Glycolytic Muscle Research Articles

Beta adrenergic agonist induces a shift in mitochondria metabolism in porcine skeletal muscle

During phases of skeletal muscle growth, the metabolic processes regulating muscle tissue exhibit remarkable adaptability. This metabolic flexibility is an essential component of maintaining the health and functionality of skeletal muscle, which allows the capacity to transition between oxidative and glycolytic metabolic pathways. Within the occurrence of these metabolic shifts, the exact underlying mechanisms that orchestrate these transitions within the context of mitochondrial function remain unresolved. Therefore, our objective is to determine the role of mitochondria during a shift in metabolism, through measuring functional capacity, gene expression, and protein abundance. To investigate this shift, we utilized β-adrenergic agonists (BAA) supplementation, due to its ability to increase the proportion of fast-twitch fibers in skeletal muscle. To assess the role of the mitochondria, we utilized a mutated pig, containing a constitutively active adenosine monophosphate activated protein kinase (AMPKγ3R200Q) that contains a greater oxidative capacity in a habitually glycolytic skeletal muscle. In this current study, mature pigs (wildtype and AMPKγ3R200Q) were fed BAA (0 and 9ppm) for 1 week, then euthanized and longissimus dorsi muscle samples were collected. Mitochondria of AMPK mutated pigs had a higher ( P = 0.08) oxygen consumption rate with pyruvate/malate substrates when stimulated with ADP when compared to their wildtype (WT) counterpart. When given succinate/rotenone substrates, there was an interaction ( P = 0.04) noted for basal respiration, where WT pigs fed control diets had lower oxygen consumption compared to that of AMPK mutated pigs and those fed BAA. These data suggest that BAA has more of an effect on WT pigs than AMPK mutated pigs, possibly due to the inherent increased oxidative metabolism in mutant pigs. In addition, skeletal muscle mitochondria of AMPK mutated pigs had higher ( P = 0.04) maximal respiration compared to that of their WT counterpart. After 1 week of feeding BAA, there were no differences in mitochondrial DNA content, but there was an increase in β1-adrenergic receptor gene expression in pigs fed BAA (Diet, P = 0.06; Interaction P = 0.08). Oxidative protein abundance increased for succinate dehydrogenase ( P < 0.01) and citrate synthase ( P = 0.08) in the skeletal muscle of AMPK mutated pigs. Glycolytic proteins were increased for lactate dehydrogenase ( P < 0.05) and glyceraldehyde 3-phosphate dehydrogenase ( P < 0.01) in the muscle of WT pigs. No interactions between genotype and diet on the protein abundance level was noted. Together, these data show that mitochondria function is altered in porcine skeletal muscle when pigs are supplemented with 1 week of BAA; however, it is not detected in protein levels after 1 week. These data suggest part of the mechanism by which BAA supplementation augments muscle growth in pigs lies within the regulation of β1-adrenergic receptors and changes in mitochondrial function. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of Myostatin Significantly and Preferentially Improves Muscle Function in a Sex-Dependent Manner in Both an Obese and Aging Mouse Model

Skeletal muscle mass and function are essential components of health and homeostasis. In addition to its role in balance and locomotion, skeletal muscle is the body’s largest metabolic reservoir, absorbing significant amounts of glucose and amino acids from the blood stream. The loss of muscle mass is associated with impaired glucose tolerance, threatens vascular health and contributes to cardiovascular disease in obese and aging (sarcopenic) populations, along with driving overall mortality. The myokine myostatin (GDF-8) is a potent negative regulator of skeletal muscle growth that is upregulated in humans and animal models of obesity and aging. Loss of myostatin is known to increase the number of glycolytic muscle fibers and has been shown to correct indices that govern cardiometabolic health in obesity and aging, improving endothelial function and glucose homeostasis, and protecting against renal injury and hypertension. However, these studies have largely been conducted using male mice, leaving the question regarding the effectiveness of myostatin deletion on skeletal muscle function, independent of sex, largely unexplored. As such, our hypothesis is that the deletion of myostatin will improve skeletal muscle function in both male and female mice, regardless of the disease phenotype being obesity or aging. Young (3-5 mo.) lean and obese mice were used, along with aged (18-24 mo.) mice. The obese db/db model was utilized as it is a well characterized model of hyperphagia that results in obesity and subsequent metabolic disease, including significant whole-body adiposity and reduced muscle mass. On this background we have crossed mice harboring constitutive genetic deletion of myostatin to reverse muscle loss. Thus, myostatin deletion was crossed onto the relevant backgrounds in combination with aging or obesity and included both sexes. Muscle function was assessed in all groups using in vivo plantarflexion. Young male mice demonstrated an ~20% increase in force production compared to the females. Results show that aging and obesity inhibited skeletal muscle function in both sexes, with female mice showing a lessened impact of obesity or aging. Myostatin deletion improved muscle function in male mice, returning both obesity-derived weakness and aging-induced sarcopenia back to levels of young control. However, female mice without myostatin experienced a relative gain-of-function, with force production significantly exceeding that of the young control. The implication of these results is that weight loss may be more effective in protecting muscle function in diseases in the male population whereas myostatin inhibition, either pharmaceutically or with prescription exercise, may result in greater improvements in the female sex comparatively. This research gratefully acknowledges support from the NIA (K01 AG064121), OCAST (HR21-045-1) and the Niblack Research Scholars Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of different myofiber types in pigs muscles and construction of regulatory networks

