Indiana University-Purdue University Indianapolis (IUPUI) / Study has shown that plasma membrane (PM) cholesterol and cortical filamentous actin (F-actin) influence skeletal muscle glucose transport. Of fundamental and clinical interest is whether diabetogenic insults promote membrane/cytoskeletal dysfunction amendable for therapy. As exposure to excess fatty acid (FA)s induce glucose intolerance by mechanisms imperfectly understood, we tested if PM cholesterol/F-actin changes could contribute to FA-induced glucose transporter GLUT4 dysregulation in skeletal muscle. High-fat fed, insulin-resistant animals displayed elevated levels of skeletal muscle PM cholesterol and a loss in cortical F-actin, compared to normal-chow fed animals. Consistent with a PM cholesterol component of glucose intolerance, human skeletal muscle biopsies revealed an inverse correlation between PM cholesterol and whole-body glucose disposal. Mechanistically, exposure of L6 myotubes to the saturated FA palmitate induced an increase in PM cholesterol that destabilized actin filaments and decreased insulin-stimulated PM GLUT4 and glucose transport, which could be reversed with cholesterol lowering. Next, study tested if the lipid-lowering action of the antidiabetic AMP-activated protein kinase (AMPK) had a beneficial influence on PM cholesterol balance. Consistent with AMPK inhibition of 3-hydroxy-3-methylglutaryl CoA reductase, a rate-limiting enzyme of cholesterol synthesis, we found that AMPK activation promoted a significant reduction in PM cholesterol and amplified basal and insulin-stimulated GLUT4 translocation. A similar loss of PM cholesterol induced by β-cyclodextrin caused an analogous enhancement of GLUT4 regulation. Interestingly, PM cholesterol replenishment abrogated the AMPK effect on insulin, but not basal, regulation of GLUT4 translocation. Conversely, AMPK knockdown prevented the enhancement of both basal and insulin-stimulated GLUT4 translocation. As a whole these studies show PM cholesterol accrual and cortical F-actin loss uniformly in skeletal muscle from glucose-intolerant mice, swine, and humans. In vivo and in vitro dissection demonstrated this membrane/cytoskeletal derangement induces insulin resistance and is promoted by excess FAs. Parallel studies unveiled that the action of AMPK entailed lowering PM cholesterol that enhanced the regulation of GLUT4/glucose transport by insulin. In conclusion, these data are consistent with PM cholesterol regulation being an unappreciated aspect of AMPK signaling that benefits insulin-stimulated GLUT4 translocation during states of nutrient excess promoting PM cholesterol accrual.

Current study demonstrated the impact of heat therapy on skeletal muscle function in a model of Duchenne muscular dystrophy (DMD). The aim of this study was to: (1) examine the impact of treatment temperature on the skeletal muscle adaptation in DBA/2J mice; and (2) determine the impact of repeated HT for 3 consecutive weeks on body composition and skeletal muscle function in D2.mdx, a model of DMD. From study 1, we revealed that HT at 39℃ for 3 weeks significantly promoted relative muscle mass of both EDL and soleus muscle in DBA/2J mice. However, from study 2, HT at 39℃ for 3 weeks does not improve muscle function or increase muscle mass in a mouse model of DMD.

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In liver tissue, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of LKB1, MO25α/β, and STRADα/β proteins; however, this complex has yet to be characterized in skeletal muscle. In this report, we demonstrate the expression of the LKB1-MO25-STRAD AMPKK complex in adult skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these complex-forming proteins. To facilitate this characterization we used control (ctrl) and muscle-specific LKB1 knockout (LKB1-/-) mice. LKB1 detection in untreated ctrl and LKB1-/- muscle lysates revealed two protein bands at approximately 50 and 60 kDa; although, only the heavier band was significantly diminished in LKB1-/- samples (ctrl: 55±2.5 AU; LKB1-/-: 13±1.5 AU; p<0.01), suggesting that LKB1 is not represented at 50 kDa as cited previously. Detection of LKB1 at the higher molecular weight was further confirmed following purification of the AMPKK complex using polyethylene glycol (PEG) (ctrl: 43±5 AU; LKB1-/-: 8.4±4 AU; p<0.01). Following ion-exchange-fast protein liquid chromatography (FPLC) the low protein band was undetectable in ctrl and LKB1-/- fractions. Mass spectrometry of PEG-treated ctrl lysates confirmed LKB1 protein detection in the 60 kDa protein band while none was detected in the 50 kDa band. Co-immunoprecipitation assays demonstrated associations between all combinations of LKB1, MO25, and STRAD in LKB1-positive samples, confirming proper complex formation. Quantitative-PCR revealed significantly reduced expression of MO25α and STRADβ in LKB1-/- muscle. Lastly, detection of CaMKKα/β protein in ctrl and LKB1-/- muscle lysates confirmed the presence of another AMPKK in muscle. Interestingly, CaMKKβ protein is increased in LKB1-/- muscle (ctrl: 19±4.3 AU; LKB1-/-: 47±9.2 AU; p<0.05) without an increase in mRNA levels, suggesting compensation for null LKB1 expression. In all, these findings confirm the presence of the LKB1-MO25-STRAD complex in adult skeletal muscle, suggest a novel post-translational modification of LKB1, and identify a potential compensatory mechanism for loss of LKB1 protein in skeletal muscle.

