitrogen availability (Blouin et al., 2002).

phosphorylation of S6K1 and increase its activity (Tesseraud et al., 2003).
S6K1 phosphorylates 40S ribosomal protein S6 that can increase the transla-
tion of elongation factors and ribosomal proteins in a selective manner. Tesser-
aud et al. (2003) concluded that S6K1 phosphorylation was mediated through
mammalian target of rapamycin (mTOR) PI3-kinase activity. This level of detail
about the effects of AA on protein synthesis is necessary to increase our
understanding of protein and energetic interactions in ruminant muscle.
Another thoughtfully designed experiment by Tesseraud et al. (2000)
utilized chicks obtained from either a fast (FGL) or slow growing line (SGL),
to examine the basis of genetic regulation of muscle protein deposition. The
FGL line had greater total body weight and pectoralis muscle weight than the
SGL at 1 and 2 weeks of age. As observed with mammals (Lobley, 1993),
Interactions between Protein and Energy Metabolism 409
K
s
declined with age in their experiment (Table 15.3), but was similar for the
pectoralis major muscle between genotypes. In their experiment, fractional
degradation rate (K
d
) in the FGL was less than the SGL between 1 and 2 weeks
of age, which would favour muscle protein accretion. This implies that selection
for enhanced growth may affect the K
d
rate at a young age, which could result in a
more metabolically efficient use of energy and protein. An important route for
protein degradation, the ubiquitin-mediated proteolytic pathway, continues to
be the subject of intensive research efforts. Tesseraud et al. (2000) noted that
mechanisms associated with genetic differences in muscle protein degradation
are poorly understood, and in the two lines of chickens selected for growth such a
possibility could account for the differences detected in fractional protein deg-

the alteration of muscle protein metabolism and that mechanisms may exist to
facilitate differential protein turnover rates in specific muscle tissues.
Energy supplied in the diet can also have a significant effect on protein
metabolism in the whole animal. When the energy intake of sheep was in-
creased from a medium to high level, both protein synthesis and degradation of
the hind limb increased, but the magnitude of increase was greater for protein
410 T.C. Wright et al.
Table 15.3. Pectoralis major muscle protein metabolism (mean from n ¼ 6 and SE) in chickens at 1 and 2 weeks of age from genetic lines
selected for fast (FGL) or slow (SGL) growth over 33 generations (from Tesseraud et al., 2000).
1-week old 2-week old
SGL FGL SGL FGL Main effect
Item Mean
SE
Mean
SE
Mean
SE
Mean
SE
Line Age L*A
Pectoralis major muscle
Weight (g) 0.61 0.03 2.17 0.17 2.17 0.07 5.67 0.18 <0.001 <0.001 <0.001
Relative weight (g/kg BW) 12.6 0.5 23.2 1.6 26.2 0.7 32.8 0.5 <0.001 <0.001 0.06
Absolute rates
Protein deposition (mg/day) 17 1 60 1 31 1 85 2 <0.001 <0.001 <0.01
Protein synthesis (mg/day) 32 3 90 4 78 6 162 13 <0.001 <0.001 <0.05
Protein breakdown (mg/day) 15 3 29 5 46 5 76 12 <0.001 <0.001 0.18
Fractional rates
Protein gain (% per day) 22.2 2.3 24.2 2.6 11.3 0.6 11.7 0.2 0.64 <0.001 0.75
Protein synthesis (% per day) 40.2 3.6 35.0 2.2 28.0 2.4 22.0 1.3 0.14 <0.001 0.92

