Journal of the Science of Food and Agriculture J Sci Food Agric 85:2401–2406 (2005) DOI: 10.1002/jsfa.2252

Role of AMP-activated kinase in the of postmortem muscle Qingwu W Shen and Min Du∗ Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA

Abstract: AMP-activated protein kinase (AMPK) is a newly identified kinase controlling energy in vivo. The objective of this study was to show the role of AMPK in postmortem glycolysis. Rapid and excessive postmortem glycolysis is directly related to the incidence of PSE (pale, soft and exudative) meat in pork, chicken and turkey, while insufficient glycolysis leads to dark cutters in beef and lamb, which causes significant loss to the meat industry. A total of 24 two-month-old C57BL/6J mice were assigned to three treatments: (1) wild-type mice without pre-slaughter treatment; (2) wild-type mice with a 2 min swim before slaughter; and (3) wild-type mice intraperitoneally injected with AICAr (50 mg kg−1), a specific activator of AMPK, to stimulate the activity of AMPK. In addition, 16 two- month-old C57BL/6J mice with AMPK knockout were assigned to two treatments: (4) AMPK knockout mice without pre-slaughter treatment; and (5) AMPK knockout mice with a 2 min swim before slaughter. The longissimus dorsi muscle was sampled at 0, 1 and 24 h postmortem for pH and enzyme activity measurements. Results showed that AMPK activity had a major role in determining the ultimate muscle pH. Pre-slaughter stress induced by swimming significantly accelerated the glycogenolysis in postmortem muscle through activating glycogen phosphorylase. AMPK is important for maintaining the activity of glycogen phosphorylase and pyruvate kinase, and glycogenolysis/glycolysis in postmortem muscle. Thus, AMPK has an important role in the control of postmortem glycolysis and is crucial for a lower ultimate pH in postmortem muscle. However, the activation of AMPK cannot fully account for the initial rapid glycogenolysis/glycolysis induced by stress and another mechanism must exist for the accelerated glycolysis induced by pre-slaughter stress.  2005 Society of Chemical Industry

Keywords: AMPK; muscle; postmortem; glycolysis; pH value; PSE

INTRODUCTION beef and lamb is another problem associated with PSE (pale, soft, exudative) meat has a high drip loss, a postmortem glycolysis, which, however, is due to low cooking yield and a dry texture after cooking. Fast insufficient glycolysis. Owing to its inferior quality, glycolysis in postmortem muscle causes PSE syndrome the dark cutter in beef also causes significant loss in pork, turkey and chicken while excessive glycolysis to the meat industry.8 In order to solve these is related to ‘acid meat’ in Hampshire pigs.1–3 The problems, it is necessary to understand the underlying Pork Chain Quality Audit stated that ‘10.2% of the mechanisms. carcasses were classified as having PSE muscle’.4 For AMP-activated protein kinase (AMPK) is mainly turkey, observations in the slaughterhouse (especially recognized as a crucial kinase controlling energy in the USA) have shown that PSE meat could metabolism.9 Recent biomedical studies have indi- represent from 5% to 30% of the slaughtered turkeys.5 cated that AMPK plays a crucial role in the initiation of In chicken, using colour L∗ values of 3 h and 24 h glycogenolysis/glycolysis in skeletal and cardiac mus- postmortem fillets as an indicator, ∼47% of the 3554 cle in vivo.9 AMPK, a heterotrimeric enzyme with α, fillets tested were pale and could potentially exhibit β and γ subunits, is mainly recognized as a critical poor water-holding capacity.6 The high incidence of regulator of energy metabolism.9 Each subunit exists PSE syndrome causes significant losses to the meat as isoforms encoded by two or three genes (α1, α2, industry.6,7 However, the mechanisms associated with β1, β2, γ 1, γ 2, γ 3).10 The α subunit is the catalytic this abnormal glycolysis in postmortem muscle and the unit, the γ subunit has a regulatory function, and incidence of PSE meat are largely unclear. The dark the β unit provides anchorage sites for α and γ .11 cutter, or DFD (dark, firm and dry), syndrome in The γ 3 subunit is expressed at high levels in skeletal

