Adenosine 5'-monophosphate-activated protein kinase (AMPK), composed of three different subunits (α, β, and γ), is considered as a key regulator of energy metabolism [1] and is activated in response to the increased ratio of adenosine monophosphate (AMP) to adenosine triphosphate (ATP) [2]. Previous studies indicated that AMPK plays a crucial role in glycolysis of postmortem skeletal muscle [3,4]. After slaughter, the circulation oxygen supply was reduced, followed by the main metabolism of skeletal muscle from aerobic to anaerobic. In this case, the glycogen stored in postmortem muscle was transferred into lactic acid (LD), resulting in an accumulation of LD in muscle and a sharp decline in the final pH of meat at postmortem 24 h [5]. Additionally, accumulated evidences revealed that pre-slaughter stress is one of the two main factors leading to the occurrence of pale, soft, exudative (PSE) meat [6,7]. Pre-slaughter stress accelerated glycolysis metabolism and upregulated the activities of hexokinase (HK), pyruvate kinase (PK) and lactate dehydrogenase (LDH), which are involved in glycolysis of postmortem muscle [5,8]. Moreover, it was reported that pre-slaughter handling might be associated with glycolysis in postmortem muscle mediated by protein acetylation [9].

Guanidinoacetic acid (GAA), acting as a natural precursor of creatine (Cr), can generate ATP through the Cr/phosphocreatine (PCr) system [10]. GAA is mainly synthesized in the kidney and transported into the liver to yield Cr, followed by the release of Cr into the circulation and the transport of Cr into the target tissue [11]. Moreover, it has been demonstrated that GAA can be more effective than Cr at increasing Cr stores in tissues [12,13]. GAA administration can also amplify the availability of Cr in skeletal muscle through enhancing Cr biosynthesis [13]. In addition, it was reported that the concentrations of GAA and Cr in serum were enhanced after a six-week oral GAA administration [14]. However, previous studies in broilers suggested that the concentrations of Cr and PCr dramatically decreased due to acute and chronic physiological stimuli, such as heat stress and transport stress [15,16]. It has been documented that GAA administration contributed to enhancing the Cr/PCr content in muscle [12,13] and attenuating the pre-slaughter transport-induced muscle energy expenditure and rapid glycolysis of muscle [17]. Therefore, under the condition of stress, it may be an effective measure to elevate muscle energy stores in order to compensate for stress-induced high energy consumption and alleviate the glycolysis of postmortem muscle to decrease the occurrence of PSE meat. However, to the best of our knowledge, the regulation of GAA on energy metabolism of postmortem muscle subjected to pre-slaughter stress is still unclear. Therefore, we hypothesized that GAA could regulate postmortem glycolysis of antemortem-stress muscle associated with AMPK signaling and protein acetylation.

In the present study, a model using mice was conducted with GAA administration combined with/without some activator or acetyltransferase was used to investigate the effect of GAA administration on postmortem energy status and the glycolysis in antemortem-stressed muscle, and further reveal the mechanism of GAA attenuating postmortem meat glycolysis through AMPK signaling pathway and protein acetylation, which could provide theoretical basis for nutrition regulation of energy metabolism in postmortem muscle of livestock animals subjected to pre-slaughter stress and contribute to providing guarantees for high-quality meat production.

Following exsanguination, the oxygen supply via blood circulation was cut off and the tissue metabolism was mainly transferred from an aerobic into an anaerobic condition. The drop of postmortem muscle pH could be ascribed to the accumulation of hydrogen ions (H⁺). The rate of postmortem pH decline is controlled by the rate of ATP hydrolysis catalyzed by muscle ATPs. The depletion of ATP yields H⁺ and results in a progressive fall in pH to the ultimate value of postmortem meat [22]. Nevertheless, it has been demonstrated that Cr monohydrate or GAA administration could enhance the levels of PCr in muscle [2,5], which could transfer a phosphate group from PCr to ADP to generate ATP, thus preventing a depletion of ATP levels [23] and further alleviating the rate of pH decline. In the present study, the values of pH_45min and pH_24h in the oral GAA administration group were higher than those in the control group. Similar to this, in our previous studies in broilers, GAA supplementation increased the value of pH_24h in broiler muscle compared with those subjected to a 3 h pre-slaughter transport stress [2,15]. In comparison with the GAA group, AICAR injection decreased muscle pH value, but no differences were observed between the GAA group and the GAA+AICAR+HAT II group. Similarly, Li et al [7] reported that AICAR injection led to a decrease in muscle pH, however, there were no differences between AICAR+HAT II treatment and the control group. Shen et al [20] also suggested that AICAR treatment induced a significant drop in muscle pH_24h in comparison with the control group. These results mentioned above indicate that GAA administration could contribute to alleviating the rate of muscle pH decline.

