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The size of the satellite cell pool is reduced in estradiol (E2)-deficient female mice and humans. Here, we use a combination of in vivo and in vitro approaches to identify mechanisms, whereby E2 deficiency impairs satellite cell maintenance. By measuring satellite cell numbers in mice at several early time points postovariectomy (Ovx), we determine that satellite cell numbers decline by 33% between 10 and 14 days post-Ovx in tibialis anterior and gastrocnemius muscles. At 14 days post-Ovx, we demonstrate that satellite cells have a reduced propensity to transition from G0/G1 to S and G2/M phases, compared with cells from ovary-intact mice, associated with changes in two key satellite cell cycle regulators, ccna2 and p16INK4a. Further, freshly isolated satellite cells treated with E2 in vitro have 62% greater cell proliferation and require less time to complete the first division. Using clonal and differentiation assays, we measured 69% larger satellite cell colonies and enhanced satellite cell-derived myoblast differentiation with E2 treatment compared with vehicle-treated cells. Together, these results identify a novel mechanism for preservation of the satellite cell pool by E2 via promotion of satellite cell cycling.

The importance of defining sex differences across various biological and physiological mechanisms is more pervasive now than it has been over the past 15–20 years. As the muscle biology field pushes to identify small molecules and interventions to prevent, attenuate, or even reverse muscle wasting, we must consider the effect of sex as a biological variable. It should not be assumed that a therapeutic will affect males and females with equal efficacy or equivalent target affinities under conditions where muscle wasting is observed. With that said, it is not surprising to find that we have an unclear or even a poor understanding of the effects of sex or sex hormones on muscle wasting conditions. Although recent investigations are beginning to establish experimental approaches that will allow investigators to assess the impact of sex-specific hormones on muscle wasting, the field still needs rigorous scientific tools that will allow the community to address critical hypotheses centered around sex hormones. The focus of this review is on female sex hormones, specifically estrogens, and the roles that these hormones and their receptors play in skeletal muscle wasting conditions. With the overall review goal of assembling the current knowledge in the area of sexual dimorphism driven by estrogens with an effort to provide insights to interested physiologists on necessary considerations when trying to assess models for potential sex differences in cellular and molecular mechanisms of muscle wasting.

Elevated mitochondrial hydrogen peroxide (H(2)O(2)) emission and an oxidative shift in cytosolic redox environment have been linked to high-fat-diet-induced insulin resistance in skeletal muscle. To test specifically whether increased flux through mitochondrial fatty acid oxidation, in the absence of elevated energy demand, directly alters mitochondrial function and redox state in muscle, two genetic models characterized by increased muscle β-oxidation flux were studied. In mice overexpressing peroxisome proliferator-activated receptor-α in muscle (MCK-PPARα), lipid-supported mitochondrial respiration, membrane potential (ΔΨ(m)), and H(2)O(2) production rate (JH(2)O(2)) were increased, which coincided with a more oxidized cytosolic redox environment, reduced muscle glucose uptake, and whole body glucose intolerance despite an increased rate of energy expenditure. Similar results were observed in lipin-1-deficient, fatty-liver dystrophic mice, another model characterized by increased β-oxidation flux and glucose intolerance. Crossing MCAT (mitochondria-targeted catalase) with MCK-PPARα mice normalized JH(2)O(2) production, redox environment, and glucose tolerance, but surprisingly, both basal and absolute insulin-stimulated rates of glucose uptake in muscle remained depressed. Also surprising, when placed on a high-fat diet, MCK-PPARα mice were characterized by much lower whole body, fat, and lean mass as well as improved glucose tolerance relative to wild-type mice, providing additional evidence that overexpression of PPARα in muscle imposes more extensive metabolic stress than experienced by wild-type mice on a high-fat diet. Overall, the findings suggest that driving an increase in skeletal muscle fatty acid oxidation in the absence of metabolic demand imposes mitochondrial reductive stress and elicits multiple counterbalance metabolic responses in an attempt to restore bioenergetic homeostasis. NEW & NOTEWORTHY Prior work has suggested that mitochondrial dysfunction is an underlying cause of insulin resistance in muscle because it limits fatty acid oxidation and therefore leads to the accumulation of cytotoxic lipid intermediates. The implication has been that therapeutic strategies to accelerate β-oxidation will be protective. The current study provides evidence that genetically increasing flux through β-oxidation in muscle imposes reductive stress that is not beneficial but rather detrimental to metabolic regulation.

