Antemortem Stress Regulates Postmortem Glycolysis in Muscle by Deacetylation of Pyruvate Kinase M1 at K141.

It is well known that preslaughter (antemortem) stress such as rough handling, transportation, a negative environment, physical discomfort, lack of consistent routine, and bad feed quality has a big impact on meat quality. The antemortem-induced poor meat quality is characterized by low pH, a pale and exudative appearance, and a soft texture. Previous studies indicate that antemortem stress plays a key role in regulating protein acetylation and glycolysis in postmortem (PM) muscle. However, the underlying molecular and biochemical mechanism is not clearly understood yet. In this study, we investigated the relationship between antemortem and protein acetylation and glycolysis using murine longissimus dorsi muscle isolated from ICR mice and murine muscle cell line C2C12 treated with epinephrine hydrochloride. Because adrenaline secretion increases in stressed animals, epinephrine hydrochloride was intraperitoneally injected epinephrine into mice to simulate pre-slaughter stress in this study to facilitate experimental operations and save experimental costs. Our findings demonstrated that protein acetylation in pyruvate kinase M1 (PKM1) form is significantly reduced by antemortem, and the reduced acetylation subsequently leads to an increase in PKM1 enzymatic activity which causes increased glycolysis in PM muscle. By using molecular approaches, we identified lysine 141 in PKM1 as a critical residue for acetylation. Our results in this study provide useful insight for controlling or improving meat quality in the future.

Dietary supplementation of Eucommia leaf extract to growing-finishing pigs alters muscle metabolism and improves meat quality.

OBJECTIVE: The objective of this study was to investigate the influence of dietary supplementation of Eucommia ulmoides leaf extract (ELE) on muscle metabolism and meat quality of pigs with and without pre-slaughter transportation. METHODS: In a 43-day feeding experiment, a total of 160 pigs with an initial body weight 60.00±2.00 kg were randomly assigned into four groups in a completely randomized design with 10 replicates. Pigs in groups A and C were fed a basal diet and pigs in groups B and D were fed a basal diet supplemented with 0.5% ELE. Pigs were slaughtered with (group B and D) or without (group A and C) pre-slaughter transport. Muscle chemical composition, postmortem glycolysis, meat quality and muscle metabolome were analyzed. RESULTS: Dietary ELE supplementation had no effect on the proximate composition of porcine muscle, but increased free phenylalanine, proline, citruline, norvaline, and the total free amino acids in muscle. In addition, dietary ELE increased decanoic acid and eicosapentaenoic acid, but decreased heptadecanoic acid, oleic acid, trans-oleic acid, and monounsaturated fatty acids in muscle. Meat quality measurement demonstrated that ELE improved meat water holding capacity and eliminated the negative effects of pre-slaughter transport on meat cooking yield and tenderness. Dietary ELE reduced muscle glycolytic potential, inhibited glycolysis and muscle pH decline in the postmortem conversion of muscle to meat and increased the activity of citrate synthase in muscle. Metabolomics analysis by liquid chromatographic tandem mass spectrometric showed that ELE enhanced muscle energy level, regulated AMP-activated protein kinase (AMPK) signaling, modulated glycogenolysis/glycolysis, and altered the metabolism of carbohydrate, fatty acids, ketone bodies, amino acids, purine, and pyrimidine. CONCLUSION: Dietary ELE improved meat quality and alleviated the negative effect of preslaughter transport on meat quality by enhancing muscle oxidative metabolism capacity and inhibiting glycolysis in postmortem muscle, which is probably involved its regulation of AMPK.

Low-protein diets supplemented with glycine improves pig growth performance and meat quality: An untargeted metabolomic analysis.

For the purpose to improve meat quality, pigs were fed a normal diet (ND), a low protein diet (LPD) and a LPD supplemented with glycine (LPDG). Chemical and metabolomic analyses showed that LPD increased IMF deposition and the activities of GPa and PK, but decreased glycogen content, the activities of CS and CcO, and the abundance of acetyl-CoA, tyrosine and its metabolites in muscle. LPDG promoted muscle fiber transition from type II to type I, increased the synthesis of multiple nonessential amino acids, and pantothenic acid in muscle, which should contributed to the improved meat quality and growth rate. This study provides some new insight into the mechanism of diet induced alteration of animal growth performance and meat quality. In addition, the study shows that dietary supplementation of glycine to LPD could be used to improved meat quality without impairment of animal growth.