BackgroundSkeletal muscle is composed of muscle fibers with different physiological characteristics, which plays an important role in regulating skeletal muscle metabolism, movement and body homeostasis. The type of skeletal muscle fiber directly affects meat quality. However, the transcriptome and gene interactions between different types of muscle fibers are not well understood.ResultsIn this paper, we selected 180-days-old Large White pigs and found that longissimus dorsi (LD) muscle was dominated by fast-fermenting myofibrils and soleus (SOL) muscle was dominated by slow-oxidizing myofibrils by frozen sections and related mRNA and protein assays. Here, we selected LD muscle and SOL muscle for transcriptomic sequencing, and identified 312 differentially expressed mRNA (DEmRs), 30 differentially expressed miRNA (DEmiRs), 183 differentially expressed lncRNA (DElRs), and 3417 differentially expressed circRNA (DEcRs). The ceRNA network included ssc-miR-378, ssc-miR-378b-3p, ssc-miR-24-3p, XR_308817, XR_308823, SMIM8, MAVS and FOS as multiple core nodes that play important roles in muscle development. Moreover, we found that different members of the miR-10 family expressed differently in oxidized and glycolytic muscle fibers, among which miR-10a-5p was highly expressed in glycolytic muscle fibers (LD) and could target MYBPH gene mRNA. Therefore, we speculate that miR-10a-5p may be involved in the transformation of muscle fiber types by targeting the MYHBP gene. In addition, PPI analysis of differentially expressed mRNA genes showed that ACTC1, ACTG2 and ACTN2 gene had the highest node degree, suggesting that this gene may play a key role in the regulatory network of muscle fiber type determination.ConclusionsWe can conclude that these genes play a key role in regulating muscle fiber type transformation. Our study provides transcriptomic profiles and ceRNA interaction networks for different muscle fiber types in pigs, providing reference for the transformation of pig muscle fiber types and the improvement of meat quality.

Mitochondrial Transplantation's Role in Rodent Skeletal Muscle Bioenergetics: Recharging the Engine of Aging.

Mitochondria are the 'powerhouses of cells' and progressive mitochondrial dysfunction is a hallmark of aging in skeletal muscle. Although different forms of exercise modality appear to be beneficial to attenuate aging-induced mitochondrial dysfunction, it presupposes that the individual has a requisite level of mobility. Moreover, non-exercise alternatives (i.e., nutraceuticals or pharmacological agents) to improve skeletal muscle bioenergetics require time to be effective in the target tissue and have another limitation in that they act systemically and not locally where needed. Mitochondrial transplantation represents a novel directed therapy designed to enhance energy production of tissues impacted by defective mitochondria. To date, no studies have used mitochondrial transplantation as an intervention to attenuate aging-induced skeletal muscle mitochondrial dysfunction. The purpose of this investigation, therefore, was to determine whether mitochondrial transplantation can enhance skeletal muscle bioenergetics in an aging rodent model. We hypothesized that mitochondrial transplantation would result in sustained skeletal muscle bioenergetics leading to improved functional capacity. Fifteen female mice (24 months old) were randomized into two groups (placebo or mitochondrial transplantation). Isolated mitochondria from a donor mouse of the same sex and age were transplanted into the hindlimb muscles of recipient mice (quadriceps femoris, tibialis anterior, and gastrocnemius complex). The results indicated significant increases (ranging between ~36% and ~65%) in basal cytochrome c oxidase and citrate synthase activity as well as ATP levels in mice receiving mitochondrial transplantation relative to the placebo. Moreover, there were significant increases (approx. two-fold) in protein expression of mitochondrial markers in both glycolytic and oxidative muscles. These enhancements in the muscle translated to significant improvements in exercise tolerance. This study provides initial evidence showing how mitochondrial transplantation can promote skeletal muscle bioenergetics in an aging rodent model.

