We determined the effects of "periodized nutrition" on skeletal muscle and whole body responses to a bout of prolonged exercise the following morning. Seven cyclists completed two trials receiving isoenergetic diets differing in the timing of ingestion: they consumed either 8 g/kg body mass (BM) of carbohydrate (CHO) before undertaking an evening session of high-intensity training (HIT) and slept without eating (FASTED), or consumed 4 g/kg BM of CHO before HIT, then 4 g/kg BM of CHO before sleeping (FED). The next morning subjects completed 2 h of cycling (120SS) while overnight fasted. Muscle biopsies were taken on day 1 (D1) before and 2 h after HIT and on day 2 (D2) pre-, post-, and 4 h after 120SS. Muscle [glycogen] was higher in FED at all times post-HIT (P < 0.001). The cycling bouts increased PGC1α mRNA and PDK4 mRNA (P < 0.01) in both trials, with PDK4 mRNA being elevated to a greater extent in FASTED (P < 0.05). Resting phosphorylation of AMPK(Thr172), p38MAPK(Thr180/Tyr182), and p-ACC(Ser79) (D2) was greater in FASTED (P < 0.05). Fat oxidation during 120SS was higher in FASTED (P = 0.01), coinciding with increases in ACC(Ser79) and CPT1 as well as mRNA expression of CD36 and FABP3 (P < 0.05). Methylation on the gene promoter for COX4I1 and FABP3 increased 4 h after 120SS in both trials, whereas methylation of the PPARδ promoter increased only in FASTED. We provide evidence for shifts in DNA methylation that correspond with inverse changes in transcription for metabolically adaptive genes, although delaying postexercise feeding failed to augment markers of mitochondrial biogenesis.

Carbohydrate-protein supplementation has been found to increase the rate of training adaptation when provided postresistance exercise. The present study compared the effects of a carbohydrate and protein supplement in the form of chocolate milk (CM), isocaloric carbohydrate (CHO), and placebo on training adaptations occurring over 4.5 weeks of aerobic exercise training. Thirty-two untrained subjects cycled 60 min/d, 5 d/wk for 4.5 wks at 75–80% of maximal oxygen consumption (VO2 max). Supplements were ingested immediately and 1 h after each exercise session. VO2 max and body composition were assessed before the start and end of training. VO2 max improvements were significantly greater in CM than CHO and placebo. Greater improvements in body composition, represented by a calculated lean and fat mass differential for whole body and trunk, were found in the CM group compared to CHO. We conclude supplementing with CM postexercise improves aerobic power and body composition more effectively than CHO alone.

DNA methylation is altered by environmental factors. We hypothesized that DNA methylation is altered in skeletal muscle in response to either insulin or glucose exposure. We performed a genome-wide DNA methylation analysis in muscle from healthy men before and after insulin exposure. DNA methylation of selected genes was determined in muscle from healthy men and men with type 2 diabetes before and after a glucose tolerance test. Insulin altered DNA methylation in the 3' untranslated region of the calcium pump gene. Insulin increased DNA methylation in the gene body of, a gene involved in cell proliferation, apoptosis, and autophagy. methylation was reduced in patients with type 2 diabetes. Carbohydrate ingestion reduced DNA methylation in healthy men and men with type 2 diabetes, suggesting glucose may play a role. Supporting this, DNA methylation was inversely correlated with the 2-h glucose concentration. Whereas glucose incorporation to glycogen was unaltered by small interfering RNA against, palmitate oxidation was increased. In conclusion, insulin and glucose exposure acutely alter the DNA methylation profile of skeletal muscle, indicating that DNA methylation constitutes a rapidly adaptive epigenetic mark. Furthermore, insulin and glucose modulate DNA methylation in a reciprocal manner, suggesting a feedback loop in the control of the epigenome.

