Mechanisms controlling pork quality development: The

biochemistry controlling postmortem energy metabolism

T.L. Scheer, D.E. Gerrard
*
Department of Animal Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907, United States
Received 23 March 2007; received in revised form 2 April 2007; accepted 2 April 2007
Abstract
Pale, soft and exudative (PSE) pork represents considerable economic losses for the industry due to its limited functionality and unde-
sirable appearance. During the past several decades, exhaustive research covering various aspects of the food chain has established geno-
typing procedures, recommended handling practices, and quality indicators in order to reduce the incidence of inferior pork quality.
Despite these eorts, there is still a relatively high occurrence of PSE pork. Development of pork quality attributes is largely governed
by the rate and extent of postmortem pH decline. The combination of high temperature at low pH or abnormally low ultimate pH causes
denaturation of sarcoplasmic and myobrillar proteins, resulting in paler color and reduced water holding capacity. The pH decline is
closely related to muscle energy availability and demand at or around slaughter. The postmortem degradation of glycogen through gly-
cogenolysis and glycolysis provides ATP to help meet energy demand and decreases pH by generating lactate and H+. Therefore, the ux
of metabolites through glycolysis, the involvement of energy signaling pathways that modulate glycolytic activity, and the inherent
metabolism of dierent ber types are critical factors inuencing pH decline and pork quality. Further, recent work implicates adenosine
monophosphate-activated protein kinase (AMPK) as a major energy sensor for the cell, and thus may be involved in the control of post-
mortem metabolism. The intent of this paper is to review the biochemistry controlling postmortem energy metabolism in pig muscle and
explore new information generated using genetic mutations in order to dene the fundamental mechanisms controlling the transforma-
tion of muscle to meat.
2007 Elsevier Ltd. All rights reserved.
Keywords: Pork; Biochemistry; Glycolysis
1. Introduction
Increased health-consciousness among consumers has
led to signicant changes in pork carcass attributes over
the past several decades. Swine producers have focused
selection pressure on improving carcass merit, resulting in
increased lean growth and higher yielding carcasses. How-
ever, extreme variation in pork quality, ranging from dark,
rm, and dry (DFD) to pale, soft and exudative (PSE)
pork, has also become more prevalent. DFD pork has a
dark, unattractive appearance and a rm, dry, and sticky
texture due to enhanced water binding capability. Con-
versely, PSE pork is characterized by pale color, soft tex-
ture, and low water-holding capacity, and has limited
functionality in further processing. Surveys indicated that
in pork produced in the USA in 2003, 15.5% was PSE
(Stetzer & McKeith, 2003). The inferior quality attributes
of PSE pork are estimated to cost the pork industry $100
million annually (Carr, Kauman, Meeker, & Meisinger,
1997).
Due to the considerable economic losses, PSE pork has
been a signicant source of research attention. Early stud-
ies established that the development of PSE pork was lar-
gely due to an increased rate of early postmortem
glycolysis, indicated by elevated muscle temperature and
rapid pH decline (Briskey, 1964). More recently, the extent
0309-1740/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2007.04.024
*
Corresponding author. Tel.: +1 765 494 8280; fax: +1 765 494 6816.
E-mail address: dgerrard@purdue.edu (D.E. Gerrard).
www.elsevier.com/locate/meatsci
Meat Science 77 (2007) 716
MEAT
SCIENCE
of glycolysis and ultimate pH have been implicated (Sellier
& Monin, 1994). Further research has established measur-
able indicators of quality, and yielded molecular diagnostic
techniques and control methods. Yet, despite the extensive
eorts to improve pork quality, Cassens (2000) concluded
that little progress has been made. A greater understanding
of postmortem metabolism during the conversion of mus-
cle to meat and the mechanisms of enhanced and extended
glycolysis are critical to characterizing pork quality
development.
2. Conversion of muscle to meat
Transitions between rest and exercise require that mus-
cle is a dynamic tissue with the ability to adapt to dramatic
changes in energy expenditure. Muscle contraction is a
rapid, energetically demanding process that requires the
splitting of adenosine triphosphate (ATP) in order to meet
energy requirements for muscle contraction and relaxation,
sequestration of calcium and maintenance of ion gradients.
The most ecient means of generating ATP is through
mitochondrial oxidative metabolism, but the ATP concen-
tration in muscle supplies enough energy for only a few
twitches. Therefore, additional reactions must be able to
buer energy levels when other metabolic processes are
not able to meet ATP demand.
Phosphocreatine (PCr) is present at a higher concentra-
tion (1819 lmole/g) than ATP (6.66.8 lmole/g) in pig
longissimus (Bendall, 1973) and can be quickly utilized in
a chemical reaction to rephosphorylate ADP to ATP by
the enzyme creatine kinase. Additionally, myokinase cata-
lyzes the conversion of two adenosine diphosphate (ADP)
to adenosine monophosphate (AMP) and ATP. Together,
these reactions allow muscle to quickly increase energy pro-
duction in order to keep cellular ATP constant and main-
tain homeostasis. Aerobic metabolism and PCr are able
to satisfy energy demand when oxygen is adequate and
muscle is working slowly, but if contraction proceeds rap-
idly, oxygen becomes limiting and the muscle will resort to
anaerobic glycolysis to supply the energy needed for con-
traction. Similarly, the removal of blood during the slaugh-
tering process eliminates the oxygen supply and forces
metabolically active muscles to adapt to new physiological
circumstances.
The basic biochemical reactions and physical changes
underlying the conversion of muscle to meat are well recog-
nized. ATP production is necessary to keep the muscle in
the relaxed state, but postmortem muscle has a high rate
of ATP turnover (Bate-Smith & Bendall, 1949). Initially,
ATP is replenished by the creatine kinase and myokinase-
catalyzed reactions. Once 70% of the PCr pool has been
degraded, ATP levels rapidly decline (Bendall, 1951), and
muscle glycogen must be degraded (glycogenolysis) and
metabolized in anaerobic glycolysis in order to rephospho-
rylate ADP to ATP and prevent the formation of perma-
nent actomyosin crossbridges. Anaerobic glycolysis also
produces lactate, H
+
, and heat. Because postmortem mus-
cle does not have the means to remove waste products, lac-
tate and H
+
accumulate and lower the pH. As the
breakdown of ATP exceeds its synthesis by glycolysis, less
ATP is available and the formation of actomyosin bonds
shortens sarcomeres and increases muscle tension, signal-
ing the onset of rigor mortis. Rigor mortis is complete
when the ATP supply is exhausted; thus, actomyosin cross-
bridges cannot be broken and the muscle is relatively inex-
tensible. Muscle tension will eventually decrease with
postmortem storage as a result of degradation of myobr-
illar proteins and loss of structural integrity.
The rate and extent of pH decline during the conversion
of muscle to meat signicantly impact the development of