Type selective ablation of postnatal slow and fast fatigue-resistant motor neurons in mice induces late onset kinetic and postural tremor following fiber-type transition and myopathy

Animals on Earth need to hold postures and execute a series of movements under gravity and atmospheric pressure. VAChT-Cre is a transgenic Cre driver mouse line that expresses Cre recombinase selectively in motor neurons of S-type (slow-twitch fatigue-resistant) and FR-type (fast-twitch fatigue-resistant). Sequential motor unit recruitment is a fundamental principle for fine and smooth locomotion; smaller-diameter motor neurons (S-type, FR-type) first contract low-intensity oxidative type I and type IIa muscle fibers, and thereafter larger-diameter motor neurons (FInt-type, FF-type) are recruited to contract high-intensity glycolytic type IIx and type IIb muscle fibers. To selectively eliminate S- and FR-type motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which the cytotoxic diphtheria toxin A fragment (DTA) was expressed in Cre-expressing neurons. The VAChT-Cre;NSE-DTA mice were born normally but progressively manifested various characteristics, including body weight loss, kyphosis, kinetic and postural tremor, and muscular atrophy. The progressive kinetic and postural tremor was remarkable from around 20 weeks of age and aggravated. Muscular atrophy was apparent in slow muscles, but not in fast muscles. The increase in motor unit number estimation was detected by electromyography, reflecting compensatory re-innervation by remaining FInt- and FF-type motor neurons to the orphaned slow muscle fibers. The muscle fibers gradually manifested fast/slow hybrid phenotypes, and the remaining FInt-and FF-type motor neurons gradually disappeared. These results suggest selective ablation of S- and FR-type motor neurons induces progressive muscle fiber-type transition, exhaustion of remaining FInt- and FF-type motor neurons, and late-onset kinetic and postural tremor in mice.

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.

Sucrose-Enriched and Carbohydrate-Free High-Fat Diets Distinctly Affect Substrate Metabolism in Oxidative and Glycolytic Muscles of Rats.

Skeletal muscle substrate preference for fuel is largely influenced by dietary macronutrient availability. The abundance of dietary carbohydrates promotes the utilization of glucose as a substrate for energy production, whereas an abundant dietary fat supply elevates rates of fatty acid (FA) oxidation. The objective of this study was to determine whether an obesogenic, high-fat, sucrose-enriched (HFS) diet or a carbohydrate-free ketogenic diet (KD) exert distinct effects on fat, glucose, and ketone metabolism in oxidative and glycolytic skeletal muscles. Male Wistar rats were fed either a HFS diet or a KD for 16 weeks. Subsequently, the soleus (Sol), extensor digitorum longus (EDL), and epitrochlearis (Epit) muscles were extracted to measure palmitate oxidation, insulin-stimulated glucose metabolism, and markers of mitochondrial biogenesis, ketolytic capacity, and cataplerotic and anaplerotic machinery. Sol, EDL, and Epit muscles from KD-fed rats preserved their ability to elevate glycogen synthesis and lactate production in response to insulin, whereas all muscles from rats fed with the HFS diet displayed blunted responses to insulin. The maintenance of metabolic flexibility with the KD was accompanied by muscle-fiber-type-specific adaptive responses. This was characterized by the Sol muscle in KD-fed rats enhancing mitochondrial biogenesis and ketolytic capacity without elevating its rates of FA oxidation in comparison with that in HFS feeding. Conversely, in the Epit muscle, rates of FA oxidation were increased, whereas the ketolytic capacity was markedly reduced by the KD in comparison with that by HFS feeding. In the EDL muscle, the KD also increased rates of FA oxidation, although it did so without altering its ketolytic capacity when compared to HFS feeding. In conclusion, even though obesogenic and ketogenic diets have elevated contents of fat and alter whole-body substrate partitioning, these two dietary interventions are associated with opposite outcomes with respect to skeletal muscle metabolic flexibility.