∗ Correspondence to: Min Du, Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA E-mail: [email protected] Contract/grant sponsor: USDA Cooperative State Research, Education, and Extension Service; contract/grant number: National Research Initiative Competitive Grant USDACSRE45101 Contract/grant sponsor: University of Wyoming (Received 9 December 2004; revised version received 15 February 2005; accepted 15 March 2005) Published online 13 July 2005  2005 Society of Chemical Industry. J Sci Food Agric 0022–5142/2005/$30.00 2401 QW Shen, M Du muscle only.12 AMPK is switched on by an increase Enzyme activity measurements in the AMP/ATP ratio in muscle cells, which leads Muscle homogenate to the phosphorylation of AMPK at Thr172 by an Frozen longissimus dorsi muscle samples were unidentified kinase.9 Once activated, AMPK switches cut into small pieces and mixed. Longissimus on glycogenolysis/glycolysis.10,11 The important role dorsi muscles (0.01 g) were weighed and then of AMPK in the control of glycolysis in vivo has been homogenized in a Polytron homogenizer (7 mm demonstrated by several studies. Administration of diameter generator) (IKA Works Inc, Wilming- AICAr, a specific activator of AMPK, to stimulate ton, NC, USA) with 5 vol. of ice-cold lysis −1 −1 AMPK activity dramatically increases the lactic acid buffer (137 mmol L NaCl, 1 mmol L MgCl2, 13 −1 content in muscle. Also, knockout AMPK signifi- 1mmolL CaCl2, 1% nonylphenyl-polyethylene − cantly reduced the pH decline in ischaemic cardiac glycol, 10% glycerol, 2 mmol L 1 phenylmethane- − muscle.14 No study has been conducted on the role of sulfonyl fluoride, 10 mmol L 1 sodium pyrophos- − AMPK in the glycolysis of postmortem muscle. It is phate, 2.5 mmol L 1 ethylenediaminetetraacetic acid − − hypothesized that AMPK plays an important role in (EDTA), 10 µgmL 1 aprotinin, 10 µgmL 1 leu- − the glycolysis of postmortem muscle and the incidence peptin, 100 mmol L 1 NaF). Following centrifuging of PSE meat. Using transgenic mice, the objective of at 12 000 × g for 5 min at 4 ◦C, the supernatant was this study was to show the role of AMPK in the used for enzyme activity measurements. glycolysis of postmortem muscle. AMPK AMPK activity was measured using a method EXPERIMENTAL based on its specific phosphorylation of a SAMS Dietary treatments peptide.16,17 Briefly, a SAMS peptide substrate A total of 24 two-month-old C57BL/6J mice† (half was used (His-Met-Arg-Ser-Ala-Met-Ser-Gly-Leu- male, half female) were assigned to three treatments His-Leu-Val-Lys-Arg-Arg; Invitrogen, Carlsbad, CA, (4 male and 4 female mice per treatment): (1) normal USA). The muscle homogenate obtained above was mice without any treatment; (2) mice with a 2 min centrifuged at 13 000 × g for 5 min at 4 ◦C. The super- swim before slaughter; and (3) mice intraperitoneally natant (10 µL) was incubated for 10 min at 37 ◦C − injected with AICAr (50 mg kg 1) (Calbiochem, La in 40 mmol L−1 4-2-hydroxyethyl-1-piperazineethane- Jolla, CA 92 039, USA), a