fresh meat quality attributes (Fig. 1). Normally, pH
declines gradually from 7.4 in living muscle to roughly
5.65.7 within 68 h of postmortem and then has an ulti-
mate pH at 24 h (pHu) of about 5.35.7 (Briskey & Wis-
mer-Pedersen, 1961). However, muscles with a hastened
pH decline exhibit rapid glycolysis and produce large
amounts of heat, which slows carcass chilling. This results
in a rapid muscle pH decline to less than 6.0 during the rst
hour after slaughter and an ultimate pH of 5.35.7. The
onset of rigor mortis at high temperature and low pH
causes the denaturation of approximately 20% of the sarco-
plasmic and myobrillar proteins (Honikel & Kim, 1986),
and the reduction in myosin head length is sucient to
draw thick and thin laments closer together, leading to
increased expulsion of water (Oer et al., 1989). Greater
precipitation of sarcoplasmic proteins is largely responsible
for paler pork color, while denaturation of myobrillar
proteins explains the reduced water holding capacity in
PSE muscle (Joo, Kauman, Kim, & Park, 1999). In con-
trast, an extended pH decline proceeds at a normal rate
but continues to a low pHu of roughly 5.35.5, resulting
in acid meat. Abnormally low pH reduces the net charge
of myobrillar proteins, and the attraction moves laments
closer together and forces water out of the myolament lat-
tice (Irving, Swatland, & Millman, 1989). Moreover, sarco-
plasmic protein solubility declines with decreasing pHu and
contributes to paler pork color (Joo et al., 1999). The rate
and extent of postmortem pH decline signicantly inuence
Fig. 1. Various pH declines occurring postmortem and associated pork
quality characteristics. Modied from Briskey (1964).
8 T.L. Scheer, D.E. Gerrard / Meat Science 77 (2007) 716
protein characteristics and thus critically aect pork quality
development.
3. Major genes and aberrant postmortem metabolism
Postmortem acidication is closely related to the energy
status of the muscle at slaughter. The decline in pH
depends on the initial concentration of PCr and glycogen
(Bendall, 1951), which may vary widely under dierent
experimental and commercial conditions. A variety of fac-
tors, such as stress, stunning method, muscle type, and diet,
aect muscle energy level and its utilization postmortem.
However, in order to develop more comprehensive and
sophisticated strategies for reducing the variation found
in pork quality, the biochemistry underlying postmortem
metabolism must be fully dened.
3.1. The halothane gene
Early studies utilized certain breeds and lines within
breeds in order to model PSE development. This tendency
to produce PSE pork was closely associated with porcine
stress syndrome, a condition synonymous with human
malignant hyperthermia (MH). Eikelenboom and Mink-
ema (1974) demonstrated that MH susceptible swine dis-
play hypermetabolism, elevated body temperature, and
muscle rigidity upon exposure to the anesthetic halothane.
Stress and excitement are also sucient to provoke an MH
episode, and can result in the enhanced rate of postmortem
metabolism associated with PSE pork. Exposure to halo-
thane gas was used to screen for stress susceptible swine,
and the genetic component has since been referred to as
the halothane (HAL) gene. The HAL locus has two alleles:
the normal dominant allele (N) and the mutant recessive
allele (n).
MH susceptibility associated with the HAL gene is due
to a defect in the ability of the muscle to adequately regu-
late myoplasmic Ca
2+
concentration. The causative poly-
morphism is the single substitution of T for C at
nucleotide 1843 in the skeletal muscle ryanodine receptor
(RYR1), leading to an amino acid change from arginine
to cysteine (Fujii et al., 1991). The RYR1, or Ca
2+
-release
channel, is the primary mechanism by which Ca
2+
stored in
the sarcoplasmic reticulum terminal cisternae is released
into the sarcoplasm to initiate muscle contraction. The
Ca
2+
release channels in MH susceptible pigs are hypersen-
sitive to agents that stimulate opening (OBrien, 1986), thus
allowing longer open time probability and resulting in
enhanced Ca
2+
release and greater twitch tension (Mickel-
son et al., 1988; Mickelson et al., 1989). The abnormal
channels ood the cell with Ca
2+
and overpowers the sar-
coplasmic reticulum ATPase pumps that resequester cyto-
plasmic Ca
2+
, resulting in sustained contraction and
metabolism.
This defect in Ca
2+
concentration has important conse-
quences for production and meat quality traits. Pigs homo-
zygous for the HAL gene have higher carcass yield and
lean percentage (Aalhus, Jones, Robertson, Tong, &
Sather, 1991; Herfort Pedersen et al., 2001; Leach, Ellis,
Sutton, McKeith, & Wilson, 1996; Rempel, Lu, Mickelson,
& Louis, 1995). The leaky Ca
2+
release channels of HAL
mutant pigs may contribute to leanness and heavy mus-
cling by causing spontaneous muscle contraction and
greater energy utilization, leading to work induced muscle
hypertrophy and limiting fat deposition (MacLennan &
Phillips, 1992).
The positive eect of the mutant allele on performance is
negated by an increased risk for stress-induced death and
high susceptibility to acute stress prior to slaughter, which
may be manifested in an accelerated rate of pH decline and
the production of PSE pork. An MH episode triggers a
massive stimulation of aerobic and anaerobic metabolism
that depletes energy resources, generates metabolic waste
products, and disrupts cellular and extracellular ion bal-
ances. The larger muscle ber area and lower capillary den-
sity of homozygous mutant pigs (Essen-Gustavsson,
Karlstrom, & Lundstrom, 1992) further compromises their
ability to cope with metabolic stress, and contributes to an
enhanced rate of glycogenolysis and glycolysis, evidenced
by reduced ATP, PCr, and glycogen levels, and higher lac-
tate levels and lower muscle pH at exsanguination (Essen-
Gustavsson et al., 1992; Fernandez, Neyraud, Astruc, &
Sante, 2002; Lundstrom, Essen-Gustavsson, Rundgren,
Edfors-Lilja, & Malmfors, 1989). Clearly, the mutant
HAL genotype increases the frequency of PSE meat by
accelerating the early postmortem rate of glycogenolysis
and glycolysis, leading to a more rapid pH decline.
3.2. The RN gene
Greater availability of glycogen may contribute to an
extended pH decline by providing additional substrate for
glycolysis. Sayre, Briskey, and Hoekstra (1963) showed
the Hampshire breed had more than twice the amount of
muscle glycogen compared to other breeds. Monin and Sel-
lier (1985) suggested that the higher muscle glycogen stores
were mainly responsible for an extended pH decline post-
mortem, resulting in pale meat with a low pHu, low water
holding capacity, and reduced technological yield, and
referred to this as the Hampshire eect in order to distin-
guish it from the PSE meat produced from a rapid pH
decline at elevated temperatures. Monin & Sellier (1985)
recommended that the muscle metabolites glycogen, glu-
cose, glucose 6-phosphate, and lactate should be combined
into a single measure termed glycolytic potential (GP) in
order to reect all of the compounds in the muscle capable
of being converted to lactate, thus indicating the muscles
capacity for extended postmortem glycolysis. A single
point mutation, Rendement Napole (RN), is responsible
for the elevated muscle GP (>180200 lmol/g muscle),
extended pH decline, and greatly reduced technological
yield that was rst found in the Hampshire breed (Le
Roy et al., 2000). There are two alleles, the dominant allele
(RN

