Study of the AMP-activated protein kinase role in energy metabolism changes during the postmortem aging of yak longissimus lumborum

To explore the postmortem physiological mechanism of muscle, activity of adenosine monophosphate activated protein kinase (AMPK) as well as its role in energy metabolism of postmortem yaks were studied. In this experiment, we injected 5-amino-1-beta-d-furanonyl imidazole-4-formamide (AICAR), a specific activator of AMPK, and the specific AMPK inhibitor STO-609, to observe the changes in glycolysis, energy metabolism, AMPK activity and AMPK gene expression (PRKA1 and PRKA2) in postmortem yaks during maturation. The results showed that AICAR could increase the expression of the PRKKA1 and PRKAA2 genes, activate AMPK and increase its activity. The effects of AICAR include a lower concentration of ATP, an increase in AMP production, an acceleration of glycolysis, an increase in the lactic acid concentration, and a decrease in the pH value. In contrast, STO-609 had the opposite effect. Under hypoxic adaptation, the activity of the meat AMPK increased, which accelerated glycolysis and metabolism, and more effectively regulated energy production.


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Yaks adapted to high altitudes because of the colder climate. This process included metabolism is strengthened; ATP consumption is increased; the ATP concentration is 44 decreased; the AMP production is increased, and a high concentration of 5'-AMP and 45 AMPK gamma subunits interact to activate AMPK. Ding et al. (2014) studied the 46 activity of lactate dehydrogenase (LDH) in yaks at three different altitudes, and its 47 activity positively correlated with altitude. LDH is the key enzyme for anaerobic 48 glycolysis, indicating that yaks at higher altitudes are more dependent on energy 49 metabolism (Chen, 2003;Wojtaszewski, 2000;Musi, 2001;Park, 2002) 50 Under hypoxic conditions, the body is under stress; metabolism is strengthened; 51 ATP consumption is increased; the ATP concentration is decreased; AMP production 52 is increased, and a high concentration of 5'-AMP and AMPK gamma subunits interact 53 to activate AMPK (Park et al., 2002). Research on the activity of lactate dehydrogenase 54 (LDH) in yaks at three different altitudes indicated that the enzyme is positively 55 correlated with altitude. LDH is the key enzyme for anaerobic glycolysis, indicating 56 that yaks at higher altitudes are more dependent on the metabolism of energy (Ding et 57 al., 2014). Thus, it is necessarily to additionally study changes of energy metabolism 58 and AMPK activity in a hypoxic environment. 59 The enzyme AMPK is a heterotrimer consisting of α, β, and γ subunits. Its primary 60 role is thought to be the critical regulation of energy metabolism (Hardie, 2004;Carling,  Any type of cellular stress can cause AMPK activation. Physiological AMP/ADP 68 elevation occurs as a result of stress, such as low nutrients or prolonged exercise. As 69 previous studies have found, the initiation of glycolysis in ischemic heart AMPK 70 activation plays an important role (Sambandam & Lopaschuk, 2003). Thus, the data 71 accumulated confirms that hypoxia is a characteristic of post-mortem skeletal muscle 72 and ischemic heart disease. Therefore, post-mortem glycolysis may be regulated by 73 AMPK.

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Previous research demonstrated the activation of AMPK in pork loins, which 75 develop into PSE meat. This finding suggests that a key role of AMPK in regulation of 76 postmortem glycolysis (Shen, Means, Underwood, et al., 2006). Therefore, the role of 77 AMPK may be the regulation of glycolysis in post-mortem skeletal muscle. If so, the 78 enzyme may be a logical target to manipulate to intervene in the process of PSE 79 development and cause its reduction, since AMPK activity depends on the postmortem 80 skeletal muscle pH values. Therefore, we further studied AMPK role in muscle 81 glycolysis regulation in post-mortem meat, using specific AMPK activators and 82 inhibitors to detect whether the induction of AMPK by 5-amino-1-β-D-ribofuranosyl- accessed using different methods. These methods included a muscle preparation that 90 had been isolated and incubated, a hindquarter preparation that had been perfused, or 91 tissues analyses following a euglycemic clamp or treatment. To our knowledge, the 5 92 effect of reactive AICAR on the AMP-activated protein kinase of mice longissimus 93 lumborum has only been examined in one study. Among other studies, we found that 94 injecting a dose of 250 mg/kg AICAR had no effect on the glycogen binding in the 95 diaphragm (respiratory muscle) of mice fed or fasting (Vincent et al., 1996). At the 96 same time, the effects on glucose transport in yak skeletal muscle due to AICAR 97 treatment remain unclear. In comparison, the inhibitory effect of STO-609, an AMPK 98 inhibitor, was used to inhibit food intake and therefore weight gain in mammals (Hayes  The M. longissimus lumborum is the LL, the 12 th rib that is anterior to the last lumbar 109 vertebrae, and they were randomly extracted from a slaughterhouse (Yushu Tibetan    (w/v) SDS, and 0.01% bromophenol blue (boiled for 5 min prior to electrophoresis).

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The gels were cast using a BioRad mini-gel system (Richmond, CA, USA) that 170 was also used to perform the SDS-PAGE electrophoresis. Gradient gels of 5%-20% 171 were used to separate the proteins. When the electrophoresis was complete, proteins 172 separated on the gels were moved to nitrocellulose membranes using buffer that   However, at 12 h postmortem and through the remaining sampling times, the muscle 218 pH of the control yak was higher (P < 0.05) than that of the AICAR-treated yak but 219 lower than STO-609-treated yak. At 24 h postmortem, the pH of the AICAR-injected 220 yak muscle remained less than 6.0, showing that the glycolytic rate was strongly 221 activated ( Fig. 1).

Lactic acid concentration 223
Increased glycolysis in skeletal muscle of yak injected with AICAR was 224 confirmed by increased lactic acid accumulation rate (Fig. 2). In addition to lowering  The nucleotide concentration in the yak LL muscle was measured in this study.

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After the 0 h control, there were no significant differences the in nucleotide 240 concentration observed between the control and treatment groups ( inhibited glycolysis in the postmortem muscle (Figures 2, 3). As a result of this 245 inhibition, less ATP was produced, and the ATP concentration increased within 12 246 hours following death ( Table 2). The IMP in the muscles of the yaks was also 247 significantly higher than the AMP following the slaughter of the animals, and these  (Figures 1, 2), resulting in lower amounts of ATP production and therefore, a decrease 257 in ATP concentration 12 h (Table 2). In addition, the data showed that the levels of IMP      (Figures 1, 2). In  However, this study did not enable us to identify the transcription factors that were 434 responsible for regulation increase in the gene expression of AMPK induced by AICAR.

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Therefore, addition research to identify the transcription factors and cis-elements that 436 are involved in the response to AICAR is merited. to AICAR activation and STO-609 inhibition. This suggests that the increased 443 expression of the PRKAA1 and PRKAA2 genes will increase the activity of AMPK.

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After AMPK is activated, the direct phosphorylation glycolysis pathway increases the 445 glycolysis activity, which promotes the glycolysis process and produces a large amount