specific activator of AMPK, sulfonic acid (HEPES), 0.2mmolL−1 SAMS pep- to stimulate the activity of AMPK, and then a wait of tide, 0.2 mmol L−1 AMP, 80 mmol L−1 NaCl, 8% 15 min to let the AICAr go into muscle cells before (w/v) glycerol, 0.8 mmol L−1 EDTA, 0.8mmolL−1 −1 slaughter. In addition, 16 two-month-old C57BL/6J dithiothreitol (DDT), 5 mmol L MgCl2,and mice with AMPK knockout were assigned to two 0.2mmolL−1 ATP + 2 µCi [32P]ATP, pH 7.0, in a treatments (4 male and 4 female mice per treatment): final volume of 50 µL. An aliquot (20 µL) was removed (4) AMPK knockout mice without any treatment; and and spotted on a 2 cm × 2 cm piece of Whatman P81 (5) AMPK knockout mice with a 2 min swim before filter paper. The [32P]ATP was removed with six slaughter. The AMPK knockout mice were origi- washes in 1% phosphoric acid, and the radioactiv- nally obtained from Dr MJ Birnbaum (Department ity was quantified after immersing the filter paper in of Medicine and Howard Hughes Medical Institute, 3 mL Scintiverse (Fisher Scientific, Hanover Park, IL, University of Pennsylvania) and bred in our labora- USA). The activity was expressed as the phosphory- tory. These AMPK knockout mice express a dominant lation of nanomolar peptide per minute per gram of negative AMPKα2 under the control of the muscle- muscle. specific creatine kinase promoter.15 The mice were anaesthetized by CO2 and then killed immediately. Glycogen phosphorylase ∼ Within 4 min, the pelt was removed and 0.2 g each of Glycogen phosphorylase-a activity was measured by upper-right and lower-left longissimus dorsi muscle was the incorporation of [U-14C] glucose into glyco- ∼ removed and combined. Part of this muscle ( 0.1g) gen when a high level of glucose-1-phosphate was was used for pH measurement and the rest was snap- present.18 Briefly, 200 µL of the supernatant of frozen in liquid nitrogen for analysis of enzyme activity. muscle homogenate obtained above was diluted Carcasses were eviscerated and suspended in a glass −1 ◦ 1:1 with ice-cold solution B (50 mmol L 2-(N- chamber at 4 C. Subsequent samples was taken in a morpholino)ethanesulfonic acid (MES), 50 mmol L−1 similar fashion from the middle part of both sides of the KF and 60 mmol L−1 β-mercaptoethanol, pH 6.1). muscle after 1 h and from the remaining lower-right The diluted supernatant (60 µL) was mixed with and upper-left region after 24 h. Fat and connective 100 µL of solution C (400 mmol L−1 KF, 2 mg mL−1 tissue were removed from muscle samples which were glycogen, 27.88 mg mL−1 glucose-1-phosphate and then snap-frozen in liquid nitrogen and used for the 0.5 µCi mL−1 [U-14C] glucose-1-phosphate) (Amer- analysis of key enzymes involved in glycolysis. sham, Piscataway, NJ, USA) and incubated for 1 h at 30 ◦C. A portion of the mixture (80 µL) was removed † The animal experimentation was approved by the University of andspottedona20mm× 20 mm piece of 3M Wyoming Animal Care and Use Committee on 7 January 2004. chromatography paper. The paper was immediately