Hexokinase-linked glycolytic overload and unscheduled glycolysis in hyperglycemia-induced pathogenesis of insulin resistance, beta-cell glucotoxicity, and diabetic vascular complications.

Hyperglycemia is a risk factor for the development of insulin resistance, beta-cell glucotoxicity, and vascular complications of diabetes. We propose the hypothesis, hexokinase-linked glycolytic overload and unscheduled glycolysis, in explanation. Hexokinases (HKs) catalyze the first step of glucose metabolism. Increased flux of glucose metabolism through glycolysis gated by HKs, when occurring without concomitant increased activity of glycolytic enzymes-unscheduled glycolysis-produces increased levels of glycolytic intermediates with overspill into effector pathways of cell dysfunction and pathogenesis. HK1 is saturated with glucose in euglycemia and, where it is the major HK, provides for basal glycolytic flux without glycolytic overload. HK2 has similar saturation characteristics, except that, in persistent hyperglycemia, it is stabilized to proteolysis by high intracellular glucose concentration, increasing HK activity and initiating glycolytic overload and unscheduled glycolysis. This drives the development of vascular complications of diabetes. Similar HK2-linked unscheduled glycolysis in skeletal muscle and adipose tissue in impaired fasting glucose drives the development of peripheral insulin resistance. Glucokinase (GCK or HK4)-linked glycolytic overload and unscheduled glycolysis occurs in persistent hyperglycemia in hepatocytes and beta-cells, contributing to hepatic insulin resistance and beta-cell glucotoxicity, leading to the development of type 2 diabetes. Downstream effector pathways of HK-linked unscheduled glycolysis are mitochondrial dysfunction and increased reactive oxygen species (ROS) formation; activation of hexosamine, protein kinase c, and dicarbonyl stress pathways; and increased Mlx/Mondo A signaling. Mitochondrial dysfunction and increased ROS was proposed as the initiator of metabolic dysfunction in hyperglycemia, but it is rather one of the multiple downstream effector pathways. Correction of HK2 dysregulation is proposed as a novel therapeutic target. Pharmacotherapy addressing it corrected insulin resistance in overweight and obese subjects in clinical trial. Overall, the damaging effects of hyperglycemia are a consequence of HK-gated increased flux of glucose metabolism without increased glycolytic enzyme activities to accommodate it.

Effects of phosphoglycerate kinase 1 and pyruvate kinase M2 on metabolism and physiochemical changes in postmortem muscle

The objective of this work was to investigate the influence of phosphoglycerate kinase-1 (PGK1) and pyruvate kinase-M2 (PKM2) activity on glycolysis, myofibrillar proteins, calpain system, and apoptosis pathways of postmortem muscle. The activity of PGK1 and PKM2 was regulated by their inhibitors and activators to construct the postmortem glycolysis vitro model and then incubated at 4 °C for 24 h. The results showed that compared to PGK1 and PKM2 inhibitors groups, the addition of PGK1 and PKM2 activators could accelerate glycogen consumption, ATP and lactate production, while declining pH value. Moreover, the addition of PGK1 and PKM2 activators could increase desmin degradation, μ-calpain activity, and caspase-3 abundance. Interestingly, troponin-T degradation was significantly increased both in PKM2 inhibitor and activator groups. It was suggested that PGK1 and PKM2 might be used as robust indicators to regulate meat quality by affecting the glycolysis, myofibrillar proteins, μ-calpain and apoptosis pathways in postmortem muscle.

Multi-omics reveals changed energy metabolism of liver and muscle by caffeine after mice swimming.

In recent years, numerous studies have investigated the effects of caffeine on exercise, and provide convincing evidence for its ergogenic effects on exercise performance. However, the precise mechanisms underlying these ergogenic effects remain unclear. In this study, an exercise swimming model was conducted to investigate the effects of orally administered with caffeine before swimming on the alterations of proteome and energy metabolome of liver and muscle after swimming. We found proteins in liver, such as S100a8, S100a9, Gabpa, Igfbp1 and Sdc4, were significantly up-regulated, while Rbp4 and Tf decreased after swimming were further down-regulated in caffeine group. The glycolysis and pentose phosphate pathways in liver and muscle were both significantly down-regulated in caffeine group. The pyruvate carboxylase and amino acid levels in liver, including cysteine, serine and tyrosine, were markedly up-regulated in caffeine group, exhibiting a strong correlation with the increased pyruvic acid and oxaloacetate levels in muscle. Moreover, caffeine significantly decreased the lactate levels in both liver and muscle after swimming, potentially benefiting exercise performance.

