Hao Shi

Mitogen-activated protein kinase signaling is necessary for the maintenance of skeletal muscle mass

The signal transduction cascades that maintain muscle mass remain to be fully defined. Herein, we report that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in vitro decreases myotube size and protein content after 3-day treatment with a MEK inhibitor. Neither p38 nor JNK inhibitors had any effect on myotube size or morphology. ERK1/2 inhibition also upregulated gene transcription of atrogin-1 and muscle-specific RING finger protein 1 and downregulated the phosphorylation of Akt and its downstream kinases. Forced expression of enhanced green fluorescent protein-tagged MAPK phosphatase 1 (MKP-1) in soleus and gastrocnemius muscles decreased both fiber size and reporter activity. This atrophic effect of MKP-1 was time dependent. Analysis of the reporter activity in vivo revealed that the activities of nuclear factor-kappaB and 26S proteasome were differentially activated in slow and fast muscles, suggesting muscle type-specific mechanisms may be utilized. Together, these findings suggest that MAPK signaling is necessary for the maintenance of skeletal muscle mass because inhibition of these signaling cascades elicits muscle atrophy in vitro and in vivo.

Modulation of skeletal muscle fiber type by mitogen-activated protein kinase signaling

Skeletal muscle is composed of diverse fiber types, yet the underlying molecular mechanisms responsible for this diversification remain unclear. Herein, we report that the extracellular signal‐regulated kinase (ERK) 1/2 pathway, but not p38 or c‐Jun NH2‐terminal kinase (JNK), is preferentially activated in fast‐twitch muscles. Pharmacological blocking of ERK1/2 pathway increased slow‐twitch fiber type‐specific reporter activity and repressed those associated with the fast‐twitch fiber phenotype in vitro. Overexpression of a constitutively active ERK2 had an opposite effect. Inhibition of ERK signaling in cultured myotubes increased slow‐twitch fiber‐specific protein accumulation while repressing those characteristic of fast‐twitch fibers. Overexpression of MAP kinase phosphatase‐1 (MKP1) in mouse and rat muscle fibers containing almost exclusively type IIb or IIx fast myosin heavy chain (MyHC) isoforms induced de novo synthesis of the slower, more oxidative type IIa and I MyHCs in a time‐dependent manner. Conversion to the slower phenotype was confirmed by up‐regulation of slow reporter gene activity and down‐regulation of fast reporter activities in response to forced MKP1 expression in vivo. In addition, activation of ERK2 signaling induced up‐regulation of fast‐twitch fiber program in soleus. These data suggest that the MAPK signaling, most likely the ERK1/2 pathway, is necessary to preserve the fast‐twitch fiber phenotype with a concomitant repression of slow‐twitch fiber program.—Shi, H., Scheffler, J. M., Pleitner, J. M., Zeng, C., Park, S., Hannon, K. M., Grant, A. L., Gerrard, D. E. Modulation of skeletal muscle fiber type by mitogen‐activated protein kinase signaling. FASEB J. 22, 2990–3000 (2008)

Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle

Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.

Excess glycogen does not resolve high ultimate pH of oxidative muscle

Skeletal muscle glycogen content can impact the extent of postmortem pH decline. Compared to glycolytic muscles, oxidative muscles contain lower glycogen levels antemortem which may contribute to the higher ultimate pH. In an effort to explore further the participation of glycogen in postmortem metabolism, we postulated that increasing the availability of glycogen would drive additional pH decline in oxidative muscles to equivalent pH values similar to the ultimate pH of glycolytic muscles. Glycolysis and pH declines were compared in porcine longissimus lumborum (glycolytic) and masseter (oxidative) muscles using an in vitro system in the presence of excess glycogen. The ultimate pH of the system containing longissimus lumborum reached a value similar to that observed in intact muscle. The pH decline of the system containing masseter samples stopped prematurely resulting in a higher ultimate pH which was similar to that of intact masseter muscle. To investigate further, we titrated powdered longissimus lumborum and masseter samples in the reaction buffer. As the percentage of glycolytic sample increased, the ultimate pH decreased. These data show that oxidative muscle produces meat with a high ultimate pH regardless of glycogen content and suggest that inherent muscle factors associated with glycolytic muscle control the extent of pH decline in pig muscles.

Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity

Objective Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. Methods We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. Results We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). Conclusions Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders.

A mitochondrial protein increases glycolytic flux

The purpose of this study was to determine the role of mitochondria in postmortem muscle metabolism. Isolated mitochondria were incorporated into a reaction buffer that mimics postmortem glycolysis with or without mitochondrial electron transport chain inhibitors. Addition of mitochondria lowered pH values at 240 and 1440min regardless of inhibitors. Reduction in pH was accompanied by enhanced glycogen degradation and lactate accumulation. To explore the mechanism responsible for this exaggerated metabolism, mitochondrial preparations were mechanically disrupted and centrifuged. Resulting supernatants and pellets each were added to the in vitro model. Mitochondrial supernatants produced similar effects as those including intact mitochondria. To narrow further our target of investigation, mitochondrial supernatants were deproteinized with perchloric acid. The effect of mitochondrial supernatant was lost after perchloric acid treatment. These data indicate that a mitochondrial-based protein is capable of increasing glycolytic flux in an in vitro model and may partially explain acid meat development in highly oxidative AMPKγ3R200Q mutated pigs.

