Browsing by Author "Shi, Hao"
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- Defining the role of mitochondria in fresh meat quality developmentMatarneh, Sulaiman K. (Virginia Tech, 2017-07-12)During postmortem metabolism, hydrogen ions accumulate in the muscle and gradually lower the pH from 7.2 to an ultimate pH near 5.6. The ultimate pH of meat is widely valued as an indicator of fresh meat quality as it directly affects the quality characteristics of color, texture, and water holding capacity. Therefore, our research was conducted to identify the processes responsible for determining ultimate pH. Pigs harboring the AMPK�•3R200Q mutation produce meat with extremely low ultimate pH (pH ~ 5.3) that is detrimental to quality. This phenomenon is often attributed to a greater glycogen content in muscle from the mutant pigs compared to wild-type pigs. However, our research indicated that greater glycolytic flux in muscle from these pigs causes a lower ultimate pH rather than greater tissue glycogen deposition. On the other hand, however, AMPK�•3R200Q pigs contain more mitochondria and retain greater oxidative capacity. Hence, we hypothesized that mitochondria may contribute to the lower ultimate pH in muscle of these pigs. To test our hypothesis, isolated mitochondria were incorporated into an in vitro system the mimics postmortem glycolysis. Mitochondria enhanced glycolytic flux and pH decline in the in vitro system similar to that of AMPK�•3R200Q pigs. After a series of experiments, we found that the causative agent for enhanced glycolytic flux is a soluble mitochondrial protein. In other experiments, mitochondrial F1-ATPase was found to be responsible for the majority of this effect, principally through promoting greater ATP hydrolysis at lower pH values, thereby allowing for greater flux through glycolysis. These data suggest that variations in ultimate pH may be more thoroughly explained and predicted by the abundance of mitochondria. Broiler pectoralis major muscle, which is a highly glycolytic muscle, possesses high ultimate pH (pH ~ 5.9) compared to pork and beef. We postulated that rapid carcass chilling reduces the flux through glycolysis, thereby causing premature termination of postmortem metabolism. Yet, chilling was only partially responsible for the high ultimate pH of pectoralis major muscle. However, we showed that pectoralis major of broiler chicken exhibits lower phosphofructokinase-1 activity compared to porcine longissimus lumborum muscle, which limits the flux through glycolysis.
- Determining the Underlying Factors of Fresh Ham Color VariationElgin, Jennifer May (Virginia Tech, 2019-07-10)Consumers associate meat color with quality. In some cases, especially in fresh and cured hams, the surface of a ham, whole, boneless or sectioned and formed displays a color gradient, which is unsightly and generally is considered of lower quality and must be discounted or processed different where color is less critical to the ultimate value of the resulting product. This disparity in color uniformity across fresh and cured products is sometimes known as two-toning and is most often found in the semimembranosus (SM) and associated muscles of fresh hams and is exacerbated with curing. The underlying color of fresh meat may be a function of postmortem metabolism or the underlying characteristics of those muscles involved. Therefore, the objective of this study is to determine the changes in underlying muscle type and postmortem metabolism in those muscles responsible for fresh ham color variation. Semimembranosus (SM) muscles of 15 mixed bred pigs were collected at 30 min and 1440 min postmortem, and muscle color was determined and muscles were collected and snap frozen for various energy metabolism analyses. Differences in color (L*, a* and b*) were noted across the face of the muscle by zone and time (P < 0.0001) but no differences were detected in pH and lactate, glucose, glucose-6-phosphate, and glycogen metabolisms. Glycolytic potential was also measured on a lactate basis and showed no differences across zone (P = 0.0746) but increased over time (P < 0.006). Lactate and pH were plotted and showed a linear relationship linear relationship (R2 = 0.928337) at 30 min (P < 0.0001) and at 1440 min (R2 = 0.161412; P < 0.0015). Muscle type characteristics showed no difference between zones and time. Buffering capacity showed a significant difference at pH 6 (P < 0.0359) and with time across all pH measured (P < 0.0001). These data suggest inherent differences, such as location and function, in the semimembranosus muscle may be more critical in developing fresh color than aberrations in postmortem metabolism.