Effect of heat stress on meat quality: A review

One of the stressful situations for livestock and poultry is heat stress (HS), which has a detrimental effect on animal physiology, production, and product quality. Because of their quick development and metabolism, high production, and species-specific characteristics including decreased sweating, the protective layer of skin, and rumen fermentation, some varieties of livestock are more vulnerable to heat stress than others. Muscle that is pale, soft and exudative (PSE) and lacks the ability to retain water can be produced by short-term heat stress that occurs pre-slaughter. This stress increases muscle glycogenolysis. In contrast, animals that experience prolonged heat stress have lower muscle glycogen reserves, which results in meat that is dark, firm, and dry (DFD), has a high final pH and water retention capacity. Moreover, HS causes shortened shelf life, oxidative stress. According to a recent study, HS might plausibly modify the rate and amount of postmortem muscle glycolysis and the resulting pH, which can impact the physiological and metabolic disturbances that occur in live animals as well as the features of carcasses and meat quality. This review reflects the effect of heat stress on meat quality.

Downregulation of mitochondrial metabolism is a driver for fast skeletal muscle loss during mouse aging

Skeletal muscle aging is characterized by the loss of muscle mass, strength and function, mainly attributed to the atrophy of glycolytic fibers. Underlying mechanisms driving the skeletal muscle functional impairment are yet to be elucidated. To unbiasedly uncover its molecular mechanisms, we recurred to gene expression and metabolite profiling in a glycolytic muscle, Extensor digitorum longus (EDL), from young and aged C57BL/6JRj mice. Employing multi-omics approaches we found that the main age-related changes are connected to mitochondria, exhibiting a downregulation in mitochondrial processes. Consistent is the altered mitochondrial morphology. We further compared our mouse EDL aging signature with human data from the GTEx database, reinforcing the idea that our model may recapitulate muscle loss in humans. We are able to show that age-related mitochondrial downregulation is likely to be detrimental, as gene expression signatures from commonly used lifespan extending interventions displayed the opposite direction compared to our EDL aging signature.

D-galactose might mediate some of the skeletal muscle hypertrophy-promoting effects of milk-A nutrient to consider for sarcopenia?

Sarcopenia is a process of progressive aging-associated loss of skeletal muscle mass (SMM) recognized as a serious global health issue contributing to frailty and increased all-cause mortality. Exercise and nutritional interventions (particularly intake of dairy products and milk) demonstrate good efficacy, safety, and broad applicability. Here, we propose that at least some of the well-documented favorable effects of milk and milk-derived protein supplements on SMM might be mediated by D-galactose, a monosaccharide present in large quantities in milk in the form of disaccharide lactose (milk sugar). We suggest that ingestion of dairy products results in exposure to D-galactose in concentrations metabolized primarily via the Leloir pathway with the potential to (i) promote anabolic signaling via maintenance of growth factor (e.g., insulin-like growth factor 1 [IGF-1]) receptor mature glycosylation patterns; and (ii) provide extracellular (liver glycogen) and intracellular substrates for short (muscle glycolysis) and long-term (muscle glycogen, intramyocellular lipids) energy availability. Additionally, D-galactose might optimize the metabolic function of skeletal muscles by increasing mitochondrial content and stimulating glucose and fatty acid utilization. The proposed potential of D-galactose to promote the accretion of SMM is discussed in the context of its therapeutic potential in sarcopenia.