ObjectiveWe sought to identify AMPK-regulated genes via bioinformatic analysis of microarray data generated from skeletal muscle of animal models with genetically altered AMPK activity. We hypothesized that such genes would play a role in metabolism. Ganglioside-induced differentiation-associated protein 1 (GDAP1), a gene which plays a role in mitochondrial fission and peroxisomal function in neuronal cells but whose function in skeletal muscle is undescribed, was identified and further validated. AMPK activation reduced GDAP1 expression in skeletal muscle. GDAP1 expression was elevated in skeletal muscle from type 2 diabetic patients but decreased after acute exercise.MethodsThe metabolic impact of GDAP1 silencing was determined in primary skeletal muscle cells via siRNA-transfections. Confocal microscopy was used to visualize whether silencing GDAP1 impacted mitochondrial network morphology and membrane potential.ResultsGDAP1 silencing increased mitochondrial protein abundance, decreased palmitate oxidation, and decreased non-mitochondrial respiration. Mitochondrial morphology was unaltered by GDAP1 silencing. GDAP1 silencing and treatment of cells with AMPK agonists altered several genes in the core molecular clock machinery.ConclusionWe describe a role for GDAP1 in regulating mitochondrial proteins, circadian genes, and metabolic flux in skeletal muscle. Collectively, our results implicate GDAP1 in the circadian control of metabolism.

AMPK controls glucose- and lipid- metabolism and modulates inflammatory responses to maintain metabolic and inflammatory homeostasis during low cellular energy levels. The AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) interferes with inflammatory pathways in skeletal muscle, but the mechanisms are undefined. We hypothesized that AMPK activation reduces cytokine mRNA levels by blocking transcription through one or several transcription factors. Three skeletal muscle models were used to study AMPK effects on cytokine mRNA: human skeletal muscle strips obtained from healthy men incubated in vitro, primary human muscle cells and rat L6 cells. In all three skeletal muscle systems, AICAR acutely reduced cytokine mRNA levels. In L6 myotubes treated with the transcriptional blocker actinomycin D, AICAR addition did not further reduce Il6 or Lif mRNA, suggesting that AICAR modulates cytokine expression through regulating transcription rather than mRNA stability. A cross-species bioinformatic approach identified novel transcription factors which may regulate LIF and IL6 mRNA. The involvement of these transcription factors was studied after targeted gene-silencing by siRNA. siRNA silencing of the transcription factors Nfyc, Sp1 and Zbtb14, or AMPK α1/α2 subunits, increased constitutive levels of Il6 and Lif. Our results identify novel candidates in the regulation of skeletal muscle cytokine expression and identify AMPK, Nfyc, Sp1, Zbtb14 and as novel regulators of immunometabolic signals from skeletal muscle.

During exercise, skeletal muscles release cytokines, peptides, and metabolites that exert autocrine, paracrine, or endocrine effects on glucose homeostasis. In this study, we investigated the effects of secreted protein acidic and rich in cysteine (SPARC), an exercise‐responsive myokine, on glucose metabolism in human and mouse skeletal muscle. SPARC‐knockout mice showed impaired systemic metabolism and reduced phosphorylation of AMPK and protein kinase B in skeletal muscle. Treatment of SPARC‐knockout mice with recombinant SPARC improved glucose tolerance and concomitantly activated AMPK in skeletal muscle. These effects were dependent on AMPK‐γ3 because SPARC treatment enhanced skeletal muscle glucose uptake in wild‐type mice but not in AMPK‐γ3–knockout mice. SPARC strongly interacted with the voltage‐dependent calcium channel, and inhibition of calcium‐dependent signaling prevented SPARC‐induced AMPK phosphorylation in human and mouse myotubes. Finally, chronic SPARC treatment improved systemic glucose tolerance and AMPK signaling in skeletal muscle of high‐fat diet–induced obese mice, highlighting the efficacy of SPARC treatment in the management of metabolic diseases. Thus, our findings suggest that SPARC treatment mimics the effects of exercise on glucose tolerance by enhancing AMPK‐dependent glucose uptake in skeletal muscle.—Aoi, W., Hirano, N., Lassiter, D. G., Björnholm, M., Chibalin, A. V., Sakuma, K., Tanimura, Y., Mizushima, K., Takagi, T., Naito, Y., Zierath, J. R., Krook, A. Secreted protein acidic and rich in cysteine (SPARC) improves glucose tolerance via AMP‐activated protein kinase activation. FASEB J. 33, 10551–10562 (2019). http://www.fasebj.org