- Driving an Oxidative Phenotype Protects Myh4 Null Mice From Myofiber Loss During Postnatal GrowthZeng, Caiyun; Shi, Hao; Kirkpatrick, Laila T.; Ricome, Aymeric; Park, Sungkwon; Scheffler, Jason M.; Hannon, Kevin M.; Grant, Alan L.; Gerrard, David E. (Frontiers, 2022-02-24)Postnatal muscle growth is accompanied by increases in fast fiber type compositions and hypertrophy, raising the possibility that a slow to fast transition may be partially requisite for increases in muscle mass. To test this hypothesis, we ablated the Myh4 gene, and thus myosin heavy chain IIB protein and corresponding fibers in mice, and examined its consequences on postnatal muscle growth. Wild-type and Myh4(-/-) mice had the same number of muscle fibers at 2 weeks postnatal. However, the gastrocnemius muscle lost up to 50% of its fibers between 2 and 4 weeks of age, though stabilizing thereafter. To compensate for the lack of functional IIB fibers, type I, IIA, and IIX(D) fibers increased in prevalence and size. To address whether slowing the slow-to-fast fiber transition process would rescue fiber loss in Myh4(-/-) mice, we stimulated the oxidative program in muscle of Myh4(-/-) mice either by overexpression of PGC-1 alpha, a well-established model for fast-to-slow fiber transition, or by feeding mice AICAR, a potent AMP kinase agonist. Forcing an oxidative metabolism in muscle only partially protected the gastrocnemius muscle from loss of fibers in Myh4(-/-) mice. To explore whether traditional means of stimulating muscle hypertrophy could overcome the muscling deficits in postnatal Myh4(-/-) mice, myostatin null mice were bred with Myh4(-/-) mice, or Myh4(-/-) mice were fed the growth promotant clenbuterol. Interestingly, both genetic and pharmacological stimulations had little impact on mice lacking a functional Myh4 gene suggesting that the existing muscle fibers have maximized its capacity to enlarge to compensate for the lack of its neighboring IIB fibers. Curiously, however, cell signaling events responsible for IIB fiber formation remained intact in the tissue. These findings further show disrupting the slow-to-fast transition of muscle fibers compromises muscle growth postnatally and suggest that type IIB myosin heavy chain expression and its corresponding fiber type may be necessary for fiber maintenance, transition and hypertrophy in mice. The fact that forcing muscle metabolism toward a more oxidative phenotype can partially compensates for the lack of an intact Myh4 gene provides new avenues for attenuating the loss of fast-twitch fibers in aged or diseased muscles.
- Dual effects of obesity on satellite cells and muscle regenerationGeiger, Ashley E.; Daughtry, Morgan R.; Yen, Con-Ning; Kirkpatrick, Laila T.; Shi, Hao; Gerrard, David E. (2020-08)Obesity is a complex metabolic disorder that often leads to a decrease in insulin sensitivity, chronic inflammation, and overall decline in human health and well-being. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury; however, the mechanism underlying these changes has yet to be determined. To test whether there is a negative impact of obesity on satellite cell (SC) decisions and behaviors, we fed C57BL/6 mice normal chow (NC, control) or a high-fat diet (HFD) for 10 weeks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller size (p < .001) compared to those from NC mice, and this decreased proliferation was confirmed (p < .05) by BrdU incorporation. Moreover, in vitro assays showed that HFD SCs exhibited diminished (p < .001) fusion capacity compared to NC SCs. In single fiber explants, a higher ratio of SCs experienced apoptotic events (p < .001) in HFD mice compared to that of NC-fed mice. In vivo lineage tracing using H2B-GFP mice showed that SCs from HFD treatment also cycled faster (p < .001) than their NC counterparts. In spite of all these autonomous cellular effects, obesity as triggered by high-fat feeding did not significantly impair muscle regeneration in vivo, as reflected by the comparable cross-sectional area (p > .05) of the regenerating fibers in HFD and NC muscles, suggesting that other factors may mitigate the negative impact of obesity on SCs properties.