After slaughter, muscle attempts to maintain cellular ATP levels within homeostatic set points, therefore additional metabolite catabolism is necessary. Pre-slaughter stress accelerated glycolysis metabolism to generate ATP for the increased muscle energy demands in response to the limited oxygen supply in anaerobic glycolysis and then became the predominant energy source for muscle ATP supply [2]. The activities of HK, PK and PFK which are key enzymes involved in glycolysis in postmortem muscle, were decreased in the GAA group, implying that the rate of glycolysis reaction was delayed owing to GAA administration. But compared with the GAA group, AICAR treatment significantly increased the activities of PK, HK and PFK in muscle. It is well-known that AICAR is an activator of AMPK. Accordingly, a previous study suggested that AICAR treatment resulted in an increase in PK activity and AMPK activity, however, PK activity was lowered by AMPK knockout [20]. These results suggest that AMPK might be involved in the muscle energy metabolism of mice subjected to pre-slaughter stress.

ATP, a high-energy compound in muscle, performs vital cellular functions, such as muscle contraction, cell signaling and biosynthesis of macromolecules [24]. The phosphagen system can be immediately activated in an attempt to maintain ATP stable. During early postmortem metabolism, muscle ATP concentration remains stable through the utilization of a high-energy phosphate compound known as PCr [25]. It has been demonstrated that dietary GAA supplementation could increase the content of PCr in postmortem muscle, which donates its phosphate group to ADP to form ATP [26]. Accordingly, in our present study, an increased ATP content and a decreased AMP content as well as a reduced ratio of AMP to ATP were observed in response to GAA administration when compared with the control group. Similar results were also observed in our previous studies, in which broilers supplemented with GAA exhibited higher ATP content in postmortem muscle [2,15], indicating that GAA could elevate energy store and contribute to delay the rate of ATP depletion. No differences were observed in ATP, ADP and the ratio of AMP to ATP in mice treated with HAT II injection. Similarly, Yan et al [27] demonstrated that the ATP content in the longissimus lumborum muscle of pigs receiving curcumin (an inhibitor of acetyltransferase) treatment was not different from those in the control group. But the AMP/ATP ratio was increased by HAT II injection. Moreover, in comparison with those only receiving GAA administration, AICAR injection increased ATP consumption, evidenced by a higher ratio of AMP to ATP. Additionally, it was well clarified that AMPK can be switched on in response to the increase in AMP/ATP ratio [3], implying that AICAR and HAT II treatment might affect the energy metabolism via the AMPK signaling pathway.