Mitochondrial F1-ATPase extends glycolysis and pH decline in an in vitro model

The experiment was conducted to identify the mitochondrial protein responsible for enhancing glycolytic flux. We hypothesized that mitochondrial F1-ATPase promotes ATP hydrolysis and thereby the flux through glycolysis. Porcine longissimus muscle mitochondria were incorporated into an in vitro system designed to recapitulate postmortem glycolysis with or without Na-azide to specifically inhibit the β-subunit of mitochondrial F1-ATPase that catalyzes ATP hydrolysis. Addition of mitochondria enhanced ATP hydrolysis, glycogen degradation, lactate accumulation, and pH decline in the in vitro system. However, the majority of mitochondria-mediated enhancement in glycolytic flux was abolished in the presence of Na-azide. To investigate further, myofibrillar and mitochondrial proteins were added to the in vitro system after 240min from the initiation of the reaction. Greater pH decline and lactate accumulation were observed in system containing mitochondrial protein compared to their myofibrillar counterpart. In conclusion, mitochondrial F1-ATPase is capable of increasing glycolytic flux through promoting greater ATP hydrolysis at lower pH.

Muscle characteristics only partially explain color variations in fresh hams

Fresh hams display significant lean color variation that persists through further processing and contributes to a less desirable cured product. In an attempt to understand the underlying cause of this color disparity, we evaluated the differences in muscle characteristics and energy metabolites across semimembranosus (SM) muscles differing in color variation. The L* (lightness) and a* (redness) values were highest and lowest (P<0.001), respectfully in the most caudal aspects of the muscle while the ultimate pH was the lowest (P<0.001). Correspondingly, this region possessed highest (P<0.01) glycolytic potential (GP) and lactate dehydrogenase (LDH) levels but did not differ in the amount of myoglobin or myosin heavy chain type I isoform. These data show that differences in muscle may contribute to ham color variation but suggest other factors may mitigate or exacerbate these variances.

Phosphofructokinase and mitochondria partially explain the high ultimate pH of broiler pectoralis major muscle

&NA; During postmortem metabolism, muscle pH gradually declines to reach an ultimate pH near 5.6 across most meat species. Yet, broiler pectoralis major (P. major) muscle generates meat with high ultimate pH (pH ˜ 5.9). For better understanding of the underlying mechanism responsible for this phenomenon, we evaluated the involvement of breast muscle chilling on the extent of postmortem metabolism. Broiler breast muscles were either subjected to chilling treatment (control) or left at room temperature (RT) for 120 min. P. major muscle from the RT treatment had lower ultimate pH, greater glycogen degradation and lactate accumulation. While these findings suggest that carcass chilling can contribute to the premature termination of postmortem metabolism, chilling did not fully explain the high ultimate pH of P. major muscle. Our results also revealed that glucose‐6‐phosphate (G6P) was very low at 24 h, and therefore we hypothesized that G6P was limiting. To test this hypothesis, muscle samples from P. major and porcine longissimus lumborum (LL) muscle were homogenized into a reaction buffer that mimics postmortem glycolysis with or without 0.5 mg/mL isolated mitochondria. While samples containing porcine LL muscle reached the normal level of ultimate pH, P. major muscle samples reached a value similar to that observed in vivo even in the presence of excess G6P, indicating that G6P was not limiting. Mitochondria enhanced the glycolytic flux and pH decline in systems containing muscle from both species. More importantly, however, was that in vitro system containing chicken with mitochondria reached pH value similar to that of samples containing LL muscle without mitochondria. To investigate further, phosphofructokinase (PFK) activity was compared in broiler P. major and porcine LL muscle at different pH values. PFK activity was lower in P. major muscle at pH 7, 6.5, and 6.2 than LL muscle. In conclusion, carcass chilling can partially contribute to the high ultimate pH of broiler P. major muscle, while low PFK activity and mitochondria content limit the flux through glycolysis.

Presence of oxygen and mitochondria in skeletal muscle early postmortem

Anaerobic glycolysis dominates energy metabolism postmortem. Even so, however, recent studies suggest mitochondria can modify postmortem energy metabolism and may contribute to pH decline, possibly affecting the transformation of muscle to meat and fresh meat quality development. Because oxygen is a necessary component of mitochondrial function, oxygenation of porcine and bovine longissimus thoracis et lumborum was determined postmortem using NIR spectroscopy. The ratio of oxy- to deoxymyoglobin decreased with time postmortem in both species. Metabolic analyses of muscle samples collected over the same timeframe also revealed fluctuations in TCA intermediates. Finally, mitochondria collected from muscle of electrically stimulated carcasses differed from those of non-stimulated muscle. Collectively, these data support the thesis that muscle mitochondria function early postmortem and may play a more active part in pH decline and possibly meat quality development.