Early Markers of Cardiac and Skeletal Muscle Metabolic Derangement in the Apc(min/+) Male Mouse

Existing techniques for the non-invasive invivo study of dynamic changes in skeletal muscle metabolism are subject to several limitations, for example, poor signal-to-noise ratios which result in long scan times and low temporal resolution. Hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy (HP-MRS) allows the real-time visualization of invivo metabolic processes and has been used extensively to study cardiac metabolism, but has not resolved oxidative phosphorylation in contracting skeletal muscle. Combining HP-MRS with an invivo muscle hindlimb electrical stimulation protocol that modelled voluntary exercise to exhaustion allows the simultaneous real-time assessment of both metabolism and function. The aim of this work was to validate the sensitivity of the method by assessing pyruvate dehydrogenase (PDH) flux in resting vs. working muscle: measuring the production of bicarbonate (H13CO3 -), a byproduct of the PDH-catalysed conversion of [1-13C]pyruvate to acetyl-CoA. Mice (n = 6) underwent two hyperpolarized [1-13C]pyruvate injections with 13C MR spectra obtained from the gastrocnemius muscle to measure conversion of pyruvate to lactate and bicarbonate, one before the stimulation protocol with the muscle in a resting state and one during the stimulation protocol. The muscle force generated during stimulation was also measured, and 13C MRS undertaken at a point of ~50% fatigue. We observed an increase in the bicarbonate/pyruvate ratio by a factor of ~1.5×, in the lactate/pyruvate ratio of ~2.7×, together with an increase in total carbon (~1.5×) that we attribute to perfusion. This demonstrates profound differences in metabolism between the resting and exercising states. These data therefore serve as preliminary evidence that hyperpolarized 13C MRS is an effective invivo probe of PDH flux in exercising skeletal muscle and could be used in future studies to examine changes in muscle metabolism in states of disease and altered nutrition.

What is the central question of this study? Cachexia causes severe changes in skeletal muscle metabolism and function and is a key predictor of negative outcomes in cancer patients: what are the changes in whole animal energy metabolism and mitochondria in skeletal muscle? What is the main finding and its importance? There is decreased whole animal energy expenditure in mice with cachexia. They displayed highly dysmorphic mitochondria and mitophagy in skeletal muscle. Cachexia causes changes in skeletal muscle metabolism. Mice with MDA-MB-231 breast cancer bone metastases and cachexia have decreased whole animal energy metabolism and increased skeletal muscle mitophagy. We examined whole animal energy metabolism by indirect calorimetry in mice with MDA-MB-231 breast cancer bone metastases, and showed decreased energy expenditure. We also examined skeletal muscle mitochondria and found that mitochondria in mice with MDA-MB-231 bone metastases are highly dysmorphic and have altered protein markers of mitochondrial biogenesis and dynamics. In addition, LC3B protein was increased in mitochondria of skeletal muscle from cachectic mice, and colocalized with the mitochondrial protein Tom20. Our data demonstrate the importance of mitophagy in cachexia. Understanding these changes will help contribute to defining treatments for cancer cachexia.