) associated with elevated muscle glycogen and infe-

T.L. Scheer, D.E. Gerrard / Meat Science 77 (2007) 716 9
rior quality, and the wild-type recessive allele (rn
+
)
(Le Roy, Naveau, Elsen, & Sellier, 1990). Additional alleles
in the PRKAG3 gene may play a signicant role in glyco-
gen variation, including the 199I allele which is correlated
with lower glycogen and lactate, higher ham and loin pH
and improved color scores, resulting in improved meat
quality (Ciobanu et al., 2001).
The mutation responsible for the RNgene is an R200Q
substitution in the PRKAG3 gene that encodes for the
muscle specic isoform of the regulatory c-subunit of aden-
osine monophosphate activated protein kinase (AMPK)
(Milan et al., 2000). AMPK monitors the energy charge
of the muscle ber and prevents high energy phosphate
depletion. AMPK is allosterically inhibited by PCr and
ATP and activated by 5
0
AMP. Therefore, increased rates
of ATP utilization during muscle contraction activate
AMPK, which is hypothesized to phosphorylate proteins
involved in triggering fatty acid oxidation and glucose
uptake (reviewed by Winder, 2001). Transfection experi-
ments performed with mice revealed that the PRKAG3
R225Q is a loss of function mutation that eliminates allo-
steric regulation by ATP/AMP resulting in increased basal
AMPK activity (Barnes et al., 2004). Additionally, the
adenosine monophosphate kinase gamma-3 subunit
(AMPKc3) is more highly expressed in mouse fast twitch
white glycolytic muscle compared to fast twitch red oxida-
tive muscle, but is virtually undetectable in red muscle (Yu,
Fujii, Hirshman, Pomerleau, & Goodyear, 2004).
The expression proling of AMPKc3 supports evidence
that the RN mutation inuences the compositional and his-
tochemical traits and metabolic enzyme activities in a mus-
cle-type dependent manner. Lebret et al. (1999) found the
RN mutation had no eect on red semispinalis capitis mus-
cle. However, enhanced glucose uptake due to an increase
in the translocation of GLUT4 to surface membranes
(Kurth-Kraczek, Hirshman, Goodyear, & Winder, 1999)
and faster resynthesis of glycogen after exercise (Anders-
son, 2003) may explain the up to 70% increase in muscle
glycogen localized mostly to abnormally enlarged sarco-
plasmic compartments of type IIB white bers observed
in RN