Aims/hypothesis Insulin-mediated signals and AMP-activated protein kinase (AMPK)-mediated signals are activated in response to physiological conditions that represent energy abundance and shortage, respectively. Focal adhesion kinase (FAK) is implicated in insulin signalling and cancer progression in various non-muscle cell types and plays a regulatory role during skeletal muscle differentiation. The role of FAK in skeletal muscle in relation to insulin stimulation or AMPK activation is unknown. We examined the effects of insulin or AMPK activation on FAK phosphorylation in human skeletal muscle and the direct role of FAK on glucose and lipid metabolism. We hypothesised that insulin treatment and AMPK activation would have opposing effects on FAK phosphorylation and that gene silencing of FAK would alter metabolism. Methods Human muscle was treated with insulin or the AMPK-activating compound 5-aminoimadazole-4-carboxamide ribonucleotide (AICAR) to determine FAK phosphorylation and glucose transport. Primary human skeletal muscle cells were used to study the effects of insulin or AICAR treatment on FAK signalling during serum starvation, as well as to determine the metabolic consequences of silencing the FAK gene, PTK2. . Silencing PTK2 in primary human skeletal muscle cells increased palmitate oxidation and reduced glycogen synthesis. Conclusions/interpretation AMPK regulates FAK signalling in skeletal muscle. Moreover, siRNA-mediated FAK knockdown enhances lipid oxidation while impairing glycogen synthesis in skeletal muscle. Further exploration of the interaction between AMPK and FAK may lead to novel therapeutic strategies for diabetes and other chronic conditions associated with an altered metabolic homeostasis.

Dedication This work is dedicated to those who have supported me outside of my graduate studies over the last few years. My family deserves as much credit as anyone for the work generated as part of this study. Namely, Kristin Bagby, Christine Lassiter, Mechelle Lassiter, Bette Rose Ryan, and Michele Poague deserve recognition; without them I would not have been able to work as tirelessly as I have on this project. v Acknowledgements Many individuals contributed significantly to the completion of this project. Though the thesis is my own, the project is a result of a team effort. First and foremost Lynne Kammer helped me every step of the research process, from study design, to data collection, and proofreading. Zhenping Ding played another critical role in training and overseeing the completion of the biochemistry techniques used as part of this work. Assays were completed is listed last, his help on this and other projects that I have been a part of is understated and I commend him for being such a hard-working and loyal individual. My graduate studies have been made possible through the concerted efforts of the entire kinesiology staff at the University of Texas and I would like to recognize the contributions to my education made by Ivy have served as readers for my thesis and for this I owe them recognition. Lastly I would like to thank the sponsor of my research, SWSS law, and the participants who were willing to ride their bikes in the lab for the sake of expanding our scientific understanding. vi Abstract Effect of an Energy Drink on Physical and Cognitive Performance in Trained Cyclists David Gray Lassiter, MS KIN The University of Texas at Austin, 2012 Supervisor: John Ivy This study investigated the effectiveness of an energy drink (ED) in enhancing cycling time-trial performance, and cognitive performance at rest, during moderate-intensity exercise, and after exercise. The protocol was double-blind, randomized, placebo-controlled, two-period, and within-subjects. The treatments were ED containing caffeine and carbohydrate, and a caffeine-free non-caloric flavored placebo beverage (PLA). Exercise performance was measured by time to finish a simulated 35 km time-trial course. Cognitive performance was measured by a Stroop task, a tapping task, a reaction time task, and an executive function task consisting of both tapping and reaction time. The effects of ED on blood markers were also assessed. Race performance was enhanced by an average of 3% when participants had ED compared to PLA without a difference in rating of perceived exertion (RPE). Performance was improved by ED even in participants that arrived to the lab with elevated blood caffeine concentrations. Both before and after the exercise, ED resulted in more taps per second in the tapping task. After receiving ED, plasma insulin spiked, there was a fall in free fatty acids (FFA) and vii blood glucose remained unchanged. Exercise onset caused a drop in blood glucose when participants consumed ED, though glucose returned to a level that was not di...