´
et al. (2003) speculated that sensing of increased extra-
cellular essential AA concentration was stimulatory to muscle protein synthesis.
Mutsvangwa et al. (2004) investigated the effects of a nutritionally induced
chronic metabolic acidosis in dairy cattle on the ATP–ubiquitin-mediated pro-
teolytic pathway. Under conditions of metabolic acidosis, ureagenesis de-
creases and glutamine synthesis increases. In this situation, liver metabolism
adjusts to effect retention of bicarbonate. Chronic metabolic acidosis has been
tied to increase in the levels of skeletal muscle degradation, via the ubiquitin-
mediated proteolytic pathway, which is the primary route for the degradation
of myofibrillar proteins of skeletal muscle in non-ruminants. Mutsvangwa et al.
(2004) noted that these events are less clearly understood in ruminant animals.
Lobley et al. (1995) achieved a chronic metabolic acidosis in sheep using
NH
4
Cl but did not note changes in muscle protein degradation or synthesis.
Mutsvangwa et al. (2004) reported increased (P < 0:05) skeletal muscle
mRNA abundance for ubiquitin-mediated protein degradation components,
including ubiquitin, the 14-kDa E2 and the C8 subunit, although there was
no effect of acidosis on the C9 subunit. The relative importance of these
components to the regulation of this protein degradation pathway is not well
understood at either the tissue or the species level (Mutsvangwa et al., 2004).
The muscle of interest in their study was the longissimus dorsi, and it would be
interesting if other muscles were similarly affected by chronic acidosis, in light
of the data from Tesseraud et al. (2001) who reported different protein
412 T.C. Wright et al.
turnover rates in different chicken muscles. Our understanding of skeletal
muscle protein turnover and associated energetics would improve with detailed
knowledge of its determinants and by examining the possibility for differential
regulation between muscles. Models similar to the one used by Mutsvangwa

i
þ AMP þ PP
i
However, the true net cost for ureagenesis remains unclear because of the
potential for fumarate to be converted to aspartate in the urea cycle. This
conversion produces 1 NADH, which generates 3 ATP in the process of
oxidative phosphorylation, for a potential net ureagenesis cost of 1 ATP,
after accounting for the use of four high-energy phosphate bonds in urea
synthesis (Newsholme and Leech, 1983). Biologically, the cost associated
with ureagenesis extends beyond the ATP cost of NH
3
detoxification, because
of the practical requirement for deamination of AA-N to provide a second N
atom for urea synthesis. Lobley et al. (1995) aptly described the absorption of
NH
3
from the gastrointestinal tract as a ‘double penalty’, because feed nitrogen
would be unavailable in an anabolic form, and the detoxification may require a
net utilization of AA that could otherwise be used for protein synthesis.
The experimental results that provided evidence of urea synthesis in enter-
ocytes in the weaned pig are noteworthy. Wu (1995) first reported urea
Interactions between Protein and Energy Metabolism 413
synthesis from arginine, glutamine and NH
3
in these cells from weaned, but not
from suckling pigs. Data from Wu (1995) are shown in Table 15.4, illustrating
enhanced capability for urea synthesis with age and substrate concentration. All
enzymes of the urea cycle were present and the author speculated that the
small intestine might function as a first line of defence against physiologically
harmful concentrations of ammonia.

1 mM Gln 5 mM Gln
0.5 mM NH
4
Cl
þ2 mM Orn*
þ2 mM Asp*
2mMNH
4
Cl
þ2 mM Orn*
þ2 mM Asp*
0–21 ND ND ND ND ND
29 ND 6.3 + 0.74
c
15.2 + 1.28
b
13.4 + 1.56
b
21.6 + 2.07
a
58 ND 7.9 + 0.82
c
16.5 + 1.43
b
14.6 + 1.28
b
23.4 + 3.25
a
Values are mean +
SE

(Kim et al., 2004).
Block et al. (2001) examined plasma leptin concentrations in periparturi-
ent dairy cattle and noted that the functional consequences of reduced plasma
leptin concentrations post-calving were unclear. However, the regulation of
energy balance during this period required tight metabolic control, without
which there would be detrimental consequences for reproduction, immune
function and animal health. Understanding the contribution of leptin to this
regulation will improve our understanding of nutrition and metabolism. The
temporal changes of leptin, insulin, GH and IGF-1 for transition dairy cows are
shown in Table 15.5. The characteristic surge in GH post-calving and the drop
in IGF-1 concentrations are evident. The differential response in GH receptor
noted by Kim et al. (2004) for liver and muscle tissues coincides with the GH
surge post-calving.
An excellent review by Burrin et al. (2003) raised intriguing questions
about the physiological effects of glucagon-like peptide 2 (GLP-2) in domestic
animals. GLP-2 has been associated with intestinal mucosal growth and cell
proliferation in several species, though not in ruminant animals. This hormone
is influenced primarily by nutritional factors, although hormonal and neural
stimulation have been reported. Understanding the role of this hormone in
affecting the development of the small intestine would be particularly important
Interactions between Protein and Energy Metabolism 415