carriers (Monin, Brard, Vernin, & Naveau, 1992;

Estrade, Vignon, Rock, & Monin, 1993). Furthermore, a
twofold increase in branching enzyme activity and a ten-
dency toward increased glycogen synthase activity may
contribute to elevated glycogen levels and dierences in
the molecular structure of glycogen (Estrade, Ayoub, Tal-
mant, & Monin, 1994).
The relatively high glycogen content of RN

carriers
might be expected to further enhance glycolytic metabolism
in IIB bers. Curiously, RN

carriers possess enhanced

oxidative metabolism, indicated by higher citrate synthase
and b-hydroxyacyl coenzyme A dehydrogenase activities,
decreased lactate dehydrogenase activity, higher relative
area of IIA bers and lower relative area of IIB bers (Leb-
ret et al., 1999). Mice with the PRKAG3 R225Q mutation
also exhibited higher skeletal muscle oxidative capacity
without altered ber type composition, suggesting that
the elevated glycogen content may be an indirect conse-
quence of altered muscle oxidation, and the role of the
AMPKc3 isoform may be to ensure that glycogen content
in muscle is restored by maintaining a high glycolytic
potential through shifting the metabolic fate of fuel toward
fat oxidation and glycogen storage (Barnes et al., 2004).
Nonetheless, high glycogen levels in RN

carriers do not
appear to alter the rate of glycogen utilization. RN

carri-
ers and wild type pigs exhibit similar glycogen degradation
during exercise (Andersson, 2003) as well as similar rates of
early postmortem pH decline (Monin & Sellier, 1985).
Estrade et al. (1994) reported no dierences in either glyco-
gen phosphorylase or debranching enzyme activity, further
supporting that the RN