To determine effects of iron deficiency on AMPK activation and signaling, as well as the AMPKα subunit composition in skeletal muscle, rats were fed a control (C=50-58 mg/kg Fe) or iron deficient (ID=2-6 mg/kg Fe) diet for 6-8 wks. Their respective hematocrits were 47.5% ± 1.0 and 16.5% ± 0.6. Iron deficiency resulted in 28.3% greater muscle fatigue (p<0.01) in response to 10 min of stimulation (1 twitch/sec) and was associated with a greater reduction in phosphocreatine (C: Resting 24.1 ± 0.9 micromol/g, Stim 13.1 ± 1.5 micromol/g; ID: Resting 22.7 ± 1.0 micromol/g, Stim 3.2 ± 0.7 micromol/g; p<0.01) and ATP levels (C: Resting 5.89 ± 0.48 micromol/g, Stim 6.03 ± 0.35 micromol/g; ID: Resting 5.51 ± 0.20 micromol/g, Stim 4.19 ± 0.47 micromol/g; p<0.05). AMPK activation increased with stimulation in muscles of C and ID animals. A reduction in Cytochrome c and other iron-dependent mitochondrial proteins was observed in ID animals (p<0.01). The AMPK catalytic subunit (alpha) was also examined because both isoforms are known to play different roles in responding to energy challenges. In ID animals, AMPK alpha2 subunit protein content was reduced to 71.6% of C (p<0.05), however this did not result in a significant difference in resting AMPK alpha2 activity. AMPK alpha1 protein was unchanged, however an overall increase in AMPK alpha1 activity was observed (C: 0.91 pmol/mg/min; ID: 1.63 pmol/mg/min; p<0.05). Resting phospho Acetyl CoA Carboxylase (pACC) was unchanged. This study indicates that chronic iron deficiency causes a shift in the expression of AMPK alpha subunit composition and potentially altered sensitivity to cellular energy challenges.

AMP-activated protein kinase (AMPK), a metabolic regulator, acts in opposition to many of the effects of aging and may provide insights into the development of sarcopenia. However, the effect of aging on AMPK activation is unclear. The purpose of this dissertation was to: 1) clarify the controversy concerning the activation of AMPK in response to endurance-like exercise in aged skeletal muscle; 2) address mechanisms for the age-associated alterations in AMPK activation; and 3) address the known benefits of chronic AICAR treatment in aged skeletal muscle. First, to clarify the effect of age on AMPK activation, young adult (YA) (8 mo.) and old (O) (30 mo.) male Fischer344 x Brown Norway F1 hybrid rats received an in situ bout of endurance-type contractions produced via electrical stimulation of the sciatic nerve (STIM). AMPK activation was attenuated in aging muscle as demonstrated by decreased AMPKα phosphorylation and AMPKα2 protein content and activity in O vs. YA muscle after STIM. In contrast, AMPKα1 content was greater in O vs. YA muscle, and α1 activity increased with STIM in O but not YA muscles. Second, the effect of age on the AMPK heterotrimer composition and nuclear localization was assessed as mechanisms for the altered AMPK activation. The AMPK heterotrimer composition was altered in aging skeletal muscle with lower AMPKγ2 and γ3 content and decreased association of AMPKγ3 with AMPKα1 and α2. Furthermore, activation of AMPK is known to increase translocation of AMPK to the nucleus in YA muscle; however, translocation of phosphorylated AMPK, AMPKα2, and AMPKγ3 were impaired in the aging rat muscle after STIM. Finally, chronic activation of AMPK with 5'-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is known to increase mitochondrial content, activate autophagy, and repress protein synthesis; pathways that are altered with aging. The known benefits of chronic AICAR treatment were assessed in YA (5 mo.) and O (23 mo.) male C57Bl/6 mice. Mice were treadmill tested prior to and after one month of AICAR treatment. In vitro muscle contractions were performed following AICAR treatment. AICAR treatment improved the O mice treadmill endurance and the YA mice rate of fatigue and recovery. Additionally, AICAR increased citrate synthase activity, decreased SQSTM1/p62 protein content , and decreased Myf6 protein content in both the YA and O mice suggesting increased mitochondrial activity, autophagy, and decreased muscle regeneration. Therefore, chronic AICAR treatment may alter metabolic pathways to improve the exercise response in both YA and O mice.