- Harvest practices cause aberrations in pork qualityDaniels, Rachel Page (Virginia Tech, 2019-08-16)Consumers are often confused and reluctant to purchase products from fresh retail meat counters that exhibit significant color variations. This hesitation to purchase results in annual profit loss to the pork industry. Processes to minimize color variation remain a major focus of the industry and many meat science programs across the globe. Previously, we found inherent muscle characteristics contribute to variations in pork quality but these characteristics fail to explain the high frequency of two-toning and other pork quality defects routinely occurring in many hog processing facilities. Therefore, we hypothesized harvesting practices, such as scald alter color across muscles of the ham. Scald time was initially investigated using 32 carcasses subjected to either a 4 (n=16) or 8 (n=16) min scald time. Samples were collected before or after scalding and at 24 hrs. A 50% reduction in scald time resulted in (p < 0.0001) lighter muscle color (L*) early postmortem, although the 8 min scald treatment was lighter (p < 0.005) at 24 hrs. Although differences in pH (p < 0.0001) and color were noted, ultimate carcass temperature was not affected. To that end, we moved to validate our hypothesis in an industrial plant setting. Carcasses (n=200) were assigned treatments of 6.5 or 7.5 min scald times, and SM muscle samples were collected at 24 hrs. Surprisingly, the shorter scald time resulted in (p < 0.05) a lighter color, contradicting our first study. To explore this color issue further, we uncoupled scald from the dehairing process. To achieve this goal, carcasses (n=24) were assigned to either an 8 or 16 min time to dehair, with or without scalding. Protracted time to dehair resulted in higher ultimate pH (p < 0.005) and less color variation across the muscle (p < 0.05). Though a color gradient remained, the variation across the muscle was reduced by increasing time to dehair. These data show time to dehair affects pork quality development and suggest that delaying time to physical manipulation of the carcass may improve pork color, thus increasing consumer acceptance.
- Impacts of dietary obesity on muscle stem cell behaviorsGeiger, Ashley Elizabeth (Virginia Tech, 2019-02-22)Occurrence of obesity has steadily increased in the human population and, along with it, associated health complications such as systemic insulin resistance, which can lead to the development of type 2 diabetes mellitus. Obesity is a complex metabolic disorder that often leads to chronic inflammation and an overall decline in human and animal health. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury, however, the mechanism underlying these changes in satellite cell (SC) biology have yet to be explored. To test the negative impacts of obesity on SC behaviors, we fed C57BL/6 mice normal chow (NC, control) or high-fat diet (HFD) for 10 wks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller numbers (P < 0.001) compared to those isolated from NC mice, and this observation was confirmed (P < 0.05) by BrdU incorporation. Moreover, in vitro differentiation assays consisting of equally seeded SCs derived from NC and HFD muscles showed that HFD SCs exhibited compromised (P < 0.001) differentiation capacity compared to NC SCs. Immunocytochemical staining of cultured SCs demonstrated that the percentage of Pax7+/MyoD- (self-renewed) SC subpopulation decreased (P < 0.001) with HFD treatment group compared to the control. In single fiber explants, a higher ratio of SCs experienced apoptotic events as revealed by the expression of cleaved caspase 3 (P < 0.001). To investigate further the impact of obesity on SC quiescence and cycling properties in vivo, we used an inducible H2B-GFP mouse model to trace the turnover rate of GFP and thus cell division under normal and obese conditions. Flow cytometric analysis revealed that SCs from HFD treatment cycled faster (P < 0.001) than their NC counterparts, as reflected by the quicker loss of the GFP intensity. To test for SC muscle regenerative capacity in vivo, we used cardiotoxin (CTX) to induce wide-spread muscle damage in the tibialis anterior muscle. After analysis we found that HFD leads to a compromised, though mild, impairment in muscle regeneration. Taken together, these findings suggest that obesity negatively affects SC quiescence, proliferation, differentiation, and self-renewal in vitro, ex vivo and in vivo.