allele increases glycogen avail-

ability without altering the rate of utilization. Thus, the
mutant RN genotype generates pork of inferior technolog-
ical quality by increasing glycolytic potential and extending
postmortem pH decline, resulting in pork with a low pHu.
4. Rate limiting enzymes
Clearly, glycogen degradation through glycolysis
impacts pork quality development. Enzymes catalyzing
the reactions of glycolysis inuence the rate and extent of
pH decline by directly controlling the conversion of metab-
olites through the pathway (Fig. 2). The properties of the
rate limiting enzymes glycogen phosphorylase, phospho-
fructokinase, and pyruvate kinase have been studied in
an eort to explain the aberrant glycolysis leading to PSE
development.
Glycogen phosphorylase and glycogen debranching
enzyme catalyze the complete degradation of glycogen.
Glycogen phosphorylase cleaves its substrate by addition
of inorganic phosphate at a-1,4 linkages of the outer chains
of the glycogen molecules, yielding glucose 1-phosphate.
Then, phosphoglucomutase catalyzes the isomerization of
glucose 1-phosphate to glucose 6-phosphate, which can
then proceed through glycolysis. Once phosphorylase
reaches the fourth glucose from the branch point, a trans-
ferase shifts the maltotriosyl group to the main chain, and
glycogen debranching enzyme breaks the a-1,6 linkages,
releasing free glucose. The main chain and remaining outer
branches are again susceptible to breakdown by
phosphorylase.
Sayre et al. (1963) found similar levels of phosphorylase
in swine exhibiting fast and slow rates of glycolysis
postmortem. However, in skeletal muscle, glycogen
phosphorylase exists in two forms: the less active, non-
phosphorylated form (GP b) and the more active, phos-
phorylated form (GP a). Hence, dierences in the fraction
of phosphorylase in the a form could plausibly explain the
abnormal glycolysis observed in PSE muscle. Early in vitro
studies indicated a strong relationship between pHu and
the amount of phosphorylase in the a form (Scopes,
1974). In support, Ono, Topel, Christian, and Althen
(1977) demonstrated an enhanced GP a activity in PSE tis-
sue, and Schwagele and Honikel (1988) determined a
10 T.L. Scheer, D.E. Gerrard / Meat Science 77 (2007) 716
higher total activity (GP a and GP b) in PSE-prone mus-
cles. However, Ensinger, Rogdakis, Muller, and Faber
(1982) could not establish dierences in the activities of
GP a and GP b in muscles of normal and PSE muscles.
More recently, Schwagele, Buesa, and Honikel (1996a)
reported no signicant dierences in the structural and
kinetic characteristics of GP a and GP b from muscles of
normal versus HAL sensitive pigs. Thus, the characteristics
of glycogen phosphorylase are not likely to be the main
factors facilitating altered metabolism in PSE muscle.
Because postmortem glycogenolysis may stop in the
presence of residual glycogen, it has been speculated that
glycogen debranching enzyme may inuence the rate and
extent of glycogenolysis and glycolysis. Kyla-Puhju, Ruus-
unen, and Puolanne (2005) reported that the activity of
debranching enzyme was only weakly aected by pH, but
activity strongly decreased when the temperature decreased
from 39 and 42 C to 4 and 15 C. Therefore, the activity of
debranching enzyme does not block rapid glycolysis and
pH decrease when the temperature is high, but rapid cool-
ing could limit the activity and thus limit glycogenolysis.
Phosphofructokinase (PFK) catalyzes the conversion of
fructose 1-phosphate and ATP to fructose 1,6-bisphos-
phate and ADP, which is the committed step in the glyco-
lytic pathway. In an in vitro glycolytic system, PFK was
still active under the extreme PSE conditions of 37 C
and pH 5.35 (Scopes, 1974). Moreover, total and specic
PFK activities were not signicantly dierent in pigs with
45 min longissimus muscle pH values ranging from 5.3 to
6.8 (Schwagele & Honikel, 1988). Therefore, activation of
PFK does not readily explain the dierences in glycolytic
rates between normal and PSE muscles. Interestingly, Alli-
son, Bates, Booren, Johnson, and Doumit (2003) observed
an inverse relationship between PFK capacity and uid
loss, and reasoned that PFK may become partially dena-
tured and inactivated by 20 min postmortem in muscles
that experience a rapid pH decline. Therefore, it appears
that increased PFK activity is not the reason for enhanced
glycolysis in PSE muscle.
Pyruvate kinase catalyzes the irreversible conversion of
phosphoenolpyruvate and ADP to pyruvate and ATP. Sch-
wagele, Haschke, Honikel, and Krauss (1996b) demon-
strated that, compared to normal muscle, pyruvate kinase
isolated from PSE muscle of halothane susceptible pigs
exhibited a tenfold increase in phosphoenolpyruvate utili-
zation. Interestingly, the activity of pyruvate kinase from
normal muscle was low at pH 5.5, whereas the enzyme
from PSE muscle maintained 70% of its maximum activity
under these acidic conditions. Schwagele et al. (1996b)
determined that this shift in pyruvate kinase activity was
due to phosphorylation of the enzyme, resulting in an addi-
tional, more acid stable isoform. It is not clear whether the
additional, more stable isoform is present in live animals or
if it is generated under postmortem conditions. Neverthe-
less, in pigs free of the HAL gene, pyruvate kinase capacity
was not correlated with longissimus pH, purge, drip loss or
paleness, and in all samples, pyruvate kinase lost more than
88% of its activity at pH 5.5 compared to pH 7.0 (Allison
et al., 2003). Although the role of pyruvate kinase in HAL
sensitive pigs is not completely understood, it appears that
enhanced pyruvate kinase capacity is not responsible for
altered postmortem metabolism in PSE muscle from pigs
free of the HAL gene.
For the most part, the inherent properties of the rate
limiting enzymes are not dierent in normal versus PSE
muscles, and thus other mechanisms must be responsible
for the aberrant glycogenolysis and glycolysis observed in
PSE muscles. Muscle possesses a complex system of both
feedforward and feedback regulation in order to precisely
modulate metabolism, and dierences in relative levels of
glycolytic regulators could contribute to altered postmor-
tem glycolysis. Specically, the modulators of rate limiting
enzymes could manipulate enzyme activity according to
Glycogen
Glucose Glucose 1 - Phosphate
glycogen phosphorylase
Glucose 6-Phosphate
hexokinase
phosphoglucose isomerase
Fructose 1,6- Bisphosphate
phosphofructokinase (PFK)
Dihydroxyacetone
phosphate
aldolase
Triose phosphate
isomerase
Bisphosphoglycerate
glyceraldehyde 3-phosphate dehydrogenase
3-Phosphoglycerate
phosphoglycerate kinase
2-Phosphoglycerate
phosphoglycerate mutase
enolase
Pyruvate
pyruvate kinase
Lactate