Duck breast muscle proteins, free fatty acids and volatile compounds as affected by curing methods.

The aim of this study was to investigate the effects of different curing methods on protein structure, protein and lipid oxidation, lypolysis and volatile compounds in duck breast meat. The results showed that compared to static brining and pulsed pressure salting, the vacuum tumbling curing significantly decreased the oxidation of proteins and lipids, and the surface hydrophobicity of proteins, increased α-helix structure but decreased the proportion of ß-sheet, and increased actomyosin dissociation, liplysis and the free fatty acid content in meat. Meanwhile, vacuum tumbling curing decreased the amount of volatile flavor compounds, hexanal, 2,3-octanone, and off-flavor compounds 1-octen-3-ol and 1-hexanol. This study suggests that concerns on healthiness and the sensory quality of processed meat products should be paid in the selection of curing methods and vacuum tumbling curing is superior in terms of both aspects.

Fatty Acid Content, Flavor Compounds, and Sensory Quality of Pork Loin as Affected by Dietary Supplementation with l-arginine and Glutamic Acid.

To investigate the effect of functional amino acid on meat flavor and eating quality, 60 growing-finishing pigs (Duroc × Large White × Landrace) were dietarily supplemented with or without 1.0% l-arginine, glutamic acid, or l-arginine plus glutamic acid for 2 months. After animals were slaughtered, the muscle fatty acid profile, flavor compounds, and meat sensory quality were comparatively investigated. The results showed that dietary supplementation with arginine, glutamic acid, or arginine plus glutamic acid had little effect on free amino acids, no effect on 5'-nucleotides and meat sensory taste traits, but supplementation with arginine plus glutamic acid significantly increased (P < 0.05) fat accumulation and fatty acid content in muscle, increased (P < 0.05) the formation of multiple fatty acid oxidation-derived volatile compounds, and improved the tenderness, juiciness, and overall eating quality of meat. This study revealed that dietary supplementation with 1.0% l-arginine and glutamic acid could be used to improve meat eating quality in pork production.

Acetylome profiling reveals extensive involvement of lysine acetylation in the conversion of muscle to meat.

Protein lysine acetylation is an post-translational modification that regulates gene expression, metabolism, cell signaling, and diseases, but its implication in the postmortem (PM) meat quality development is basically unclear. In the present study, a quantitative proteomic analysis was conducted to profile acetylome in porcine muscle within 24 h PM. In total 595 acetylation sites assigned to 163 proteins were identified in porcine muscle, of which 460 sites distributing to 110 proteins significantly changed in acetylation levels in the conversion of muscle to meat. The dynamic acetylation/deacetylaion of muscle proteins was closely associated with critical chemical-biophysical changes in PM muscle. Bioinformatic analysis revealed that protein lysine acetylation likely regulated postmortem meat quality development by regulating glycolysis and muscle pH, cell stress reponse and apoptosis, muscle contraction and rigor mortis, calcium signaling and proteolysis, IMP synthesis and meat flavor development, and even the stability of pigment proteins and meat color. This study provided the first overview of protein lysine acetylation in PM muscle and revealed its significance in the conversion of muscle to meat. Future exploration of the exact role of protein lysine acetylation at specific sites will further our understanding regarding the underlying mechanisms and be helpful for meat quality control. SIGNIFICANCE: This is the first analysis of acetylome in farm animal and postmortem muscle. Our data showed that the dynamic acetylation/deacetylation of muscle proteins was closely related to the postmortem changes of muscle that affect the final quality of raw meat. Proteins related to glucose metabolism and muscle contraction were the two largest clusters of acetylproteins identified in postmortem porcine muscle. Networks of acetylproteins involved in apoptosis, calcium signaling and IMP synthesis were identified in postmortem porcine muscle at the same time. Our results revealed that protein lysine acetylation regulated the conversion of muscle to meat. It likely regulated meat quality development by regulating postmortem glycolysis, mitochondrion initiated cell apoptosis, calcium signaling, rigor mortis, meat flavor compound sysnthesis and meat tenderization. Our study broadened our understanding of the biochemistry regulating the postmortem conversion of muscle to meat and final meat quality development, which may be helpful for future meat quality control.