Muscle function is determined by structure and morphology at the architectural level. In response to resistance training, older adults have demonstrated that the neuromuscular system has a substantial adaptability, which may compensate for muscle size and quality and lead to improved functional capacities and higher quality of life. PURPOSE: The purpose of this study was to examine the effect of six weeks of progressive resistance exercise on muscle morphology and architecture in healthy older adults. METHODS: Twenty- five healthy men and women were randomly assigned to either six weeks of progressive resistance training (RT) (n=13; age = 71.08 ± 6.75, BMI = 28.5 ± 5.22) or to serve as a control (CON) (n = 12; age = 70.17 ± 5.58, BMI = 27.52 ± 5.6). Fat mass (FM), lean mass (LM), and lean thigh mass (LTM) were evaluated using dual x-ray absorptiometry. Lower body strength was estimated by predicting maximal knee extensor strength (1RM). Muscle quality (MQ) was evaluated as strength per unit mass (kg/kg). Cross-sectional area (CSA), muscle thickness (MT), fascicle length (Lf), pennation angle (cosΘ), and echo intensity (EI) of the rectus femoris (RF) and vastus lateralis (VL) were collected using B-mode ultrasound and extended field of view (FOV) ultrasound. EI was quantified using grayscale analysis software. Strength per unit of echo intensity (REI) was determined by dividing 1RM by EI of the thigh. Physiological cross-sectional area (PCSA) was calculated as the ratio of (CSA x cosΘ) / (EI x Lf). A 2x2 (group [exercise vs. control] x time [pre vs. post]) repeated measures ANOVA was used to identify group differences and group x time interactions and stepwise regression was performed to assess variables related to strength. RESULTS: 1RM increased by 31.9% (p ≤ 0.01) in the RT group and was significantly correlated to PCSA of the thigh (r = .579; p = .003) at baseline. MQ increased 31.4% (p ≤ 0.01) in the RT group consistent iv with an REI increase of 33.3% (p ≤ 0.01). There were no significant changes in LTM in either group. VL CSA increased 7.4%, (p ≤ 0.05) and demonstrated a significant interaction (p ≤ 0.05) in the RT group. There were no significant changes in the CON group for 1RM, MQ, REI or VL CSA. PCSA demonstrated a significant (p ≤ 0.05) group x time interaction but did not significantly change in either group. EI did not significantly change in the RT or CON groups. CONCLUSION: Calculated PCSA of the thigh assessed by ultrasound was related to the force producing capacity of muscle and demonstrated a significant interaction following resistance training. Short term resistance exercise training was effective in increasing 1RM, muscle quality as relative strength, muscle quality as relative echo intensity, and muscle morphology, but not EI. In addition, ultrasonography appears to be a safe, feasible, informative and sensitive clinical technique to aid in our understanding of muscle strength, function, and quality.

Introduction: Resistance training improves muscle strength and induces myofiber hypertrophy in young males and females with blunted responses occurring in older adults. These adaptations are partially due to the function of muscle stem cells (MuSCs) and fibro-adipogenic progenitors (FAPs). It remains unknown whether middle-aged males and females respond similarly to resistance training with protein supplementation, specifically at the cellular level. Purpose: The purpose of this study is to investigate the potential sex-specific responses of middle-aged males and females to whole-body resistance training. Methods: Middle-aged adults (N=28), 40-64 years, participated in a 10-week progressive, whole-body resistance training intervention coupled with protein supplementation. Muscle biopsies were collected from the vastus lateralis and stained for fibre morphology, MuSCs, and FAPs. Results: Both sexes increased type II fibre cross-sectional area with training. Myonuclear content, myonuclear domain size, and MuSC content were not altered with training in either sex. Both males and females altered FAP content with training. Interestingly, the change in MuSCs and both FAPs were correlated in males but not females (both P<0.05). It was concluded that there were no sex-specific responses to resistance training in middle-aged males and females; however, MuSCs and FAPs appear to be correlated in males but not females.