lactate dehydrogenase
phosphoglucomutase
Fructose 6-Phosphate
Phosphenolpyruvate
Glyceraldehyde
3-Phosphate
Fig. 2. Enzymes and metabolic intermediates of the glycolytic pathway.
T.L. Scheer, D.E. Gerrard / Meat Science 77 (2007) 716 11
energy demand of the cell, and this would aect ux
through the glycolytic pathway and contribute to altered
postmortem metabolism.
Rapid rates of postmortem glycolysis are associated
with low levels of glycogen and high levels of glucose
6-phosphate, indicative of increased phosphorylase activity
(Briskey et al., 1966; Kastenschmidt, Hoekstra, & Briskey,
1968; Moesgaard, Quistor, Christensen, Therkelsen, &
Jorgensen, 1995). Glycogen phosphorylase activity is regu-
lated both by phosphorylation and allosteric mechanisms.
Phosphorylase kinase exists in two forms, phosphorylase
kinase a and b (PK a and PK b), which are both capable
of converting GP b to the active GP a form. Maximal acti-
vation of phosphorylase kinase is achieved through phos-
phorylation of PK b to PK a and Ca
2+
binding. The
hormones epinephrine and glucagon induce increases in
cAMP that lead to phosphorylation of PK b to PK a,
and PK a is active at the concentration of Ca
2+
in resting
muscle. Exposure or susceptibility to stress would permit
epinephrine mediated activation of PK a and subsequent
activation of GP a, resulting in increased glycogenolysis.
The cAMP cascade provides a link between epinephrine
and glycogen degradation, whereas Ca
2+
couples muscle
contraction with glycogenolysis (Drummond, Harwood,
& Powell, 1969). Compared to PK a, PK b requires a
higher Ca
2+
concentration for activity (Connett & Sahlin,
1996). The Ca
2+
concentration in the sarcoplasm during
contraction is sucient to activate PK b, resulting in con-
version of GP b to the more active GP a form. Preslaughter
stress places an increased demand on muscles for contrac-
tion, and this could also be responsible for increased glyco-
genolysis by mediating the conversion of GP b to GP a by
means of Ca
2+
. Similarly, in the case of the HAL gene, the
enhanced Ca
2+
release may trigger activation of phosphor-
ylase and stimulate glycogenolysis.
The relative concentration of several modulators related
to cellular energy charge and substrate availability regulate
the activity of GP b. The levels of inhibitors, including
ATP, ADP, and glucose 6-phosphate, are usually sucient
to inhibit GP b in resting muscle, but their eect may be
overcome by increases in AMP and inosine monophos-
phate (IMP), and lead to GP b activation (Connett & Sah-
lin, 1996). Furthermore, the sensitivity of GP b for each
factor is dependent on the concentration of substrate (gly-
cogen and inorganic phosphate) and product (glucose 1-
phosphate). In contrast, GP a is active in the absence of
AMP, although activity is enhanced by low concentrations
of AMP and high concentrations of IMP. The other allo-
steric modulators have little eect on GP a activity.
In muscles exhibiting normal rates of glycolysis, glucose
6-phosphate levels tend to decrease during the rst hour
postmortem and increase thereafter (Kastenschmidt et al.,
1968; Hammelman et al., 2003). This suggests that glyco-
gen phosphorylase may be unable to supply adequate glu-
cose 6-phosphate for the subsequent reactions of the
glycolytic pathway, resulting in a rapid utilization of the
glucose 6-phosphate pool. If animals exhibiting slow
rates of glycolysis were assumed to experience minimal
antemortem stress, glycogen phosphorylase would exist
predominantly in the b form. Moreover, following exsan-
guination, the relatively high levels of ATP and low levels
of AMP (Kastenschmidt et al., 1968) and IMP (Klont &
Lambooy, 1995) would be insucient to activate GP b.
After the rst hour postmortem, changes in the relative
concentrations of allosteric activators may permit GP b
activation. Therefore, the accumulation of G6P after one
hour may be the result of increased glycogen breakdown
due to glycogen phosphorylase, or a decrease in the activity
of PFK or other enzymes downstream.
The regulation of the other rate limiting enzyme, PFK is
less complex. Similar to GP b, PFK activity depends largely
on the energy status of the muscle cell. As the ATP/AMP
ratio decreases, PFK activity is stimulated. PFK is also
activated by hexose bisphosphates and inorganic phos-
phate. Moreover, ATP is required for the transfer of a
phosphate group from ATP to fructose 6-phosphate.
Rapid depletion of ATP in fast glycolyzing muscles (Kas-
tenschmidt et al., 1968) could compromise the ability of
PFK to catalyze the formation of fructose 1,6-bisphos-
phate. This would suggest PFK is exerting some glycolytic
control. However, in the case of slow glycolyzing muscles,
the imbalance between fructose 6-phosphate and fructose
1,6-bisphosphate becomes apparent after 60 min, and Kas-
tenschmidt et al. (1968) suggested this may be due to
decreasing muscle pH and partial inactivation of PFK.
In contrast, ux through pyruvate kinase appears to be
largely governed by the concentration of the substrates
phosphoenolpyruvate and ADP and product ATP. The
high levels of ATP present in slow glycolyzing muscles
may exert some control over PK early postmortem. How-
ever, in fast glycolyzing muscles, lactate production ceased
in the presence of residual glycogen, glucose 1-phosphate,
glucose 6-phosphate (G6P), and fructose 6-phosphate,
whereas subsequent intermediates were present at low lev-
els (Kastenschmidt et al., 1968). This implies PFK is more
likely than PK to be a site of glycolytic control.
Altogether, previous studies suggest that the relative lev-
els of glycolytic regulators contribute to altered postmor-
tem glycolysis. Specically, the modulators of rate
limiting enzymes manipulate enzyme activity according to
cellular energy status, and this aects ux through the
pathway. Glucose 6-phosphate levels fall during the rst
60 min, and subsequently increase, indicating an imbalance
between glycogenolysis and glycolysis. Therefore, dierent
enzymes may be rate limiting at dierent times during the
conversion of muscle to meat.
5. Control of postmortem glycolysis by AMPK
AMPK is particularly attractive for augmenting post-
mortem metabolism primarily because in vivo studies show
AMPK is activated in ischemic cardiac muscle and hypoxic
skeletal muscle (Kim, Solis, & Cartee, 2004). As outlined
above, when muscle (cardiac or skeletal) is placed in a
12 T.L. Scheer, D.E. Gerrard / Meat Science 77 (2007) 716
low oxygen environment, the primary source of energy pro-
duction defaults to anaerobic glycolysis, thereby increasing
the AMP:ATP ratio rapidly. This, in turn, allows AMP to
bind to AMPK making it a better substrate for phosphor-
ylation by its upstream kinase, AMPKK. Activated
