Deactivation of AMPK α / GSK-3 β Leads to High-Level Glycogen Synthase in Poor Pork Meat Quality

We investigated glycogen synthase and upstream regulatory proteins determining meat quality in porcine longissimus dorsi at 24 h post-mortem. The general meat quality traits of 300 muscle samples were estimated. Muscle samples were classified into two groups based on ultimate pH of meat 24 h post-mortem (pH24h). Muscle glycogen synthase belonging to the low pH24h group showed remarkably higher expression than that in the high pH24h group. AMP activated protein kinase (AMPK) and glycogen synthase kinase 3 (GSK-3) as negative regulators of glycogen synthase deactivated the suppression of glycogen synthase by phosphorylating Ser485 of AMPK and Ser9 of GSK-3β. These inhibitory kinases lead to high glycogen synthase expression. These results suggest that the accumulation of glycogen by up-regulating glycogen synthase and inhibiting AMPKα and GSK-3β was rapidly converted to lactate resulting in acidic meat. This molecular clue representing acidic meat based on post-mortem muscular pH can be used to estimate meat quality via Akt-AMPKα/GSK-3β-mediated up-regulation of glycogen synthase.


Introduction
Porcine meat quality is determined by various intrinsic factors such as meat color, water holding capacity (WHC), hardness, and fat content of muscle tissue (Rosenvold et al., 2001;Hocquette et al., 2005).Pale, soft, and exudative (PSE), which are poor meat qualities, are very problematic in the pork industry (Barbut et al., 2008).Severe PSE quality occurs during the early post-mortem stage and is triggered by a rapid pH fall and high temperature in the muscle.The pale color and WHC of meat are influenced by a number of factors including low ultimate pH and protein denaturation (Briskey & Wismer-Pedersen, 1961;Warner 1997;Bowker et al., 2000;Wang et al., 2009).Thus, post-mortem muscular pH is a critical factor determining meat quality.In general, the final pH of meat is dependent on muscular glycogen content after slaughter.muscle tissue (Bowker et al., 2000;Pösö & Puolanne, 2005).The accumulation of lactic acid leads to a rapid decline in muscular pH known as acidic meat (referred to as poor meat quality) and vice versa during the post-mortem period, particularly during the early phase (Ryu et al., 2005).Thus, post-mortem pH value has a considerable effect on meat quality.In particular, the ultimate pH of pig muscle at 24 h post-slaughter (pH 24h ) is a critical factor determining meat quality traits.In addition, pH 24h is closely related to WHC (Warner et al., 1997;Kang et al., 2010;Nam et al., 2012).
The relationship between glycogen and meat quality has been widely studied.For example, pork muscle with low glycogen and high lactate levels has a low muscular pH at 45 minute post-mortem (Choe et al., 2008).A rapid pH fall caused by accelerated glycogenolysis and ATP breakdown produces the PSE defect (Pryzbylski et al., 2006).Nevertheless, why glycogen synthases (GSs) are differentially expressed in low and high pH muscle, which refer to low and good quality meat, respectively, has not been addressed.
In this study, we evaluated pork meat quality by elucidating the molecular mechanism occurring during post-mortem metabolism.We examined the expression levels of GS and upstream regulatory proteins involved in post-mortem muscular pH affecting glycogen.In several studies, AMP-activated protein kinase (AMPK) has been shown to regulate muscular glycogen synthesis (Jørgensen et al., 2004).By quantitative immunoblot analysis, the expression levels of GS and regulatory proteins such as phosphorylated AMPK at Ser485 (inhibitor of GS) and Thr172 (activator of GS) and the AMPK-upstream regulator Akt were compared between low and high pH 24h groups.Taken together, this molecular study of post-mortem muscular pH provides possible indicators of pork meat quality traits.

Animals and Meat Samples
A total of 300 Berkshire pigs were bred under the same conditions (Da-San-Genetics Co. Ltd., Namwon, Korea) and then slaughtered in 10 batches according to standard slaughtering procedures, when their body weight reached 80-90 kg as described previously (Kang et al., 2010).Subsequently, the samples were used for meat quality trait evaluation analyses.

