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Browsing by Author "Brault, Jeffrey J."
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Item AMP deamination is sufficient to replicate an atrophy-like metabolic phenotype in skeletal muscle(Elsevier, 2021) Miller, Spencer G.; Hafen, Paul S.; Law, Andrew S.; Springer, Catherine B.; Logsdon, David L.; O’Connell, Thomas M.; Witczak, Carol A.; Brault, Jeffrey J.; Anatomy, Cell Biology and Physiology, School of MedicineBackground: Skeletal muscle atrophy, whether caused by chronic disease, acute critical illness, disuse or aging, is characterized by tissue-specific decrease in oxidative capacity and broad alterations in metabolism that contribute to functional decline. However, the underlying mechanisms responsible for these metabolic changes are largely unknown. One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3: AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP). Given the central role of AdN in signaling mitochondrial gene expression and directly regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would be sufficient to alter their metabolic phenotype similar to that of atrophic muscle. Methods: AMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics, and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression. Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity were measured. Results: TA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls (-25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly decrease ATP concentrations (-16%), increase IMP, and increase efflux of IMP catabolites into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such, pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3 at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced pAMPK/AMPK (-24%), phosphorylation of AMPK substrates (-14%), and PGC-1α protein (-22%). Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates (-55%), basal ATP synthase-dependent (-13%), and maximal uncoupled oxygen consumption (-15%). Conclusions: Increased expression of AMPD3 significantly decreased mitochondrial protein synthesis rates and broadly altered cellular metabolites in a manner similar to that of atrophic muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism is not dependent on reductions in oxidative capacity, but may be consequence of increased AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism that alters the metabolic phenotype of skeletal muscle during atrophy and could be a target to improve muscle function during muscle wasting.Item CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle(Elsevier, 2023) Negoita, Florentina; Addinsall, Alex B.; Hellberg, Kristina; Bringas, Conchita Fraguas; Hafen, Paul S.; Sermersheim, Tyler J.; Agerholm, Marianne; Lewis, Christopher T. A.; Ahwazi, Danial; Ling, Naomi X. Y.; Larsen, Jeppe K.; Deshmukh, Atul S.; Hossain, Mohammad A.; Oakhill, Jonathan S.; Ochala, Julien; Brault, Jeffrey J.; Sankar, Uma; Drewry, David H.; Scott, John W.; Witczak, Carol A.; Sakamoto, Kei; Anatomy, Cell Biology and Physiology, School of MedicineObjective: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. Methods: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. Results: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. Conclusions: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.Item Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia(Public Library of Science, 2020-04-23) Schmidt, Cameron A.; Goldberg, Emma J.; Green, Tom D.; Karneka, Reema R.; Brault, Jeffrey J.; Miller, Spencer G.; Amorese, Adam J.; Yamaguchi, Dean J.; Spangenburg, Espen E.; McClung, Joseph M.; Anatomy and Cell Biology, School of MedicineStored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct rodent hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24 hour fasting period that resulted in substantially reduced muscle carbohydrate supply. 