2402 J Sci Food Agric 85:2401–2406 (2005) AMPK and glycolysis in postmortem muscle dropped into ice-cold 66% (v/v) ethanol. Three 6.6 separate washes in ice-cold 66% ethanol were per- 6.5 a formed. The paper was incubated in acetone for ab ab ab b a a a 2–3 min before being dried for scintillation count- 6.4 ab ab ing. The activity was expressed as the incorporation of bc 6.3 glucose (mmol) per minute per gram of muscle.18 c pH 6.2 b

Pyruvate kinase activity 6.1 c Activity was measured by the coupled reaction of c pyruvate kinase with lactic dehydrogenase which 6.0 results in the formation of oxidized nicotinamide- 5.9 adenine dinucleotide (NAD+). Briefly, supernatant 0 h 1 h 24 h of the muscle homogenate obtained above (200 µL) Control Swim AICAr Knockout Swim+knockout was diluted 1:1 with a solution (0.3 mmol L−1 NADH, 1.5mmolL−1 phosphoenolpyruvic acid, Figure 1. pH values of postmortem muscle from mice that had −1 −1 −1 received various pre-slaughter treatments; n = 8, P < 0.05. 16 mmol L MgSO4, 150 mmol L KCl, 60 U mL lactic dehydrogenase and 0.1 mol L−1 Tris-HCl, pH 7.4), mixed, and incubated at 37 ◦C for 5 min. Then, The overall difference in pH at 0 h postmortem was 0.64 mmol L−1 ADP was added and the change in quite small. At 1 h postmortem, however, a much absorbance at 340 nm was recorded. The activity was greater difference in pH was observed. The pH was expressed as mmol NAD+ formation per minute per lowest in the postmortem muscle of mice stressed gram of muscle.19 by swimming, followed by AICAr injection, showing that pre-slaughter stress accelerates glycolysis. This pH measurement result is consistent with that obtained from pigs that Within 4 min of death longissimus dorsi muscle (0.1 g) had experienced pre-slaughter stress. Pre-slaughter −1 stress induces rapid initial pH decline which may was homogenized in 0.9 mL of 5 mmol L iodoacetate 22 solution, and the pH of the homogenate was measured lead to PSE meat. The pH was slightly higher directly with a pH meter.20 in the muscle from mice with AMPK knockout. At 24 h postmortem, however, the pH of muscle with AMPK knockout was dramatically higher than that of Statistical analysis wild-type mice. The pH for the postmortem muscle Data were analyzed as a complete randomized design of AMPK knockout mice was 6.38 and for AMPK using GLM (general linear model of Statistical knockout plus swimming was 6.34. These pH values Analysis System, SAS, 2000). The activities of AMPK, were much higher than the pH of muscle from control glycogen phosphorylase and pyruvate kinase, and the mice, which was 6.13. Compared with the muscle pH of longissimus dorsi muscle were analyzed. The of livestock, the initial pH of postmortem muscle differences in the mean values were compared by the measured in this study was quite low but the ultimate Fisher’s protected least significant difference (LSD) pH was high; the exact reason causing this difference test (P < 0.05). Mean values and standard errors of is unclear. The pH at 24 h was lowest for mice either the mean were reported. stressed by swimming or injected with AICAr, with pH values of 6.03 and 6.01, respectively. These results strongly suggest that AMPK has an important role in RESULTS AND DISCUSSION postmortem glycolysis. To show this, the activity of Pre-slaughter stress has long been recognized as induc- AMPK was further analyzed. ing PSE syndrome in pigs and poultry.7,21 The under- At 0 h postmortem, the AMPK activity (Fig 2) was lying mechanism, however, is unclear. The formation significantly lower in the postmortem muscle from of AMP during muscle contraction and other bio- chemical processes is expected to play a key role. Since AMPK is activated by an increase in AMP in muscle, 3.0

) a 1 2.5 we hypothesize that AMPK plays an important role in − g postmortem glycolysis. In this study, mice with AMPK 1 − 2.0 b knockout were used to elucidate the role of AMPK a min 1.5 1

− c in postmortem glycolysis and the pre-slaughter stress a a was simulated by 2 min of forced swimming. 1.0 d d AMPK activity b b

The pH values of postmortem muscle were (nmol L 0.5 significantly influenced by pre-slaughter treatments 0.0 (Fig 1). At 0 h postmortem, the pH of postmortem 0 h 1 h muscle from mice injected with AICAr, a specific Control Swim AIACr Knockout Swim+knockout activator of AMPK, was significantly lower than that of mice with AMPK knockout, showing the Figure 2. AMPK activity of postmortem muscle from mice that had important role of AMPK in postmortem glycolysis. received various pre-slaughter treatments; n = 8, P < 0.05.