The LKB1 and AMPK proteins participate in an energy sensing cascade that responds to depletion of ATP, and the LKB1/AMPK pathway has been considered as primarily an energy sensing pathway engaged by cells in response to low energy levels [28]. AMPK consists of three subunits (α, β and γ), among which, the α subunit comprises α1 and α2. Moreover, AMPKα2 but not AMPKα1 KO abolished the activity of AMPK in postmortem muscle [1], indicating that phosphorylation of the α2 subunit was more AMP-dependent [29,30]. In accordance with this, our results revealed that GAA treatment downregulated protein abundances of LKB1 and AMPKα2, whereas, AMPKα1 protein level was not affected by GAA administration, suggesting that GAA regulated muscle energy metabolism under stress through the LKB1/AMPK signaling pathway. It has been demonstrated that AMPK, as a cellular energy sensor triggered by falling energy status, can be activated by an increase in AMP/ATP ratio [31,32]. Accordingly, compared with the control group, GAA administration diminished the AMP/ATP ratio and further induced the down-regulation of AMPK signaling in our present study, evidenced by the reduced protein abundance of AMPKα2. The information mentioned above might provide that GAA could inhibit AMPK signaling through the down-regulation of AMP/ATP ratio. In a previous study, it was demonstrated that 50 μg/kg AICAR could activate AMPK signaling pathway and elevate the protein expression of AMPK [7]. Compared with GAA administration, AICAR injection upregulated protein expressions of LKB1 and AMPKα2, but the protein abundance of AMPKα1 was not affected, implying that AMPKα2 could be more sensitive to ATP content than AMPKα1 even under activation by AICAR [33]. Moreover, it was demonstrated that AMPK is a major regulator of postmortem glycolysis and leads to a reduction in the value of pH_24h postmortem muscle [1]. In consistent with this, our results found that AICAR injection lowered the value of pH_24h in muscle compared with GAA administration group, indicating that the activation of AMPK might accelerate muscle glycolysis and ultimately result in a lower pH_24h value. However, the protein expression of LKB1 and AMPKα2 was lower in AICAR+HAT II group that those in the AICAR group, indicating that the activation/inhibition effect of AICAR/HAT II on the regulation of GAA in postmortem muscle glycolysis might be associated with the LKB1/AMPK signaling pathway. Winder et al [34] found that AICAR injection increased AMPK activity in skeletal muscle. Furthermore, it was reported that AICAR treatment increased the activity of AMPKα2 and elevated AMPKα2 phosphorylation level in skeletal muscle [29]. The result of a previous study demonstrated that HAT II treatment could reduce ATP depletion and glycogen breakdown in trapezius, psoas major and semitendinosus muscles before slaughter, and further slowed down LD accumulation and pH decline, which could be due to reduced AMPK activation [7,27]. Indeed, in our present study, HAT II treatment reduced the protein abundance of LKB1 and AMPKα2 compared with those receiving only AICAR injection. Taken together, GAA administration could regulate energy metabolism of postmortem muscle via the AMPK signaling pathway and contribute to alleviating the glycolysis of postmortem muscle.

Accumulated evidence has revealed that protein acetylation was closely associated with the glycolysis of postmortem muscle [35–37] and participated in the regulation of antemortem stress on glycolysis in postmortem muscle [19,38]. Besides, it also has been demonstrated that protein acetylation is related to the regulation of AMPK signaling in the glycolysis of skeletal muscle [7]. In our present study, GAA administration downregulated the protein acetylation level in skeletal muscle of mice. Although direct evidence revealing the regulation of GAA on muscle protein acetylation is not available, it has been demonstrated that the activation of AMPK was involved in the regulation of protein acetylation [39]. Furthermore, there is convincing evidence that phosphorylating AMPK on Thr172 inhibits acetyl-CoA carboxylase and thus elevates acetyl-CoA level (a substrate for HAT enzymes) and the activity of lysine acetyltransferases (known as KATs, and previously termed histone acetyltransferases) [40,41]. Moreover, it has been demonstrated that the activation of AMPK increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation [42]. Therefore, we could speculate that GAA administration decreases phosphorylation level of AMPKα2, furtherly reduces the level of acetyl-CoA and downregulates total protein acetylation. Convincing evidence demonstrated that AICAR, the activator of AMPK molecular, increased protein level of AMPK [7,34]. Further study revealed that AICAR treatment elevated the level of protein acetylation [7]. In accordance with this, the increased protein acetylation was induced by AICAR injection in our study compared with the GAA group. However, HAT II administration diminished the increased expression of protein acetylation induced by AICAR. Consistent with this, it was reported that 185 μg/kg HAT II treatment was identified to reduce the protein acetylation level in mice muscle subjected to antemortem-stress [7]. Li et al [19] also suggested that pre-slaughter injection of HAT II lowered the abundance of protein acetylation. Moreover, compared with the control group, the protein acetylation expression in the three groups administered with antemortem GAA oral administration was lower. The information mentioned above indicated that GAA could contribute to alleviating postmortem glycolysis via inhibiting protein acetylation.

Antemortem stress increased energy expenditure, accelerated glycolysis metabolism and ultimately led to a lower ultimate pH in postmortem muscle. However, GAA administration could elevate the level of high energy compounds and reduce the ratio of AMP to ATP, further downregulate the protein abundance of AMPK signaling and protein acetylation involving in energy metabolism of postmortem muscle, and ultimately contribute to attenuating the postmortem glycolysis of antemortem-stress muscle. In conclusion, GAA could contribute to alleviating the glycolysis of antemortem-stress muscle through AMPK signaling and protein acetylation.