180 mins of acute hypoxia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product-inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Fasting and the associated reduction of the available intracellular carbohydrate pool (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics.Item Hypoxia Resistance Is an Inherent Phenotype of the Mouse Flexor Digitorum Brevis Skeletal Muscle(Oxford University Press, 2023-03-21) Amorese, Adam J.; Minchew, Everett C.; Tarpey, Michael D.; Readyoff, Andrew T.; Williamson, Nicholas C.; Schmidt, Cameron A.; McMillin, Shawna L.; Goldberg, Emma J.; Terwilliger, Zoe S.; Spangenburg, Quincy A.; Witczak, Carol A.; Brault, Jeffrey J.; Abel, E. Dale; McClung, Joseph M.; Fisher-Wellman, Kelsey H.; Spangenburg, Espen E.; Anatomy, Cell Biology and Physiology, School of MedicineThe various functions of skeletal muscle (movement, respiration, thermogenesis, etc.) require the presence of oxygen (O2). Inadequate O2 bioavailability (ie, hypoxia) is detrimental to muscle function and, in chronic cases, can result in muscle wasting. Current therapeutic interventions have proven largely ineffective to rescue skeletal muscle from hypoxic damage. However, our lab has identified a mammalian skeletal muscle that maintains proper physiological function in an environment depleted of O2. Using mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure, we observed the preservation of force production in the flexor digitorum brevis (FDB), while in contrast the extensor digitorum longus (EDL) and soleus muscles suffered loss of force output. Unlike other muscles, we found that the FDB phenotype is not dependent on mitochondria, which partially explains the hypoxia resistance. Muscle proteomes were interrogated using a discovery-based approach, which identified significantly greater expression of the transmembrane glucose transporter GLUT1 in the FDB as compared to the EDL and soleus. Through loss-and-gain-of-function approaches, we determined that GLUT1 is necessary for the FDB to survive hypoxia, but overexpression of GLUT1 was insufficient to rescue other skeletal muscles from hypoxic damage. Collectively, the data demonstrate that the FDB is uniquely resistant to hypoxic insults. Defining the mechanisms that explain the phenotype may provide insight towards developing approaches for preventing hypoxia-induced tissue damage.Item Increased Adenine Nucleotide Degradation in Skeletal Muscle Atrophy(MDPI, 2019-12-21) Miller, Spencer G.; Hafen, Paul S.; Brault, Jeffrey J.; Anatomy and Cell Biology, School of MedicineAdenine nucleotides (AdNs: ATP, ADP, AMP) are essential biological compounds that facilitate many necessary cellular processes by providing chemical energy, mediating intracellular signaling, and regulating protein metabolism and solubilization. A dramatic reduction in total AdNs is observed in atrophic skeletal muscle across numerous disease states and conditions, such as cancer, diabetes, chronic kidney disease, heart failure, COPD, sepsis, muscular dystrophy, denervation, disuse, and sarcopenia. The reduced AdNs in atrophic skeletal muscle are accompanied by increased expression/activities of AdN degrading enzymes and the accumulation of degradation products (IMP, hypoxanthine, xanthine, uric acid), suggesting that the lower AdN content is largely the result of increased nucleotide degradation. Furthermore, this characteristic decrease of AdNs suggests that increased nucleotide degradation contributes to the general pathophysiology of skeletal muscle atrophy. In view of the numerous energetic, and non-energetic, roles of AdNs in skeletal muscle, investigations into the physiological consequences of AdN degradation may provide valuable insight into the mechanisms of muscle atrophy.Item Increased AMP deaminase activity decreases ATP content and slows protein degradation in cultured skeletal muscle(Elsevier, 2020-07) Davis, Patrick R.; Miller, Spencer G.; Verhoeven, Nicolas A.; Morgan, Joshua S.; Tulis, David A.; Witczak, Carol A.; Brault, Jeffrey J.; Anatomy and Cell Biology, School of MedicineBackground: Protein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. Methods: To determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. Results: Overexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. Conclusions: Altogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.Item Insulin Resistance Is Not Sustained Following Denervation in Glycolytic Skeletal Muscle(MDPI, 2021-05-06) McMillin, Shawna L.; Stanley, Erin C.; Weyrauch, Luke A.; Brault, Jeffrey J.; Kahn, Barbara B.; Witczak, Carol A.; Anatomy and Cell Biology, School of MedicineDenervation 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.Item Intrinsic adaptations in OXPHOS power output and reduced tumorigenicity characterize doxorubicin resistant ovarian cancer cells(Elsevier, 2022) Hagen, James T.; Montgomery, McLane M.; Biagioni, Ericka M.; Krassovskaia, Polina; Jevtovic, Filip; Shookster, Daniel; Sharma, Uma; Tung, Kang; Broskey, Nickolas T.; May, Linda; Huang, Hu; Brault, Jeffrey J.; Neufer, P. Darrell; Cabot, Myles C.; Fisher-Wellman, Kelsey H.; Anatomy, Cell Biology