J Sci Food Agric 85:2401–2406 (2005) 2403 QW Shen, M Du mice with AMPK knockout, and was numerically 12 a ab higher in mice that had received an AICAr injec- b a ab 10 b b ab tion. At 1 h postmortem, the differences in AMPK )

1 bc − c were magnified. The AMPK activity was highest g 8 1 in mice that had received an AICAr injection, fol- − lowed by mice that had been stressed with swimming, 6 and then by the control mice. The AMPK activity 4 was dramatically lower in mice with AMPK knock- (mmol min out. This corresponds well with the pH values. At 2

1 and 24 h, the pH values were significantly higher Glycogen phosphorylase activity 0 in mice with AMPK knockout and lower in mice 0 h 1 h either injected with AICAr or stressed by swimming Control Swim AICAr Knockout Swim+knockout (Fig 1). This shows that the activity of AMPK is negatively associated with the pH of postmortem Figure 3. Glycogen phosphorylase activity of postmortem muscle muscle, suggesting an important role for AMPK in from mice that had received various pre-slaughter treatments; n = 8, postmortem glycolysis. The role of AMPK in glycol- P < 0.05. ysis in vivo has been well established. In ischaemic cardiac and skeletal muscles, AMPK initiates gly- the activity of glycogen phosphorylase in postmortem colysis through two possible mechanisms. Firstly, muscle (Fig 3). Stress and AICAr had no effect on the activated AMPK can phosphorylate 6-phosphofructo- activity of glycogen phosphorylase, although glyco- 2-kinase.23 Phosphorylated 6-phosphofructo-2-kinase gen phosphorylase activity was numerically higher in promotes the synthesis of fructose 2,6-bisphosphate, stressed mice and mice injected with AICAr. These a potent allosteric stimulator of 6-phosphofructo-1- results are consistent with in vivo studies showing that kinase, which is a key glycolysis-controlling enzyme. glycogen phosphorylase is activated by AMPK.18,25,26 Secondly, AMPK can inhibit the activity of glycogen However, this does not mean that AMPK activation synthase and activate phosphorylase kinase by phos- is necessary for the activation of glycogen phosphory- phorylation. Phosphorylase kinase further activates lase. At 0 h postmortem, the activity of AMPK was glycogen phosphorylase by phosphorylation, which quite low and was not fully activated (Fig 2), while promotes glycogenolysis.13,18,24 However, the activa- the glycogen phosphorylase appeared fully activated. tion of AMPK can not fully account for the initial These results show that the activation of glycogen rapid glycolysis induced by stress, since the activation phosphorylase in postmortem muscle immediately fol- of AMPK by AICAr only decreased the pH at 24 h, lowing slaughter was not induced by AMPK and that while stress decreased both the pH values at 1 and AMPK has a role in maintaining the activity of glyco- 24 h (Fig 1), suggesting another mechanism is associ- gen phosphorylase. ated with the rapid glycolysis induced by pre-slaughter If the AMPK is not responsible for the activation stress. of glycogen phosphorylase, there must be another Glycogen phosphorylase contains a and b forms. pathway leading to the activation of glycogen Phosphorylase-b is not an active form though AMP phosphorylase. This unidentified pathway may be + and Ca2 can lead to its activation. Phosphorylase-b associated with the stress induced by slaughtering is activated by protein kinase A (PKA) through phos- itself. It has been shown that the activation of glycogen phorylation at a specific Ser residue in phosphorylase- phosphorylase is controlled by muscle contraction b, leading to its conversion to phosphorylase-a, a and by catecholamines.27 Stresses instantly increase fully active form of phosphorylase. It was recently the catecholamine level in serum.28– 30 Epinephrine, found that phosphorylase-b is also phosphorylated one of the main catecholamines secreted during and activated by AMPK,13,18,24 which leads to its stress, is a potent stimulator of lactate production conversion to phosphorylase-a. To assess the role of in in vivo.31,32 In vivo, epinephrine AMPK in the activation of phosphorylase, the activ- activates β-adrenoceptor and leads to the production ity of glycogen phosphorylase-a was analyzed. At 0 h of cAMP and activation of PKA which then activates postmortem, the activity of glycogen phosphorylase-a glycogen phosphorylase, initiating glycogenolysis.31,32 in mice stressed by swimming was higher than that Therefore, epinephrine plays an important role in the of control mice (Fig 3). This result indicates that activation of glycogen phosphorylase and glycolysis stress activates glycogen phosphorylase. At this time in vivo. Owing to the loss of neural and hormone the AMPK activity was still quite low and there was regulation in postmortem muscle, it is unclear whether no difference in AMPK activity between control mice epinephrine produced by pre-slaughter stress still plays and mice that had received stress (Fig 2), suggest- an important role in postmortem glycolysis, and this ing that the glycogen phosphorylase was activated by needs further study. another mechanism. At 1 h postmortem, the activ- Pyruvate kinase is another key glycolytic enzyme. ity of glycogen phosphorylase was significantly lower At 0 h postmortem, the activity of pyruvate kinase was in AMPK knockout mice compared with wild-type lower in AMPK knockout mice stressed by swimming mice, indicating that AMPK is needed to maintain than that of control mice (Fig 4). The reason for this