Comparative effects of phosphorylation and acetylation on glycolysis and myofibrillar proteins degradation in postmortem muscle

The study investigated the different effects between protein phosphorylation and acetylation on glycolytic enzyme activity and myofibrillar protein degradation. Lamb longissimus thoracis lumborum muscles were homogenized and then inhibitors were added for incubation at 4 °C. Phosphatase inhibitor was added to produce a high phosphorylation level (PI group) and lysine deacetylase inhibitor was added to produce a high acetylation level (DI group). The lactate and ATP content in the PI group was inhibited compared with that in the DI group (P < 0.05). Phosphofructokinase (PFK) activity was negatively related with the phosphorylation level and was positively related with the acetylation level in the DI group (P < 0.05). The degradation of troponin T and desmin of the DI group were restrained when compared to that in the PI group (P < 0.05). Compared with initial PFK and desmin, the simulation of phosphorylation and acetylation of PFK and desmin showed different electrostatic potential at the surface and a more unstable structure. The phosphorylation level of the DI group was increased, suggesting that the changes of protein acetylation altered protein phosphorylation. In conclusion, compared with protein phosphorylation, protein acetylation had a greater effect on promoting glycolysis and inhibiting protein degradation.

GADD45A is a mediator of mitochondrial loss, atrophy, and weakness in skeletal muscle.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle-specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

Improved endurance capacity of diabetic mice during SGLT2 inhibition: Role of AICARP, an AMPK activator in the soleus.

Diabetes is associated with an increased risk of deleterious changes in muscle mass and function or sarcopenia, leading to physical inactivity and worsening glycaemic control. Given the negative energy balance during sodium-glucose cotransporter-2 (SGLT2) inhibition, whether SGLT2 inhibitors affect skeletal muscle mass and function is a matter of concern. However, how SGLT2 inhibition affects the skeletal muscle function in patients with diabetes remains insufficiently explored. We aimed to explore the effects of canagliflozin (CANA), an SGLT2 inhibitor, on skeletal muscles in genetically diabetic db/db mice focusing on the differential responses of oxidative and glycolytic muscles. Db/db mice were treated with CANA for 4weeks. We measured running distance and handgrip strength to assess skeletal muscle function during CANA treatment. At the end of the experiment, we performed a targeted metabolome analysis of the skeletal muscles. CANA treatment improved the reduced endurance capacity, as revealed by running distance in db/db mice (414.9±52.8 vs. 88.7±22.7m, P<0.05). Targeted metabolome analysis revealed that 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate (AICARP), a naturally occurring AMP-activated protein kinase (AMPK) activator, increased in the oxidative soleus muscle (P<0.05), but not in the glycolytic extensor digitorum longus muscle (P=0.4376), with increased levels of AMPK phosphorylation (P<0.01). This study highlights the potential role of the AICARP/AMPK pathway in oxidative rather than glycolytic skeletal muscles during SGLT2 inhibition, providing novel insights into the mechanism by which SGLT2 inhibitors improve endurance capacity in patients with type 2 diabetes.

383 Effect of Feeding Ractopamine to Finisher Lard-Type Mangalica Pigs on Longissimus Muscle Fiber Myosin-Heavy Chain Isoform Expression and Cross-Sectional Area

Abstract Heritage breeds such as Mangalica pigs are gaining popularity in US niche markets and currently sold as high-quality meat due to greater meat quality characteristics. Research has demonstrated that feeding the β-adrenergic agonist ractopamine hydrochloride (RAC), stimulates hypertrophic growth of type-2, fast-twitch glycolytic muscle fibers via mTOR signaling resulting in greater myofibrillar protein synthesis. Therefore, the current study evaluated the effect of feeding RAC to Mangalica pigs during the finisher phase on longissimus muscle fiber myosin heavy-chain (MHC) isoform expression and cross-sectional area. Mangalica pigs were randomly assigned finisher diets for 21 d with 20 ppm (RAC; n = 8) or without (CTL; n = 6). Longissimus muscle samples were collected during carcass fabrication, flash-frozen in liquid nitrogen and stored at -80 °C. Immunofluorescence staining of 5-µm-thick cryosections to identify Type-1 (Developmental Studies Hybridoma Bank (DSHB) antibody Ba-D5), Type-2a (DSHB SC-71), and 2x (DSHB BF-35) MHC positive fibers. Type 2b fibers were determined by subtraction. Fibers in each image (≥ 150) were assessed for fiber type and their cross-sectional area (CSA) at 200X magnification. Data were analyzed as a one-way ANOVA using the GLIMMIX procedure of SAS V9.4. Means were separated with the PDIFF option and declared different when P ≤ 0.05. Tendencies were declared when 0.0501 ≤ P ≤ 0.1000. Feeding RAC decreased the density of Type 2a (P = 0.0136) and Type 2b (P = 0.0186) fibers in the longissimus muscle of Mangalica pigs. A tendency for RAC to reduce the density of Type 1, slow-twitch (6.61 vs. 16.14 fiber per mm2; P = 0.0505) fibers and increase the density of primarily oxidative fibers expressing a combination of Type 1 + Type 2a MHC isoforms (10.23 vs. 2.88%; P = 0.0524) was also observed. When analyzed as a proportion of total fibers, RAC reduced the proportion of fibers expressing only Type 2a (11.8 vs. 6.70%; P = 0.0468) and Type 2b (0.19 vs. 1.32%; P = 0.0101) MHC isoforms but increased the percentage of fibers expressing both Type 1 + Type 2a MHC isoforms (4.66 vs. 1.13%; P = 0.0280). Feeding RAC did not impact the density or proportion of fibers expressing Type 2x MHC (P ≥ 0.386). Mean longissimus muscle fiber CSA was not impacted by feeding Mangalica pigs RAC for 21 d (P = 0.4815). The CSA of the smallest and largest fibers nor fiber distribution were influenced by RAC inclusion in the diet of Mangalica pigs (P ≥ 0.484). In conclusion, feeding RAC Mangalica pigs impacted fiber MHC isoform expression in the longissimus muscle without altering fiber CSA. Future studies will explore the impact of RAC on the CSA of the various fiber types as well as the impact on other muscles in the Mangalica pig.