Objective Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development. Methods A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKOism). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women. Results Twelve weeks of HFD exposure had no differential effects on body composition, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2O2 emission potential was not affected in the ERαKOism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity. Conclusions Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete., Highlights • Induced skeletal muscle specific ERαKO (ERαKOism) examines the role of ERα without confounding factors of development. • Skeletal muscle glucose uptake is not impaired in ERαKOism. • Skeletal muscle mitochondrial function is not impaired in ERαKOism. • ERαKD in human myotubes does not severely affect mitochondrial respiratory capacity.

Denervation rapidly induces insulin resistance (i.e., impairments in insulin-stimulated glucose uptake and signaling proteins) in skeletal muscle. Surprisingly, whether this metabolic derangement is long-lasting is presently not clear. The main goal of this study was to determine if insulin resistance is sustained in both oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles following long-term (28 days) denervation. Mouse hindlimb muscles were denervated via unilateral sciatic nerve resection. Both soleus and EDL muscles atrophied ~40%. Strikingly, while denervation impaired submaximal insulin-stimulated [3H]-2-deoxyglucose uptake ~30% in the soleus, it enhanced submaximal (~120%) and maximal (~160%) insulin-stimulated glucose uptake in the EDL. To assess possible mechanism(s), immunoblots were performed. Denervation did not consistently alter insulin signaling (e.g., p-Akt (Thr308):Akt, p-TBC1D1 [phospho-Akt substrate (PAS)]:TBC1D1, or p-TBC1D4 (PAS):TBC1D4) in either muscle. However, denervation decreased glucose transporter 4 (GLUT4) levels ~65% in the soleus but increased them ~90% in the EDL. To assess the contribution of GLUT4 to the enhanced EDL muscle glucose uptake, muscle-specific GLUT4 knockout mice were examined. Loss of GLUT4 prevented the denervation-induced increase in insulin-stimulated glucose uptake. In conclusion, the denervation results sustained insulin resistance in the soleus but enhanced insulin sensitivity in the EDL due to increased GLUT4 protein levels.

Estradiol (E2) influences the inflammatory response. Research has shown that skeletal muscle chemokine mRNA expression and neutrophil infiltration after a traumatic freeze injury are blunted in ovariectomized mice treated with placebo compared to E2-treated mice. How E2 deficiency alters the acute muscle inflammatory response is unclear with potential mechanisms including alterations in the number of circulating leukocytes, leukocyte chemotaxis, and/or muscle chemokine expression. To investigate these possibilities, female C57BL/6J mice underwent a sham or ovariectomy (OVX) surgery, 21–28 d later tibialis anterior (TA) muscles were freeze injured. TA muscles and blood were collected 24 h post injury. Total white blood cell (149%) and monocytes (427%) were elevated in OVX compared to Sham mice but there was no difference in the neutrophil counts (p≥0.64). To determine if E2 deficiency impacts leukocyte chemotaxis, isolated leukocytes from bone marrow of non-injured Sham or OVX mice were placed in a transwell migration assay. OVX leukocytes had greater chemotaxis towards CXCL1 (37%), CXCL2 (43%), and CXCL5 (27%) than Sham. To examine if E2 deficiency alters muscle CXCL1 and CXCL5 protein content contributing to chemotaxis, ELISA assays were performed. Surprisingly, muscle CXCL1 and CXCL5 contents were similar between OVX and Sham. Collectively, mice lacking E2 have relatively high blood leukocytes and chemotaxis suggesting a greater acute inflammatory response which appears not to be driven by changes in muscle chemokine expression. Additional studies need to consider possible negative regulators of the acute inflammatory response in OVX muscle. Supported by DAL and GW (R01AG031743 & R01AG062899), and SLM (T32AR007612)

Aerobic exercise training and resistance exercise training are both well-known for their ability to improve human health; especially in individuals with type 2 diabetes. However, there are critical differences between these two main forms of exercise training and the adaptations that they induce in the body that may account for their beneficial effects. This article reviews the literature and highlights key gaps in our current understanding of the effects of aerobic and resistance exercise training on the regulation of systemic glucose homeostasis, skeletal muscle glucose transport and skeletal muscle glucose metabolism.