Hypoxia-Inducible Factor 1 in Lower Limb Ischemia

Cancer cachexia is a metabolic and wasting disease that occurs in up to 80% of cancer patients. Currently, there are no clear diagnostic criteria, its effects are irreversible, and it cannot be treated. Most patients progress undetected to late stages of cancer cachexia, stop responding to traditional treatment, and die without hope for intervention. As such, there is a great need for accurate biomarkers. To determine the metabolic signature of cancer cachexia in muscle to assist with metabolic biomarker discovery, heart and gastrocnemius tissues from 15‐week‐old male ApcMin mice (Apc+) and litter‐matched non‐carrier mice (WT) were analyzed by non‐targeted GC/MS metabolomics. Analysis identified 136 metabolites in the hearts of cachectic (Apc+) mice with 3 metabolites significantly (P<0.05) decrease compared to WT mice: campesterol, hypoxanthine, and alanine. Variable Importance in Projection (VIP) analysis, which shows which metabolites contributes most significantly to group, and pathway analysis revealed that linoleic acid metabolism; biosynthesis of unsaturated fatty acids; arginine metabolism; taurine/hypotaurine metabolism; and arginine biosynthesis pathways were affected by cancer cachexia. Analysis also identified 135 metabolites in the gastrocnemius of cachectic (Apc+) mice with 4 significant (P<0.05) metabolites: 1,5‐anhydroglucitol and fumaric acid were decreased, lysine and ribose‐5‐phospate were increased. VIP analysis and pathway analysis showed linoleic acid metabolism; biosynthesis of unsaturated fatty acids; taurine/hypotaurine metabolism; glycolysis/gluconeogenesis metabolism; and arginine biosynthesis pathways were affected by cancer cachexia. Taken together, these data demonstrate altered fatty acid metabolism, arginine metabolism, glycolysis, taurine metabolism, and proline metabolism in hearts and skeletal muscle of cachectic mice. Interestingly, skeletal muscle shows an increase in fatty acid metabolism and decrease in glucose and 1,5‐anhydroglucitol while the heart shows an opposite response. Differently, cachectic hearts showed an increase in glycolytic metabolites compared to skeletal muscle. This indicates a non‐preferential fuel switch in the heart towards less energetically favorable glycolysis (vs fatty acid metabolism). Additionally, cachectic heart exhibited decreased levels of taurine and campesterol which have been shown to be cardioprotective. Finally, both the heart and skeletal muscle shows an increase in proline metabolism which has been shown to be upregulated during times of metabolic stress and arginine metabolism which plays a key role in inhibiting protein metabolism and promoting proteolysis. These data shed important light on the metabolic derangement associated with cancer cachexia that in turn lead to muscle and fat wasting. Such data may provide a valuable stepping stone in understanding the metabolic consequence of cancer cachexia as well as the identification of metabolic biomarkers.

Investigation of skeletal muscle denervation and reinnervation using magnetic resonance spectroscopy

Physical training improves skeletal muscle metabolism in patients with chronic heart failure

Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA-free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α-linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.