Angiogenic potential of skeletal muscle derived extracellular vesicles differs between oxidative and glycolytic muscle tissue in mice

Skeletal muscle fibers regulate surrounding endothelial cells (EC) via secretion of numerous angiogenic factors, including extracellular vesicles (SkM-EV). Muscle fibers are broadly classified as oxidative (OXI) or glycolytic (GLY) depending on their metabolic characteristics. OXI fibers secrete more pro-angiogenic factors and have greater capillary densities than GLY fibers. OXI muscle secretes more EV than GLY, however it is unknown whether muscle metabolic characteristics regulate EV contents and signaling potential. EVs were isolated from primarily oxidative or glycolytic muscle tissue from mice. MicroRNA (miR) contents were determined and endothelial cells were treated with OXI- and GLY-EV to investigate angiogenic signaling potential. There were considerable differences in miR contents between OXI- and GLY-EV and pathway analysis identified that OXI-EV miR were predicted to positively regulate multiple endothelial-specific pathways, compared to GLY-EV. OXI-EV improved in vitro angiogenesis, which may have been mediated through nitric oxide synthase (NOS) related pathways, as treatment of endothelial cells with a non-selective NOS inhibitor abolished the angiogenic benefits of OXI-EV. This is the first report to show widespread differences in miR contents between SkM-EV isolated from metabolically different muscle tissue and the first to demonstrate that oxidative muscle tissue secretes EV with greater angiogenic signaling potential than glycolytic muscle tissue.

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

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. 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. 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. 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.

Effects of the order of endurance and high-intensity interval exercise in combined training on mouse skeletal muscle metabolism.

Endurance exercise (EE) mainly improves oxidative capacity, whereas high-intensity interval exercise (HIIE) also improves glycolytic capacity. There is growing evidence that suggests that combining EE with HIIE can lead to improved athletic performance and fitness outcomes compared with either form of exercise alone. This study aimed to elucidate whether the order in which EE and HIIE are performed in combined training affects oxidative metabolism and glycolysis in mouse skeletal muscle. Male ICR mice at 7 wk of age were divided into three groups: control (CON), EE-HIIE, and HIIE-EE. The total training period was 3 wk (3 times/week). Mice performed running on a treadmill as endurance exercise and swimming with a weight load of 10% of body weight as high-intensity interval exercise. EE before HIIE (EE-HIIE) improved running performance in the maximal EE capacity test (all-out test) and partly enhanced the expression levels of molecular signals involved in glycolysis compared with HIIE before EE (HIIE-EE). The order of exercise did not, however, impact the expression of proteins related to mitochondrial dynamics, including those involved in the morphological changes of mitochondria through repeated fusion and fission, as well as oxidative energy metabolism. The findings suggest that the order of exercise has no significant impact on the expression of proteins associated with glycolytic and oxidative energy metabolism. Nevertheless, our results indicate that the order of EE-HIIE may enhance running performance.