This thesis sought to advance our understanding of the physiological and health-related adaptations to low-volume interval training. Three separate studies were conducted in previously sedentary adults who trained three times per week. High-intensity interval training (HIIT) involved ten, 60-second cycling efforts at an intensity that elicited ~90% of maximal heart rate, interspersed with 60 seconds of recovery, whereas sprint interval training (SIT) involved three, 20-second ‘all-out’ cycling efforts interspersed with 2 minutes of recovery. Both protocols involved a brief warm-up and cool-down, resulting in 25- and 10-minute sessions for HIIT and SIT, respectively. Peak oxygen uptake (VO2peak), skeletal muscle mitochondrial content as reflected by the maximal activity and protein content of mitochondrial enzymes, and glycemic control based on oral glucose tolerance tests (OGTTs), intravenous glucose tolerance tests (IVGTTs) or continuous glucose monitoring (CGM), were determined before and after training. Study 1 found that 6 weeks of HIIT in the fed or fasted state increased VO2peak and mitochondrial content in women, but insulin sensitivity based on OGTTs was unchanged. Study 2 showed that 6 weeks of SIT increased VO2peak and mitochondrial content in men and women, whereas mean 24-hour glucose based on CGM was reduced in men only. Study 3 directly compared 12 weeks of SIT to traditional moderate-intensity continuous training (MICT) in men. The two protocols elicited similar improvements in VO2peak, mitochondrial content and insulin sensitivity based on IVGTTs, despite SIT involving a five-fold lower exercise volume and time commitment. This work advances our understanding of the potency of brief, intense exercise training to induce physiological remodeling and improve cardiometabolic health. It also highlights potential sex-specific adaptations to interval training that warrant clarification. Further investigation into the mechanisms of physiological remodeling to HIIT and SIT is needed, as are large-scale randomized clinical trials that compare these protocols to MICT. / Thesis / Doctor of Philosophy (PhD) / This thesis examined physiological and health-related adaptations to interval training, which involves brief bouts of intense exercise interspersed with recovery periods. One protocol involved alternating 60-second hard and easy cycling efforts for 20 minutes; the other involved three, 20-second ‘all-out’ sprints interspersed with 2 minutes of recovery. Both protocols improved indices of cardiometabolic health in previously inactive adults who trained three times per week for 6 weeks, even though the amount of exercise performed was lower than typically recommended in public health guidelines. When the latter protocol was directly compared against traditional endurance training, the improvement in cardiometabolic health after 12 weeks was the same, despite a five-fold difference in the total amount of exercise performed. Our findings highlight the effectiveness of short bursts of high-intensity exercise for improving health. These results may appeal to individuals who cite “lack of time” as a barrier to exercise.

Previous work from our laboratory in the mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that a fraction of muscle stem cells (i.e., satellite cells) (MuSCs) progressively lose the expression of myogenic markers during the progression of the disease. In the process of characterizing this aberrant behaviour, we serendipitously discovered that MuSCs might be separated into two distinct subpopulations based on the expression of the GPI-anchored surface protein CD90. Crucially, this separation does not correlate with a divergence from the myogenic lineage; instead, it separates the pool of MuSCs into two subpopulations, both maintaining myogenic properties in healthy muscles. These two newly identified subpopulations do not overlap with any previously reported subpopulation and may be prospectively isolated; present a different response in terms of kinetics of activation and differentiation during the regenerative process induced by acute muscle damage; show a different propensity to enter in GAlert state upon distal injury; display dissimilar pAMPK activity and contain a different amount of mitochondria; are present in different proportions in distinct muscle groups; differentially express ECM encoding genes during quiescence. Moreover, one of the two subpopulations can give rise to the other and therefore appears to be upstream in the lineage hierarchy. Notably, loss of function experiments, in which CD90 was downregulated in MuSCs, diminish the difference in activation displayed by the two subpopulations. This demonstrates that CD90 is a molecular determinant of MuSCs functional diversification. Importantly, although the two subpopulations of MuSCs are numerically similar in healthy limb muscles, one of the two subpopulations is lost with time in dystrophic mdx mice. Based on these data, we are hypothesizing that an imbalance between the two newly identified subpopulations may impair regeneration in the dystrophic muscles. These observations not only increase our knowledge of the molecular and cellular dynamics that are controlling normal and pathological muscle homeostasis but also open the possibility that restoring the proper functional equilibrium between subpopulations of MuSCs may counteract the progression of the dystrophic disease.