and Physiology, School of MedicineAlthough the development of chemoresistance is multifactorial, active chemotherapeutic efflux driven by upregulations in ATP binding cassette (ABC) transporters are commonplace. Chemotherapeutic efflux pumps, like ABCB1, couple drug efflux to ATP hydrolysis and thus potentially elevate cellular demand for ATP resynthesis. Elevations in both mitochondrial content and cellular respiration are common phenotypes accompanying many models of cancer cell chemoresistance, including those dependent on ABCB1. The present study set out to characterize potential mitochondrial remodeling commensurate with ABCB1-dependent chemoresistance, as well as investigate the impact of ABCB1 activity on mitochondrial respiratory kinetics. To do this, comprehensive bioenergetic phenotyping was performed across ABCB1-dependent chemoresistant cell models and compared to chemosensitive controls. In doxorubicin (DOX) resistant ovarian cancer cells, the combination of both increased mitochondrial content and enhanced respiratory complex I (CI) boosted intrinsic oxidative phosphorylation (OXPHOS) power output. With respect to ABCB1, acute ABCB1 inhibition partially normalized intact basal mitochondrial respiration between chemosensitive and chemoresistant cells, suggesting that active ABCB1 contributes to mitochondrial remodeling in favor of enhanced OXPHOS. Interestingly, while enhanced OXPHOS power output supported ABCB1 drug efflux when DOX was present, in the absence of chemotherapeutic stress, enhanced OXPHOS power output was associated with reduced tumorigenicity.Item Liquid Chromatography Method for Simultaneous Quantification of ATP and its Degradation Products Compatible with both UV-Vis and Mass Spectrometry(Elsevier, 2022) Law, Andrew S.; Hafen, Paul S.; Brault, Jeffrey J.; Anatomy, Cell Biology and Physiology, School of MedicineATP and its degradation products are essential metabolic and signaling molecules. Traditionally, they have been quantified via high-performance liquid chromatography (HPLC) with UV-Vis detection while utilizing phosphate buffer mobile phase, but this approach is incompatible with modern mass detection. The goal of this study was to develop an ultra-performance liquid chromatography (UPLC) method free of phosphate buffer, to allow for analysis of adenine nucleotides with UV-Vis and mass spectrometry (MS) simultaneously. The final conditions used an Acquity HSS T3 premier column with a volatile ammonium acetate buffer to successfully separate and quantify ATP-related analytes in a standard mixture and in extracts from non-contracted and contracted mouse hindlimb muscles. Baseline resolution was achieved with all 10 metabolites, and a lower limit of quantification down to 1 pmol per inject was observed for most metabolites using UV-Vis. Therefore, this method allows for the reliable quantification of adenine nucleotides and their degradation products via UV-Vis and their confirmation and/or identification of unknown peaks via MS.Item Nutraceuticals and Exercise against Muscle Wasting during Cancer Cachexia(MDPI, 2020-12-09) Aquila, Giorgio; Re Cecconi, Andrea David; Brault, Jeffrey J.; Corli, Oscar; Piccirillo, Rosanna; Anatomy and Cell Biology, School of MedicineCancer cachexia (CC) is a debilitating multifactorial syndrome, involving progressive deterioration and functional impairment of skeletal muscles. It affects about 80% of patients with advanced cancer and causes premature death. No causal therapy is available against CC. In the last few decades, our understanding of the mechanisms contributing to muscle wasting during cancer has markedly increased. Both inflammation and oxidative stress (OS) alter anabolic and catabolic signaling pathways mostly culminating with muscle depletion. Several preclinical studies have emphasized the beneficial roles of several classes of nutraceuticals and modes of physical exercise, but their efficacy in CC patients remains scant. The route of nutraceutical administration is critical to increase its bioavailability and achieve the desired anti-cachexia effects. Accumulating evidence suggests that a single therapy may not be enough, and a bimodal intervention (nutraceuticals plus exercise) may be a more effective treatment for CC. This review focuses on the current state of the field on the role of inflammation and OS in the pathogenesis of muscle atrophy during CC, and how nutraceuticals and physical activity may act synergistically to limit muscle wasting and dysfunction.