Meat Quality Evaluation
Pork meat quality such as carcass weight, backfat thickness, pH 24h , meat color, WHC, chemical composition (collagen, fat, and protein), drip loss, cooking loss, and Warner-Bratzler shear force were evaluated.Samples of all Berkshire pigs (n = 300) including 133 castrations, 165 boars, and 2 sows, were divided into two groups such as the low pH value group (LpH 24 ; the lower 5% pH 24h of the 300 samples, n = 15) and the high pH value group (HpH 24h ; the upper 5% pH 24h , n = 15).

Glycogen and Lactate Contents Measurement
Muscular glycogen content was measured according to Choe et al. (2008).Ten samples were randomly chosen from the LpH 24 and HpH 24 groups.In brief, approximately 2 g of muscle tissue was minced, suspended in 10 mL of 9% (v/v) cold perchloric acid, and thoroughly homogenized.After centrifugation (15,000 × g at 4°C for 20 min), the supernatant was used for glycogen determination.Iodine color reagent was added to a glycogen standard.Linear regression equations of the glycogen standard for each set of samples were applied to determine glycogen concentration in the corresponding samples.Lactate content was determined spectrophotometrically (absorbance at 340 nm) using a commercial kit (Boeringer, Mannheim, Germany) as described previously (Choe et al., 2008) with minor modifications.Briefly, approximately 5 g of muscle sample was homogenized in 20 mL perchloric acid (1 M), and potassium hydroxide was added to neutralize the solution.The final volume was brought up to 10 mL with distilled water.Lactate concentration was measured after 20 min of refrigeration and centrifugation.

Muscle Protein Extraction and Quantification
Muscle tissues (30 mg) were homogenized in lysis buffer purchased from iNtRON (Daejeon, Korea) according to the manufacturer's protocol.Protein concentration was quantified in triplicate using the Coomassie Dye Binding Assay (Bio-Rad, Hercules, CA, USA) employing BSA as the standard (Bradford, 1976).

Meat quality Evaluation in Berkshire Muscle
Post-mortem pH values were measured from 300 Berkshire longissimus dorsi 24 hours after slaughter.The lowest 5% pH values (LpH 24 ) and highest 5% pH values (HpH 24 ) were selected based on the 24 hour post-mortem values.The mean values for the meat quality traits of the 300 Berkshires are shown in Table 1.Berkshire meat generally exhibits a high post-mortem pH value and WHC compared with those of other breeds, whereas drip and cooking losses are lower than those of other breeds (Suzuki et al., 2003;Lee et al., 2011).Our previous study indicated that porcine serum metabolites are clearly different between low and high pH groups based on the pH 24h value of the post-mortem samples by 1 H nuclear magnetic resonance and high performance liquid chromatography analyses (Kim et al., 2011).Taken together with these reports, the present results indicate that pH 24h is an important factor for evaluating meat quality.We chose individual meat samples separately based on the pH 24h corresponding to the lower 5% (n = 15, LpH 24h ) and the higher 5% group (n = 15, HpH 24h ).The average pH 24h values for the low and high pH groups were 5.52 ± 0.02 and 6.16 ± 0.09, respectively (P < 0.01) (Figure 1 and Table 2).The pH 24h value of LpH 24h ranged from 5.48-5.54,whereas that for the HpH 24h group was 6.07-6.31.Besides, middle pH 24h group (n = 15) shown 5.73 ± 0.0 which are ranged from 5.73-5.74.2005).WHC was higher as a percentage in the HpH 24h group than that in the LpH 24h group (P < 0.05), whereas drip loss (%) and cooking loss (%) were vice versa with a significant difference (P < 0.01) between the two groups.