2404 J Sci Food Agric 85:2401–2406 (2005) AMPK and glycolysis in postmortem muscle

0.6 University of Wyoming. The authors thank Dr MJ a Birnbaum, Department of Medicine and Howard 0.5

) a Hughes Medical Institute, University of Pennsylvania, 1 ab ab − ab ab for providing AMPK knockout mice.

g 0.4 b

1 ab b − b 0.3

0.2 REFERENCES

(mmol min 1 Solomon MB, Van Laack RLJM and Eastridge JS, Biophysical 0.1 basis of pale, soft, exudative (PSE) pork and poultry muscle: Pyruvate kinase activity Areview.J Muscle Foods 9:1–12 (1998). 0.0 2 van Laack RL and Kauffman RG, Glycolytic potential of 0 h 1 h red, soft, exudative pork longissimus muscle. JAnimSci Control Swim AICAr Knockout Swim+knockout 77:2971–2973 (1999). 3 Miller KD, Ellis M, Sutton DS, McKeith FK and Wilson ER, Effects of live-animal sampling procedures and sample storage Figure 4. Pyruvate kinase activity of postmortem muscle from mice on the glycolytic potential of porcine longissimus muscle that had received various pre-slaughter treatments; n = 8, P < 0.05. samples. J Muscle Foods 11:61–67 (2000). 4 Cannon JE, Morgan JB, McKeith FK, Smith GC, Sonka S, difference is unclear. At 1 h postmortem, however, the Heavner J and Meeker DL, Pork chain quality audit survey: activity of pyruvate kinase was the highest in mice quantification of pork quality characteristics. J Muscle Foods that had been injected with AICAr, with an activity 7:29–44 (1996). −1 −1 −1 −1 5 Barbut S, Estimating the magnitude of the PSE problem in of 0.48 mmol min g versus 0.35 mmol min g poultry. J Muscle Foods 9:35–49 (1998). in control mice. Since the major effect of AICAr is 6WoelfelRL,OwensCM,HirschlerEM,Martinez-DawsonR to activate AMPK, this result suggests that AMPK is and Sams AR, The characterization and incidence of pale, related to the activation of pyruvate kinase. This notion soft, and exudative broiler meat in a commercial processing was further strengthened by the low pyruvate kinase plant. Poult Sci 81:579–584 (2002). 7 Josell A, von Seth G and Tornberg E, Sensory quality and the activity in mice with AMPK knockout. It has been incidence of PSE of pork in relation to crossbreed and RN reported that the activity of pyruvate kinase from PSE phenotype. Meat Sci 65:651–660 (2003). muscles has a five-fold greater affinity for phosphoenol 8 McKenna DR, Roebert DL, Bates PK, Schmidt TB, Hale DS, pyruvate and is roughly ten times more efficient in Griffin DB, Savell JW, Brooks JC, Morgan JB, Mont- catalyzing the reaction than that of pyruvate kinase gomery TH, Belk KE and Smith GC, National Beef Quality 33,34 Audit-2000: survey of targeted cattle and carcass character- from normal meat. It was further shown that the istics related to quality, quantity, and value of fed steers and phosphorylation of pyruvate kinase was responsible for heifers. JAnimSci80:1212–1222 (2002). the increased activity in PSE meat.34 It is possible that 9 Kim J, Solis RS, Arias EB and Cartee GD, Postcontraction insulin sensitivity: relationship with contraction protocol, AMPK leads to the activation of pyruvate kinase via  phosphorylation, which needs further study. glycogen concentration, and 5 AMP-activated protein kinase phosphorylation. J Appl Physiol 96:575–583 (2004). 