Proteomic analysis reveals that lysine acetylation mediates the effect of antemortem stress on postmortem meat quality development.

To explore the involvement of protein lysine acetylation in the conversion of muscle to meat, a quantitative analysis of the acetylome in postmortem porcine muscle with or without antemortem stress was conducted. In total, 771 acetylpeptides containing 681 lysine acetylation sites mapping to 176 acetylproteins were identified. Acetylproteins were enriched in muscle contraction, carbohydrate metabolism, cell apoptosis and calcium signaling. Bioinformatic analysis suggests that preslaughter handling may be associated with glycolysis in postmortem muscle and the overall meat quality, via acetylation of multiple enzymes of glycogenolysis/glycolysis, regulate rigor mortis via acetylation of contractile, ATP production and calcium signaling-related proteins, and regulate stress response, cell apoptosis and meat tenderization via regulating the functions of heat shock proteins and permeability transition pore complex. This study provides the first overview of the acetylome in postmortem muscle as affected by preslaughter handling and broadens knowledge of the biochemistry regulating meat quality development.

Principles of gene editing techniques and applications in animal husbandry.

Gene editing techniques were developed chronologically, which include zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9). In this review, the working principles of these techniques were first introduced, their advantages and disadvantages were then discussed, their application in animal husbandry were elaborated, and finally human concerns about gene editing were presented. Compared to the two former techniques, the third-generation gene editing technique CRISPR/Cas9 has higher targeting efficiency and accuracy, less off-target effect, lower cytotoxicity and lower costs for being easier for vector design and manipulation. Although some people may concern about social or ethical issues, the benefits of gene editing certainly overweigh its demerits. The three gene editing techniques have been successfully used to improve the production and quality of livestock products, animal fertility, resistance to diseases, and welfare in animal husbandry. With legislation and the development of gene editing technology per se, it anticipatable that gene editing will have a broader utilization and make our lives happier.

Quantitative phosphoproteomic analysis among muscles of different color stability using tandem mass tag labeling.

A quantitative analysis of protein phosphorylation in ovine LTL muscle with different color stability was performed in the present study using TMT labeling in combination with TiO2 phosphopeptide enrichment. A total of 3412 phosphopeptides assigned to 1070 phosphoproteins were identified by mass spectrometry, of which 243 proteins were detected to be differentially phosphorylated between muscles of different color stability. Among these differentially phosphorylated proteins, 27 phosphoproteins were identified to be key color-related proteins by informatics analysis. Proteins involved in carbohydrate metabolism, especially glycolytic enzymes, were the largest cluster of protein determined to be color-related. In addition, the phosphorylation of myoglobin at Ser133 plays a negative role in the regulation of meat color stability. In summary, this study revealed that the phosphorylation of some glycolytic enzymes and myoglobin at specific serine residues may play critical roles in the regulation of meat color stability.

Comparative profiling of sarcoplasmic phosphoproteins in ovine muscle with different color stability.

The phosphorylation of sarcoplasmic proteins in postmortem muscles was investigated in relationship to color stability in the present study. Although no difference was observed in the global phosphorylation level of sarcoplasmic proteins, difference was determined in the phosphorylation levels of individual protein bands from muscles with different color stability. Correlation analysis and liquid chromatography - tandem mass spectrometry (LC-MS/MS) identification of phosphoproteins showed that most of the color stability-related proteins were glycolytic enzymes. Interestingly, the phosphorylation level of myoglobin was inversely related to meat color stability. As the phosphorylation of myoglobin increased, color stability based on a∗ value decreased and metMb content increased. In summary, the study revealed that protein phosphorylation might play a role in the regulation of meat color stability probably by regulating glycolysis and the redox stability of myoglobin, which might be affected by the phosphorylation of myoglobin.