The intracellular signalling kinases Akt/protein kinase B (Akt), protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) are phosphorylated in response to increased mechanical force or perfusion rate in cultured endothelial cells or isolated blood vessels. All three kinases phosphorylate endothelial nitric oxide synthase (eNOS) on serine (S) 1177, while Akt and PKA additionally phosphorylate eNOS on S617 and S635 respectively. Although these kinases might contribute to subsequent activation of eNOS during dynamic exercise, the specific mediators of exercise-induced eNOS phosphorylation and activation in vivo are unknown. We determined the impact of 50 min of treadmill running on the phosphorylation of Akt, AMPK, cyclic adenosine monophosphate response element binding protein (CREB – a target of PKA) and eNOS (S 1177, 635 and 617 and threonine (T) 495) in the presence or absence of pharmacological inhibition of PI3 kinase (PI3K) and Akt signalling using wortmannin. Compared to arteries from sedentary mice, eNOS enzyme activity was greater in vessels from treadmill-running animals and was associated with increased phosphorylation of Akt (S473), CREB (S133), AMPK (T172), and eNOS at S1177 and S617 but not at S635 or T495. These data suggest that Akt signalling is a major mediator of eNOS activation. To confirm this, treadmill-running was performed in the presence of vehicle (DMSO) or PI3K inhibition. Compared to results from sedentary mice, vascular Akt phosphorylation and eNOS phosphorylation at S617 during treadmill-running were prevented by wortmannin but not vehicle treatment, whereas exercise-related increases in AMPK and CREB phosphorylation were similar between groups. Arterial eNOS phosphorylation at S1177 increased during exercise after wortmannin treatment relative to values obtained from sedentary animals, but the elevation was blunted by ∼50% compared to results from vehicle-treated mice. These findings indicate that Akt and AMPK contribute importantly to vascular eNOS S1177 phosphorylation during treadmill-running, and that AMPK is sufficient to activate p-eNOS S1177 in the presence of PI3K inhibition.

Since instating Title IX of the Education Amendments of 1972, there has been a significant increase in sports participation and athletic opportunities among women (1). While it is still more common for younger than older women to engage in athletic competition, the participation of older women is growing, with over 50 countries sponsoring master athletes events (2). While aging is associated with a decrease in metabolic and physiologic function, competitive athletic women may experience more gradual declines. These declines can be slowed further, by combining adequate dietary intake with proper exercise training. Therefore, this chapter will 1) discuss how aging influences physiologic and metabolic adaptations of highly trained women athletes and 2) explore how nutrition recommendations may change with exercise and the possible benefit of supplementation of micronutrients to improve athletic performance.

The intracellular signalling kinases Akt/protein kinase B (Akt), protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) are phosphorylated in response to increased mechanical force or perfusion rate in cultured endothelial cells or isolated blood vessels. All three kinases phosphorylate endothelial nitric oxide synthase (eNOS) on serine (S) 1177, while Akt and PKA additionally phosphorylate eNOS on S617 and S635 respectively. Although these kinases might contribute to subsequent activation of eNOS during dynamic exercise, the specific mediators of exercise-induced eNOS phosphorylation and activation in vivo are unknown. We determined the impact of 50 min of treadmill running on the phosphorylation of Akt, AMPK, cyclic adenosine monophosphate response element binding protein (CREB - a target of PKA) and eNOS (S 1177, 635 and 617 and threonine (T) 495) in the presence or absence of pharmacological inhibition of PI3 kinase (PI3K) and Akt signalling using wortmannin. Compared to arteries from sedentary mice, eNOS enzyme activity was greater in vessels from treadmill-running animals and was associated with increased phosphorylation of Akt (S473), CREB (S133), AMPK (T172), and eNOS at S1177 and S617 but not at S635 or T495. These data suggest that Akt signalling is a major mediator of eNOS activation. To confirm this, treadmill-running was performed in the presence of vehicle (DMSO) or PI3K inhibition. Compared to results from sedentary mice, vascular Akt phosphorylation and eNOS phosphorylation at S617 during treadmill-running were prevented by wortmannin but not vehicle treatment, whereas exercise-related increases in AMPK and CREB phosphorylation were similar between groups. Arterial eNOS phosphorylation at S1177 increased during exercise after wortmannin treatment relative to values obtained from sedentary animals, but the elevation was blunted by approximately 50% compared to results from vehicle-treated mice. These findings indicate that Akt and AMPK contribute importantly to vascular eNOS S1177 phosphorylation during treadmill-running, and that AMPK is sufficient to activate p-eNOS S1177 in the presence of PI3K inhibition.

Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues ( TTr-IR −/− ) or mice with genetic ablation of Akt1 (Akt1−/−). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulin-mediated eNOS phosphorylation in TTr-IR −/− mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1−/− mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulin-stimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid-mediated impairment of eNOS phosphorylation.