- O-GlcNAcylation of SIRT1 Protects against Cold Stress-Induced Skeletal Muscle Damage via Amelioration of Mitochondrial HomeostasisCao, Yu; Zhang, Meng; Li, Ye; Lu, Jingjing; Zhou, Wanhui; Li, Xiaoshuang; Shi, Hao; Xu, Bin; Li, Shize (MDPI, 2022-11-22)Cold stress disturbs cellular metabolic and energy homeostasis, which is one of the causes of stress-induced illnesses. O-GlcNAcylation is a nutrient-sensing pathway involved in a myriad of cellular processes. It plays a key role in metabolic homeostasis. Nevertheless, a specific sensing mechanism linking skeletal muscle to O-GlcNAcylation in cold stress is unknown. In this study, O-GlcNAcylation of SIRT1 was targeted to explore the mechanism of skeletal muscle adaptation to cold stress. Ogt mKO aggravated skeletal muscle fibrosis induced by cold stress. At the same time, Ogt gene deletion accelerated the homeostasis imbalance and oxidative stress of skeletal muscle mitochondria induced by cold stress. In vitro results showed that inhibition of SIRT1’s O-GlcNAcylation accelerated mild hypothermia induced mitochondrial homeostasis in mouse myogenic cells (C2C12 cells). However, overexpression of SIRT1’s O-GlcNAcylation improved the above phenomena. Thus, these results reveal a protective role of OGT-SIRT1 in skeletal muscle’s adaptation to cold stress, and our findings will provide new avenues to combat stress-induced diseases.
- Skeletal Muscle O-GlcNAc Transferase Action on Global Metabolism Is Partially Mediated Through Interleukin-15Zumbaugh, Morgan D.; Yen, Con-Ning; Bodmer, Jocelyn S.; Shi, Hao; Gerrard, David E. (2021-07-13)Besides its roles in locomotion and thermogenesis, skeletal muscle plays a significant role in global glucose metabolism and insulin sensitivity through complex nutrient sensing networks. Our previous work showed that the muscle-specific ablation of O-GlcNAc transferase (OGT) led to a lean phenotype through enhanced interleukin-15 (IL-15) expression. We also showed OGT epigenetically modified and repressed the Il15 promoter. However, whether there is a causal relationship between OGT ablation-induced IL-15 secretion and the lean phenotype remains unknown. To address this question, we generated muscle specific OGT and interleukin-15 receptor alpha subunit (IL-15r alpha) double knockout mice (mDKO). Deletion of IL-15r alpha in skeletal muscle impaired IL-15 secretion. When fed with a high-fat diet, mDKO mice were no longer protected against HFD-induced obesity compared to wild-type mice. After 22 weeks of HFD feeding, mDKO mice had an intermediate body weight and glucose sensitivity compared to wild-type and OGT knockout mice. Taken together, these data suggest that OGT action is partially mediated by muscle IL-15 production and provides some clarity into how disrupting the O-GlcNAc nutrient signaling pathway leads to a lean phenotype. Further, our work suggests that interfering with the OGT-IL15 nutrient sensing axis may provide a new avenue for combating obesity and metabolic disorders.
- Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivityShi, Hao; Munk, Alexander; Nielsen, Thomas S.; Daughtry, Morgan R.; Larsson, Louise; Li, Shize; Hoyer, Kasper F.; Geisler, Hannah W.; Sulek, Karolina; Kjobsted, Rasmus; Fisher, Taylor; Andersen, Marianne M.; Shen, Zhengxing; Hansen, Ulrik K.; England, Eric M.; Cheng, Zhiyong; Hojlund, Kurt; Wojtaszewski, Jorgen FP P.; Yang, Xiaoyong; Hulver, Matthew W.; Helm, Richard F.; Treebak, Jonas T.; Gerrard, David E. (Elsevier, 2018-05-01)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.