Skeletal muscle is an energetically demanding tissue that catabolizes a variety of different substrates, a phenomenon that is largely activity‐dependent. Lipids are an energetically‐rich macromolecule and, in this light, are advantageous for skeletal muscle performance and maintenance. It has long been established that lipids have dichotomous lasting effects on the body that appear to be, at least in part, species‐specific. While lipid‐related pathologies are largely associated with species such as saturated fatty acids, diacylglycerols, and ceramides, other lipid species, such as various unsaturated fatty acids, confer adaptive changes and may actually protect against diseases. Lipids have previously been linked to increased aerobic capacity in skeletal muscle, though the role of fatty acid quality in this context (e.g., degree of saturation) is less clear. In the present study, we used C2C12 cells, an immortalized line of mouse skeletal muscle cells, to better understand how polyunsaturated fatty acids (PUFAs), in combination with exercise, affect metabolism and protein concentration in skeletal muscle. Following standard proliferation protocols, mature myotubes were differentiated for 7 days in either control media or media supplemented with a 5% addition of linoleic acid (PUFA); additionally, to stimulate contractile activity, a subset of cell cohorts from each media group were exposed to caffeine for 20 minutes per day during differentiation days 4–7. All cells were harvested for protein on differentiation day seven. Protein concentration across cohorts was measured using a Bradford protein assay. Metabolic changes were characterized by running functional enzyme assays for lactate dehydrogenase (LDH) and citrate synthase (CS) to compare anaerobic and aerobic changes, respectively. Preliminary data show that non‐exercised cohorts have higher protein concentration when supplemented PUFA, and that exercise, both with and without PUFA supplementation, decreases protein concentration as compared to respective non‐exercised cohorts. Regarding enzyme activity, PUFA supplementation appears to decrease LDH activity in non‐exercised cells, while PUFA supplementation appears to increase LDH activity in exercised cells; CS activity also appears to change in PUFA supplemented cohorts. Collectively, these preliminary data suggest activity‐induced adaptive changes in skeletal muscle metabolism are contingent upon specific substrate availability, whereby PUFA availability may advantageously prime skeletal muscle for a higher overall metabolic capacity.Support or Funding InformationMcNair Scholars Program and The College of Saint ScholasticaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carnitine derivatives, such as propionyl-l-carnitine (PLC), have been shown to improve walking distance in patients with obstructive peripheral artery disease (PAOD). The aim of this study was to ascertain whether technetium-99m sestamibi leg scintigraphy may be a useful tool in the evaluation of changes in skeletal muscle metabolism induced by chronic therapy with PLC. Twenty patients with clinical and instrumental evidence of PAOD were randomly assigned to a 3-month period of therapy with either PLC or placebo. Rest 99mTc-sestamibi leg scintigraphy and echo-Doppler sonography were performed on all subjects immediately before and upon completion of the treatment period. At the end of the protocol the following results were observed in patients who underwent PLC administration: (a) a significant increase in both thigh and calf 99mTc-sestamibi uptake, in comparison with baseline values (P<0.001); (b) the absence of statistically significant modifications of Doppler blood flow indices of the lower limbs. In conclusion, after chronic administration of PLC, a significant increment in skeletal muscle uptake of 99mTc-sestamibi was demonstrated without any apparent change in regional blood flow. This fact, if proven in further studies, may suggest a role for this tracer as a non-invasive probe of tissue bioenergetics.

Skeletal muscle is the largest metabolic organ system in the human body. As such, metabolic dysfunction occurring in skeletal muscle impacts whole-body nutrient homeostasis. Macronutrient metabolism changes within the skeletal muscle with aging, and these changes are associated in part with age-related skeletal muscle remodeling. Moreover, age-related changes in skeletal muscle metabolism are affected differentially between males and females and are likely driven by changes in sex hormones. Intrinsic and extrinsic factors impact observed age-related changes and sex-related differences in skeletal muscle metabolism. Despite some support for sex-specific differences in skeletal muscle metabolism with aging, more research is necessary to identify underlying differences in mechanisms. Understanding sex-specific aging skeletal muscle will assist with the development of therapies to attenuate adverse metabolic and functional outcomes.

Skeletal muscle oxidative capacity is highly plastic, strongly associated with whole-body aerobic capacity (16, 18) and state of health. Loss of muscle oxidative capacity is associated with physical inactivity, aging and chronic disease (17), and has been implicated in the pathophysiology of obesity and diabetes (21). Evaluating these changes has traditionally been limited to invasive or costly assessments (biopsy or ³¹P MRS). To address this, Hamaoka and colleagues developed an innovative, non-invasive approach using near-infrared spectroscopy (NIRS) to quantitatively measure muscle oxygen consumption (mVO₂; 12) and use this to infer muscle oxidative capacity based on the mVO₂ recovery rate constant (k) (23; later modified 26). This technique has been subsequently used to interpret relative differences in oxidative capacity across a wide range of muscles, ages and disease states (Figure 1C). The purpose of this Viewpoint is to open a discussion on the principles, insights and potential pitfalls of using NIRS to measure k and infer muscle oxidative capacity.