AMPK can impact glycolysis in two manners. First,
AMPK can activate phosphorylase kinase, which then
activates glycogen phosphorylase and promotes glycogen-
olysis. Second, AMPK can phosphorylate phosphofructo-
kinase-2 which catalyzes the formation of fructose
2,6-bisphosphate. This product is an allosteric activator
of PFK-1, a key rate-limiting enzyme of glycolysis. Thus,
AMPK activation indirectly increases ux through glycol-
ysis making it a strong candidate for modulating meat
quality development.
Shen and Du (2005) rst investigated the involvement of
AMPK in regulating, or modulating postmortem metabo-
lism by studying exercised wild-type and AMPK-knockout
mice treated with or without the AMP analog 5-amino-4-
imidazolecarboxamide riboside (AICAR). These scientists
showed heavily exercised mice and those exercised mice
treated with AICAR had signicantly lower (more rapid)
muscle pH values at 24 h postmortem (Fig. 3). Conversely,
mice lacking a functional AMPK gene, whether exercised
or not, had higher ultimate muscle pH values, even higher
than wild-type, non-exercised mice. These data closely
reected trends in AMPK activity suggesting that aug-
mented postmortem glycolysis in mouse skeletal muscle is
partially controlled by the status of AMPK phosphoryla-
tion antemortem. To eliminate the eects of AMPK activa-
tion in living muscle on postmortem metabolism, Du et al.
(personal communication) infused muscle with compound
C immediately antemortem. In good agreement with their
aforementioned data, postmortem glycolysis and muscle
pH decline was inhibited. These data strongly support the
role of AMPK in modulating postmortem muscle
metabolism.
At a glance, these data appeared to shed much light on
the 50 year debate as to what may be controlling extended
postmortem glycolysis in pig muscle. However, conditions
whereby mouse and pig muscle undergo postmortem
metabolism are quite dierent. To that end, this same
group (Shen et al., 2006a) used pre-slaughter transporta-
tion as a stressor and monitored AMPK activity and
energy status of pig muscle postmortem. Similar to the
mouse data, AMPK activity reached maximum levels
quicker in transported pig muscle than in control pigs, or
those rested after transport. Consistent with these ndings,
the (AMP + IMP):ATP ratio was greater in the trans-
ported pigs arguing AMPK activation through cytosolic
AMP concentrations. These researchers subsequently
showed an association between early postmortem AMPK
activation and PSE development in commercial pigs and
suggested that the rapid glycolysis occurring in muscle of
HAL pigs was in part from increases in AMPK activation
early postmortem (Shen et al., 2006b). These data clearly
show an association between AMPK activation and aggra-
vated metabolism postmortem. However, much more work
is needed to understand exactly how AMPK works in
dying muscle tissue.
We have attempted to study muscle metabolism further
using the HAL and RN mutations separately and in com-
bination rapid metabolism together with abundant gly-
cogen supply (HAL/RN mutant) to provide additional
insight into the control of glycogenolysis and glycolysis
(Copenhafer, Richert, Schinckel, Grant, & Gerrard,
2006). As expected, the previously documented eects of
both genes were evident: the HAL mutation elicited has-
tened metabolism, as evidenced by lower ATP levels at
0 and 30 min, and rapid degradation of glycogen and
accumulation of lactate early postmortem compared to
control; whereas the RN mutation resulted in much
greater glycolytic potential due to elevated glycogen
levels.
Additional investigation yielded more insight regarding
biochemical events within the glycolytic pathway. Free glu-
cose, released by the action of debranching enzyme on gly-
cogen, was independently aected by HAL and RN
genotype. The HAL mutants exhibited increased glucose
accumulation indicative of more rapid glycogen degrada-
tion and early postmortem metabolism, whereas the
increases in the RN mutants occurred to a greater extent
and after 60 min postmortem. Rapid rates of glycolysis
were associated with decreased glycogen as well as high
concentrations of glucose 6-phosphate, indicating
increased phosphorylase activity. G6P concentrations
decreased in the HAL mutants during the rst 30 min
and remained at a similar level thereafter. Meanwhile, in
the HAL/RN mutant, G6P concentrations tended to
increase in the rst 30 min, then decrease through 60 min,
indicating the combined eects of the HAL and RN max-
imize activation of glycogen phosphorylase by enhancing
Ca
2+
concentration and glycogen availability. Despite large
dierences in G6P between HAL and HAL/RN mutants,
pH decline and lactate accumulation were similar early
postmortem. Glycogen phosphorylase and debranching
enzyme were capable of aggressive glycogenolysis to supply
adequate G6P for the reactions of glycolysis. Additionally,
Fig. 3. Muscle pH values at various times postmortem from mice treated
dierently prior to euthanasia (Shen & Du, 2005).
T.L. Scheer, D.E. Gerrard / Meat Science 77 (2007) 716 13
high glycogen levels did not aggravate rapid postmortem
metabolism.
From 60 min to 24 h postmortem, G6P increased only in
normal and RN mutant genotypes. The accumulation of
G6P after 60 min conrmed that glycogen phosphorylase
activity was adequate to meet the demands of glycolysis
and that either glycogen phosphorylase activity was
increased or phosphofructokinase activity was reduced.
Similar residual glycogen levels as well as the slowing of
glycolysis with time, indicated that increased G6P is likely
due to reduced phosphofructokinase activity.
Interestingly, RN mutants possessed higher phosphocre-
atine concentrations than all other genotypes (Fig. 4).
Ponticos et al. (1998) observed that a fall in PCr:creatine
ratio leads to increased AMPK activity, which results in
a concomitant decrease in muscle CK activity. The pro-
posed inactivation of CK by AMPK may be an energy con-
servation mechanism that prevents CK from consuming
ATP for the rephosphorylation of creatine. Presence of
the mutant RN genotype also resulted in an increase in
ATP concentrations. The constitutively active AMPK in
the RNmutant genotype appears to decrease CK activity
and preserve energy levels more eciently in the rst
30 min postmortem. This may have several ramications
in extending postmortem proteolysis. First, it could pro-
long glycolysis by having greater adenosine levels in the tis-
sue longer. Second, this could delay maximal rate of
glycolysis, which would likely occur at a reduced carcass
temperature allowing for extended glycolysis. And third,
the increase in inorganic phosphate may increase the buf-
fering capacity of the muscle, and again, allow for extended
glycolysis.
The RN