Quantitative Protein Comparison
Berkshire breeds have excellent meat quality (Suzuki et al., 2003;Lee et al., 2011).The differences in the genetic effects between individuals such as single nucleotide polymorphisms have broad effects on meat quality traits (Dalvit et al., 2007;Gao et al., 2007;Williams, 2008).Proteome strategies have been attempted to understand the relationship between protein profiles and meat quality traits (Lametsch et al., 2003;Hwang et al., 2005;Kwaskborski et al., 2008;Te Pas et al., 2009).However, potential meat quality protein markers are not as well considered for evaluating as those of other meat quality traits such as genetic variations.Berkshire longissimus dorsi muscle has been used to quantitatively compare proteins between low and high pH 24h , in which the dominant expressions of Igc, Prep, Ldhb, Aco2 were identified in the LpH 24h group by shotgun proteome analysis (Nam et al., 2012).

Effect of GS on Meat Quality in Berkshire Muscle
Post-mortem glycogenolysis has a significant effect on meat quality (Przybylisk et al., 2006).Muscle glycogen is one of the most important energy sources for muscle contraction.Glycogen degradation during the post-mortem period has a considerable effect on meat quality.The levels of glycogen and ATP decrease when oxygen is no longer available such as at slaughter and then muscle glycogen degradation during post-mortem produces lactic acid that accumulates and lowers muscle pH (Kang et al., 2010;Przybylisk et al., 2006).The analysis of enzymes related to glycogen synthesis and degradation during the post-mortem period can be a useful way to discover protein biomarkers for evaluating meat quality.Glycogenolysis, the breakdown of glycogen to glucose-1-phosphate and glucose, is initiated by glycogen phosphorylase, and then the phosphate group is shifted to glucose-6-phosphate by phosphoglucomutase.The rate of glycogenolysis, phosphorylase activity, and lactate production rapidly increases in the perfused rat heart under anoxia, which is a surrogate to the post-mortem condition (Cornblath et al., 1963).In our previous shotgun proteomics report, lactate dehydrogenase B chain, which catalyzes the interconversion of pyruvate to lactate, was highly expressed in the low pH meat quality group compared to that in the high pH group (Nam et al., 2012).In summary, we suggest that muscle glucose was converted to glycogen by GS, followed by increased lactate concentration through glycogenolysis and glycolysis, which leads to a drop in pH at 24 h post-mortem (Figure 4).

Conclusions
Although several pork meat quality traits analyses have been conducted to analyze the metabolic rate and general proteome profile under different meat conditions, no study has investigated how they regulate GS expression under different meat conditions related to good and poor meat quality.Thus, understanding the biochemical perspective is an important issue to facilitate the signal transduction cascade analysis in different meat conditions to evaluate meat quality traits.Highly expressed GS in the low pH 24h group suggests that accumulated glycogen produced more lactic acid leading to acidic meat.AMPK and GSK-3 are negative regulators of GS.However, phospho-Akt (Ser473) phosphorylated GSK-3β (Ser9) which acts as an Akt substrate and a regulatory site.This phosphorylation inactivated the kinase leading to decreased phosphorylation and activation of GS.Furthermore, phosphorylation of AMPKα at Ser485 and inhibition of AMPKα by activated Akt deactivated the suppression of GS and increased GS expression.Thus, our results suggest that glycogen synthesis occurs in a complex manner after slaughter, where the Akt-AMPKα/GSK-3β complex plays a pivotal role under different meat conditions.Although this study is an initial biochemical investigation into meat quality during the post-mortem period, we believe that this kind of study provides new insight into the biochemical mechanism and provides biochemical protein markers for meat quality evaluations, particularly for the porcine industry.

Table 1 .
Characters of meat quality traits in Berkshire longissimus dorsi (n = 300) ** CIE L, a, and b represent meat color lightness, redness, and yellowness, respectively.

Table 2 .
Characters of meat quality traits in Berkshire longissimus dorsi groups based on pH value at 24 hour post-mortem corresponding to low pH 24h (n = 15) and high pH 24h (n = 15) groups * The pH value 24 h after slaughter.** CIE L, a, and b represent meat color lightness, redness, and yellowness, respectively.a, P < 0.05; b, P < 0.01.Lightness (L) and yellowness (b) of the meat samples were significantly lower in HpH 24h group than those in the LpH 24h group.But, redness (a) was not significantly different between the two groups.WHC of meat is one of the most important properties as it affects the quality of the end meat product (Huff-Lonergan and Lonergan,