Age-related changes in muscle architecture and metabolism in humans: The likely contribution of physical inactivity to age-related functional decline

Regional Variation in Skeletal Muscle and Adipose Tissue FDG Uptake Using PET/CT and Their Relation to BMI

To date equine skeletal muscle metabolism has centered on a limited number of candidate metabolites. As such, there is a dearth of information regarding global changes in skeletal muscle metabolism following exercise training. Thus, the purpose of this study was to determine the effects of exercise training on skeletal muscle metabolism through changes in the skeletal muscle metabolome in healthy, untrained Standardbred horses. In addition, we aimed to determine the extent to which protein metabolism is also altered through changes in the concentrations of plasma amino acids (AA). We hypothesized that training would significantly alter the skeletal muscle metabolome, as well as protein metabolism, as reflected in changes in the plasma AA profile. Eight untrained, Standardbred horses (n=4 mares, n=4 geldings; 3–9 yrs; 485 ± 13 kg, mean ± S.E.) underwent a 12‐week training program that significantly increased VO2 and exercise capacity (p&lt;0.05). Percutaneous needle biopsies were obtained from the M. gluteus medius in the untrained state (UT) and again following training (TR). Frozen muscle samples were submitted to Metabolon, Inc. for global metabolite profiling via UHPLC‐MS/MS. Blood obtained via jugular venipuncture was placed into EDTA tubes for the measurement of plasma AA concentrations via HPLC. For metabolomics analysis, differences between UT and TR samples were evaluated by two way‐ANOVA and Welch's t‐test for paired comparisons and considered significant at p&lt;0.05. For plasma AAs, differences were analyzed via Student's t‐test and considered significant at p&lt;0.05. Training resulted in significant alterations in lipid, branched‐chain amino acid (BCAA), and nucleotide metabolite classes in skeletal muscle. Of note, analysis revealed significant increases in the relative abundance of almost every identified FFA and complex lipid species in TR compared to UT muscle (p&lt;0.05), particularly the long‐chain (LC)‐acylcarnitines and diacylglycerides (DAGs) (1.5 to 2.3‐fold increases). Further, TR muscle exhibited increased fold‐changes (1.2 to 4.2‐fold) of C4 and several C5 BCAA‐derived acylcarnitines compared to UT (p&lt;0.05). In plasma, training caused a significant 36% increase in the concentration of total essential AAs (p&lt;0.05), of which and interestingly there was a significant increase in total BCAAs (42%, p&lt;0.05) and phenylalanine (27%, p&lt;0.05). In conclusion, exercise training significantly increased blood concentrations of total EAAs and BCAAs alongside significant increases in the relative abundances of certain skeletal muscle metabolites (i.e. LC‐acylcarnitines, BCAA‐derived acylcarnitines, and DAGs) that have been shown to be elevated during obesity and are proposed to play a role in the development of insulin resistance. These data underscore the complex nature of static metabolic signatures and the role that certain metabolites play in mediating health and disease. Future research should aim to utilize other sophisticated techniques that can address issues of metabolite flux and tissue utilization.Support or Funding InformationThis research was supported by: the New Jersey Institute for Food, Nutrition and Health (IFNH), USDA NIFA (NC 1184), the Rutgers University Equine Science Center (ESC), and the New Jersey Agriculture Experiment Station (NJAES).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

This Review focuses on currently available literature describing sex differences in skeletal muscle metabolism in humans, as well as highlighting current research gaps within the field. These discussions serve as a call for action to address the current lack of sufficient sex-balanced studies in skeletal muscle research, and the resulting limitations in understanding sex-specific physiological and pathophysiological responses. Although the participation of women in studies has increased, parity between the sexes remains elusive, affecting the validity of conclusions drawn from studies with limited numbers of participants. Changes in skeletal muscle metabolism contribute to the development of metabolic disease (such as type 2 diabetes mellitus), and maintenance of skeletal muscle mass is a key component for health and the ability to maintain an independent life during ageing. Exercise is an important factor in maintaining skeletal muscle health and insulin sensitivity, and offers promise for both prevention and treatment of metabolic disease. With the increased realization of the promise of precision medicine comes the need to increase patient stratification and improve the understanding of responses in different populations. In this context, a better understanding of sex-dependent differences in skeletal muscle metabolism is essential.