Dephosphorylation enhances postmortem degradation of myofibrillar proteins.

Protein degradation is primarily responsible for postmortem meat tenderization, which might be affected by phosphorylation. The objective of this study was to investigate the effect of phosphorylation on myofibrillar proteins degradation in muscle during postmortem. Here we modulated the phosphorylation status of protein by protein kinase inhibitor and phosphatase inhibitor, and the effect of these inhibitors on myofibrillar protein degradation was evaluated. Generally, myofibril fragmentation index of samples with lower phosphorylation level was higher. Troponin T and heat shock protein 27 were degraded faster in protein kinase inhibited (low phosphorylation level) muscle, compared with the other two groups, while the degradation of desmin was not affected by inhibitors. Meanwhile, myosin heavy chain, actin and tropomyosin showed limited degradation in postmortem muscle. This study showed that dephosphorylation enhances the degradation of some myofibrillar proteins, indicating that protein phosphorylation may play an important role in postmortem meat tenderization.

Effects of protein phosphorylation on color stability of ground meat.

The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin.

Histone acetyltransferase inhibitors antagonize AMP-activated protein kinase in postmortem glycolysis.

OBJECTIVE: The purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK) activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality. METHODS: A total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-ß-D-ribofuranoside (AICAR, a specific activator of AMPK), AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II) and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases). After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured. RESULTS: Activation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates. CONCLUSION: Protein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.

Phosphorylation prevents in vitro myofibrillar proteins degradation by µ-calpain.

Myofibrillar proteins degradation contributes to meat tenderisation during post-mortem ageing. Protein phosphorylation has been revealed to be associated with meat tenderness in recent years. This study was undertaken to determine the impact of myofibrillar proteins phosphorylation on the degradation susceptibility by µ-calpain. Myofibrillar proteins were first incubated with protein kinase A (PKA) or alkaline phosphatase (AP) to increase or decrease the phosphorylation level, following µ-calpain hydrolysis. Myosin heavy chain, actin, desmin and troponin T showed different levels of degradation in control, AP and PKA groups under different Ca2+ concentrations. Generally, more degradation products were detected with the increase of Ca2+ concentration. Compared to the control, the protein degradation was higher in AP-treated group and lower in PKA-treated group. This study shows that phosphorylation prevents proteolytic susceptibility of myofibrillar proteins to degradation by µ-calpain, indicating that protein phosphorylation plays an important role in meat tenderisation during post-mortem ageing.

Antidiabetic and antihyperlipidemic activities of Forsythia suspensa (Thunb.) Vahl (fruit) in streptozotocin-induced diabetes mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Forsythia suspense (Thunb.) Vahl, a well-known Chinese Materia Medica, has been traditionally used in traditional Chinese medicine for the treatment of diabetes and some other diseases, but the rational for the usage of this plant is unclear. The aim of this study was to investigate the therapeutic effect and potential mechanism of the fruit of F. suspensa using streptozotocin (STZ)-induced diabetic mice. MATERIALS AND METHODS: Crude methanol extract of F. suspense fruit was fractionated with different solvents and the ethyl acetate fraction (EAF) was selected for in vivo studies based on the in vitro α-amylase and HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl coenzyme A) inhibiting activities. For in vivo study, diabetes mellitus was induced in mice with STZ. Diabetic mice were orally administrated with 50, 100 and 200mg/kg body weight of EAF for 4 weeks. Mouse body weight, blood glucose, glucose tolerance, biochemical parameters and gene expression related to pancreas and liver function were analyzed after EAF administration. RESULTS: After 4 weeks of EAF intervention, a significant decrease in blood glucose, triglyceride, creatinine total cholesterol, acid phosphatase, alkaline phosphatase, aspartate transaminase, alanine transaminase, and hepatic lipid (triglycerides and cholesterol) content as well as a significant increase in body weight, insulin secretion and glucose tolerance was observed in EAF treated diabetic mice. qRT-PCR analysis revealed that EAF antagonized STZ-induced alteration of the expression of rate-limiting enzymes (glucokinase and phosphorenolpyruvate carboxykinase) in liver and insulin secretion related genes insulin-1, insulin-2 and duodenal homeobox factor-1 in pancreas. CONCLUSION: The ethyl acetate extract of Forsythia suspense (Thunb.) Vahl fruit has potency to develop an antihyperglycemic and antihyperlipidemic agent for the treatment of diabetes mellitus via modulation of oxidative stress, the hepatic glucose metabolism and pancreatic insulin secretion.