allele is typically associated with increased

glycolytic potential and lower ultimate pH. However, at
24 h postmortem, RN and HAL/RN mutant pigs exhibited
lower pH than the normal genotype but not the HAL
mutants. Previous studies have also indicated that the
mutant HAL allele tends to lower ultimate pH. Therefore,
substrate availability alone does not explain variation in
ultimate pH. Data from this study show that elevated gly-
cogen does not aggravate rapid early postmortem glycoly-
sis, nor does it fully explain low ultimate pH. The
diminished ATP in muscles undergoing rapid glycolysis
suggests phosphofructokinase may be rate limiting during
aggressive early postmortem metabolism. Moreover, the
accumulation of glucose and glucose 6-phosphate in mus-
cles with normal rates of glycolysis suggests phosphofruc-
tokinase may be rate limiting after 1 h postmortem.
Thus, depending on the pace of postmortem metabolism,
enzymes may become rate limiting at dierent times during
postmortem metabolism. Regardless, this is a truly valu-
able model for studying adverse pork quality development.
Further analyses of muscle tissues from the aforemen-
tioned study showed that AMPK activation was greater
in muscle from RN

pigs compared to all other genotypes

(Park et al., submitted). Curiously, classic data from Milan
et al. (2000) reported that AMPK activation in RN-pigs is
greatly reduced compared to wild type pigs. The reason for
this discrepancy is not readily apparent but may be related
to sampling time or muscle type. In our studies, samples
were collected immediately post-stunning from the longiss-
imus muscle, however, details regarding sampling proce-
dures in the Milan study are somewhat sketchy. Our
data, however, are corroborated by data from the AMPK
RN mutant mice, where increased AMPK activation is
observed in that muscle as well. Clearly, establishing
whether AMPK is activated in RN mutated pig muscle,
in either living or dying muscle is critical for our under-
standing of how AMPK may modulate postmortem muscle
metabolism.
Another intriguing observation merits discussion is that
AMPK activation is blunted in the presence of the HAL
gene (Park et al., submitted). Thus, if it is true that AMPK
modulates postmortem energy metabolism as reported by
Shen et al. (2006), then the state of AMPK prior to slaugh-
ter may be more important than what happens to the
enzyme after death. This scenario is further complicated
by the fact that we observed greater glycolytic potential
in the double mutant, in the absence of AMPK activation.
Given that increased GLUT-4 production, the primary
means of glucose uptake in the muscle, is only observed
in muscle with activated AMPK, it is dicult to imagine
how greater glycogen is accumulated in the muscle of
HAL/RN mutants. At present, the only possibility is that
phosphorylation of AMPK may not be the only mecha-
nism by which AMPK modulates energy balance in muscle.
Additional studies are needed to support this hypothesis.
Taken together, the aforementioned review shows that
postmortem metabolism in skeletal muscle is quite compli-
cated and largely aected by the state of the tissue prior to
death. Regardless of the complexity, however, numerous
genetic models, molecular techniques and commercially
0
2
4
6
8
10
12
0 min 30 min
Time
Normal
RN
HAL
HAL/RN
a
b b
b
b
c
c c
P
h
o
s
p
h
o
c
r
e
a
t
i
n
e

(
u
m
o
l
/
g
)

Fig. 4. LS means of postmortem longissimus muscle phosphocreatine
concentrations in halothane and RN genotypes. (Copenhafer et al., 2006).
14 T.L. Scheer, D.E. Gerrard / Meat Science 77 (2007) 716
available compounds exist that make studying this event
quite rewarding. If technologies are going to be developed
that allow the pork industry to predict quality, then a com-
prehensive understanding of the biochemical and molecu-
lar events controlling postmortem metabolism must be
known.
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