10 Hardie DG, AMP-activated protein kinase: a key system mediating metabolic responses to exercise. Med Sci Sports CONCLUSIONS Exerc 36:28–34 (2004). AMPK has an important role in the control of 11 Sambandam N and Lopaschuk GD, AMP-activated protein kinase (AMPK) control of fatty acid and glucose metabolism postmortem glycolysis, mainly through maintaining intheischemicheart.Prog Lipid Res 42:238–256 (2003). the activities of glycogen phosphorylase and pyruvate 12 Nielsen JN, Mustard KJ, Graham DA, Yu H, MacDonald CS, kinase, leading to sustained glycogenolysis and Pilegaard H, Goodyear LJ, Hardie DG, Richter EA and  glycolysis and a low ultimate pH value. Pre- Wojtaszewski JF, 5 -AMP-activated protein kinase activity slaughter stress induced by swimming significantly and subunit expression in exercise-trained human skeletal muscle. J Appl Physiol 94:631–641 (2003). accelerated glycogenolysis in postmortem muscle 13 Bergeron R, Russell RR 3rd, Young LH, Ren JM, Marcucci M, through activating glycogen phosphorylase, leading Lee A and Shulman GI, Effect of AMPK activation on to rapid initial glycolysis. The activation of AMPK muscle glucose metabolism in conscious rats. Am J Physiol by AICAr significantly reduced the ultimate pH 276:E938–944 (1999). of postmortem muscle. The activation of AMPK 14 Xing Y, Musi N, Fujii N, Zou L, Luptak I, Hirshman MF, Goodyear LJ and Tian R, Glucose metabolism and energy can not fully account for the initial rapid glycolysis homeostasis in mouse hearts overexpressing dominant induced by stress, and another mechanism, possibly negative alpha2 subunit of AMP-activated protein kinase. an epinephrine-dependent pathway, must exist for the J Biol Chem 278:28 372–28 377 (2003). accelerated initial glycolysis induced by pre-slaughter 15 Mu J, Brozinick JT, Jr, Valladares O, Bucan M and Birn- stress. baum MJ, A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skele- tal muscle. Mol Cell 7:1085–1094 (2001). 16 Davies SP, Carling D and Hardie DG, Tissue distribution of the ACKNOWLEDGEMENTS AMP-activated protein kinase, and lack of activation by cyclic- This work was supported by National Research AMP-dependent protein kinase, studied using a specific and sensitive peptide assay. Eur J Biochem 186:123–128 (1989). Initiative Competitive Grant USDACSRE45101 from 17 Winder WW and Hardie DG, Inactivation of acetyl-CoA the USDA Cooperative State Research, Education, carboxylase and activation of AMP-activated protein kinase in and Extension Service and Faculty-Grant-in-Aid from muscle during exercise. Am J Physiol 270:E299–304 (1996).