KLF7 Regulates Satellite Cell Quiescence in Response to Extracellular Signaling.

Retaining muscle stem satellite cell (SC) quiescence is important for the maintenance of stem cell population and tissue regeneration. Accumulating evidence supports the model where key extracellular signals play crucial roles in maintaining SC quiescence or activation, however, the intracellular mechanisms that mediate niche signals to control SC behavior are not fully understood. Here, we reported that KLF7 functioned as a key mediator involved in low-level TGF-ß signaling and canonical Notch signaling-induced SC quiescence and myoblast arrest. The data obtained showed that KLF7 was upregulated in quiescent SCs and nonproliferating myoblasts. Silence of KLF7 promoted SCs activation and myoblasts proliferation, but overexpression of KLF7 induced myogenic cell arrest. Notably, the expression of KLF7 was regulated by TGF-ß and Notch3 signaling. Knockdown of KLF7 diminished low-level TGF-ß and canonical Notch signaling-induced SC quiescence. Investigation into the mechanism revealed that KLF7 regulation of SC function was dependent on p21 and acetylation of Lys227 and/or 231 in the DNA binding domain of KLF7. Our study provides new insights into the regulatory network of muscle stem cell quiescence. Stem Cells 2016;34:1310-1320.

Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle.

Phosphorylation of myofibrillar proteins in post-mortem ovine muscle with different tenderness.

BACKGROUND: Tenderness is one of the most important quality attributes especially for beef and lamb. As protein phosphorylation and dephosphorylation regulate glycolysis, muscle contraction and turnover of proteins within living cells, it may contribute to the conversion of muscle to meat. The changes of myofibrillar protein phosphorylation in post-mortem ovine muscle with different levels of tenderness were investigated in this study. RESULTS: The protein phosphorylation level (P/T ratio) of the tender group increased from 0.5 to 12 h post mortem and then decreased. The P/T ratio of tough group increased during 24 h post mortem, increasing faster from 0.5 to 4 h post mortem than from 4 to 24 h post mortem.The global phosphorylation level of tough meat was significantly higher than tender meat at 4, 12 and 24 h post mortem (P < 0.05). Protein identification revealed that most of the phosphoproteins were proteins with sarcomeric function; the others were involved in glycometabolism, stress response, etc. The phosphorylation levels of myofibrillar proteins, e.g. myosin light chain 2 and actin, were significantly different among groups of different tenderness and at different post-mortem time points (P < 0.05). CONCLUSION: Protein phosphorylation may influence meat rigor mortis through contractile machinery and glycolysis, which in turn affect meat tenderness.

Different actions of salt and pyrophosphate on protein extraction from myofibrils reveal the mechanism controlling myosin dissociation.

BACKGROUND: Myosin is the major functional protein in muscle foods for water retention, protein binding/gelation and fat holding/emulsification. To maximize its functionality, myosin needs to be released from thick filaments. Understanding of the mechanism controlling myosin extraction will help improve quality traits of meat products. RESULTS: The data obtained show that actomyosin binding is the rate-limiting constraint for myosin release in rigor condition. Magnesium pyrophosphate (MgPPi) increased myosin extraction by weakening actomyosin interaction and maximized myosin extraction at 0.4 mol L(-1) NaCl, which was not attained at 1.0 mol L(-1) NaCl in the absence of PPi. Interaction between myosin rod domains is another critical constraint for myosin extraction, which is, rather than PPi, salt dependent. Further, our data suggest that MyBP-C (myosin binding protein C) and M-line might not be of significance in the process of NaCl-induced myosin extraction, though further study was needed. CONCLUSION: Our study provides new insight into the mechanism that controls myosin extraction from intact sarcomere, which could be applied to maximize myosin function and to improve meat quality in practice.