J Sci Food Agric 85:2401–2406 (2005) 2405 QW Shen, M Du

18 Young ME, Leighton B and Radda GK, Glycogen phospho- metabolic stress sensing to glycogen. Curr Biol 13:867–871 rylase may be activated by AMP-kinase in skeletal muscle. (2003). Biochem Soc Trans 24:268S (1996). 27 Roach PJ, Glycogen and its metabolism. Curr Mol Med 19 Bucher T and Pfleiderer G, Pyruvate kinase from muscle, in 2:101–120 (2002). Methods in Enzymology, ed by Colowick SP and Kaplan NO. 28 Sampaio-Barros MM, Farias-Silva E, Grassi-Kassisse DM and Academic Press, London, vol. 1, pp. 435–440 (1955). Spadari-Bratfisch RC, Effect of swimming session duration 20 Fernandez X, Neyraud E, Astruc T and Sante V, Effects of and repetition on metabolic markers in rats. Stress 6:127–132 halothane genotype and pre-slaughter treatment on pig meat (2003). quality. Part 1. Post mortem metabolism, meat quality 29 Skalski M, Goto M, Ravindranath T, Myers T and Zeller WP, indicators and sensory traits of m. Longissimus lumborum Meat Omega-3 polyunsaturated fatty acid enriched diet attenuates Sci 62:429–437 (2002). stress-induced lactacidemia in 10-day-old rats. Pediatr Int 21 El Rammouz R, Babile R and Fernandez X, Effect of ultimate 43:409–416 (2001). pH on the physicochemical and biochemical characteristics of 30 Apple JK, Dikeman ME, Minton JE, McMurphy RM, Fedde turkey breast muscle showing normal rate of postmortem pH MR, Leith DE and Unruh JA, Effects of restraint and fall. Poult Sci 83:1750–1757 (2004). isolation stress and epidural blockade on endocrine and 22 Warriss PD, Optimal lairage times and conditions for slaughter blood metabolite status, muscle glycogen metabolism, and pigs: a review. Vet Rec 153:170–176 (2003). incidence of dark-cutting longissimus muscle of sheep. JAnim 23 Marsin AS, Bouzin C, Bertrand L and Hue L, The stimula- Sci 73:2295–2307 (1995). tion of glycolysis by hypoxia in activated monocytes is 31 Luchette FA, Robinson BR, Friend LA, McCarter F, Frame SB mediated by AMP-activated protein kinase and inducible and James JH, Adrenergic antagonists reduce lactic acidosis 6-phosphofructo-2-kinase. J Biol Chem 277:30 778–30 783 in response to hemorrhagic shock. JTrauma46:873–880 (2002). (1999). 24 Carling D and Hardie DG, The substrate and sequence speci- 32 McCarter FD, Evans JA, Luchette FA, Friend LA, James JH, ficity of the AMP-activated protein kinase. Phosphorylation of Davis K and Frame SB, Concurrent reduction of glycogenol- glycogen synthase and phosphorylase kinase. Biochim Biophys ysis, glycolysis, and NA(+)/K(+) pump activity after hemor- Acta 1012:81–86 (1989). rhagic shock. Curr Surg 57:639–639 (2000). 25 Aschenbach WG, Hirshman MF, Fujii N, Sakamoto K, Howlett 33 Schwagele F, Lopez P, Haschke C and Honikel KO, Rapid pH KF and Goodyear LJ, Effect of AICAR treatment on glycogen drop in PSE-muscles: Enzymological investigations into the metabolism in skeletal muscle. Diabetes 51:567–573 (2002). causes. Fleischwirtschaft 74:95–99 (1994). 26 Polekhina G, Gupta A, Michell BJ, van Denderen B, Murthy S, 34 Schwagele F, Haschke C, Krauss G and Honikel KO, Compar- Feil SC, Jennings IG, Campbell DJ, Witters LA, Parker MW, ative studies of pyruvate kinase from PSE and normal pig Kemp BE and Stapleton D, AMPK beta subunit targets muscles. Z Lebensm Unters Forsch 203:14–20 (1996).

2406 J Sci Food Agric 85:2401–2406 (2005)