Browsing by Author "Hamilton, Karyn L., advisor"
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Item Open Access Adjusting attitudes about altitude: novel approaches to promote human performance in high-altitude(Colorado State University. Libraries, 2014) Scalzo, Rebecca Lynn, author; Bell, Christopher, advisor; Hamilton, Karyn L., advisor; Miller, Benjamin F., committee member; Kanatous, Shane B., committee memberMilitary personnel frequently operate in environmental extremes, such as high-altitude, without adequate time for acclimatization. Altitude mediated decrements in human physiological function jeopardize mission success and personal safety. The following dissertation describes three experiments directed at the identification of non-traditional, military specific approaches to promote human functional performance in high-altitude. The specific aims of the following experiments were: 1) to compare the difference in time trial performance in normoxia and hypoxia following oral administration of a placebo, a non-specific phosphodiesterase inhibitor/adenosine receptor antagonist (Aminophylline), a carbonic anhydrase inhibitor (Neptazane), or the combination of Aminophylline and Neptazane; 2) to assess endurance exercise performance in hypoxia following an intravenous infusion of glucose with and without prior sympathetic nervous system inhibition (clonidine); and 3) to determine endurance exercise performance in hypoxia following a high-carbohydrate meal with and without prior/concurrent administration of an oral insulin sensitizer (metformin) and to compare hypoxic endurance exercise performance with endurance exercise performance in normoxia following the same meal. When compared with normoxia, hypoxia attenuated endurance exercise performance in these experiments. In experiment 1, we found that concomitant administration of Aminophylline and Neptazane attenuated the hypoxia-mediated deficit in endurance exercise performance compared with placebo. Neither Aminophylline nor Neptazane alone ameliorated this decrement. In experiment 2, prior clonidine administration attenuated the cardiovascular response to hypoxia assessed by heart rate and blood pressure responses at rest but did not deleteriously impact endurance exercise performance in hypoxia. Finally, the preliminary data from experiment 3 suggest metformin improved the metabolic response to a high-carbohydrate meal in hypoxia, and potentially augmented skeletal muscle glycogen synthesis. Endurance exercise performance was unaffected in hypoxia following metformin administration. Collectively, the data from these experiments suggest these pharmacological treatments, compatible with military specific demands, effectively promote human physiological function in high-altitude.Item Open Access Characterization of the Dunkin Hartley guinea pig as a non-transgenic and multimorbid model of brain aging(Colorado State University. Libraries, 2024) Glennie, Kristen Skye, author; Hamilton, Karyn L., advisor; Moreno, Julie A., advisor; LaRocca, Thomas J., committee member; Santangelo, Kelly S., committee memberAlzheimer's Disease and Alzheimer's Disease Related Dementia (AD/ADRD) affect an estimated 55 million people worldwide; a staggering figure that is expected to grow in the coming years. With this projection looming, we have yet to identify any effective cures, treatments, or preventative strategies. Historically, AD/ADRD research is conducted using genetically engineered pre-clinical models, that express a specific brain aging pathology. Recent discoveries, however, have identified a dynamic whole-body "inflammaging" phenotype that exists with, and likely contributes to, AD/ADRD onset and progression. Currently, we do not have an accessible and tractable preclinical model that naturally mimics the age-related, systemic and progressive neurodegenerative phenotype present in humans. Recent findings, however, suggest the Dunkin Hartley guinea pig (HGP) may address this need. HGPs are known to develop systemic inflammation and progressive age-related comorbidities characteristic of human aging. The presence of this whole-body aging phenotype prompted investigation into the brain. Genetic and transcriptomic analyses found aged HGPs exhibit strong sequence homology, and similar protein expression patterns to human brain aging and AD. Further, immunohistochemical assessment found aged HGPs express markers of neuroinflammation and misfolded proteins in the hippocampus. To further interrogate these novel findings, we examined the histopathology of 4 brain regions often implicated in neurodegenerative decline for evidence of progressive neuropathology. Our results identify the presence of an age related neuroinflammatory and phosphorylated tau phenotype. Findings from this study contribute to the overarching hypothesis that AD/ADRD is a whole-body disease, and ultimately support the goal of closing the existing translational gap between preclinical and clinical neurodegenerative research.Item Open Access Compensatory responses to oxidant stresses in vitro and in vivo(Colorado State University. Libraries, 2013) Khademi, Shadi, author; Hamilton, Karyn L., advisor; Miller, Benjamin F., advisor; Hickey, Matthew S., committee member; Tjalkens, Ronald B., committee memberEmerging evidence shows that reactive oxygen species (ROS) are not merely damaging agents causing random destruction to cell structure and function, but that they act as modulators of physiological processes (such as cell adaptation to physical exercise) by regulating gene transcription and protein synthesis. The exact redox signaling pathways involved in cell adaptations to oxidative stress are unknown. Since various stimuli can induce oxidative stress under different conditions in vivo and in vitro, different models are warranted to study the cell signaling pathways involved in compensatory responses to oxidative stress. The following investigation comprises a series of experiments with the overall aim of elucidating the role of redox sensitive pathways in inducing cellular responses to oxidative stress in vitro and in vivo. The experiments tested the general hypothesis that changes in the redox state of the cell, through hypoxia, contractile activity or direct application of hydrogen peroxide (H2O2), would cause antioxidant compensatory responses and cell adaptations. The specific aims of the experimental series were: 1) to determine whether pulmonary edema, evoked by cerebral hypoxia in the presence of systemic normoxia, will be accompanied by sympathetic activation, increased oxidative stress, and upregulation of endogenous antioxidant pathways, 2) to determine whether electrical stimulation (Es) induced contractile activity of cultured murine myotubes would induce energetic stress, redox sensitive signaling, and mitochondrial biogenesis, and 3) to determine whether treatment with H2O2 would result in a greater rate of mitochondrial biogenesis compared to control, and whether the increase would be maintained during co-treatment with either an exogenous antioxidant (vitamin C) or a nuclear erythroid 2 -related factor 2 (Nrf2) activator that increases transcription of endogenous antioxidants. Studies in aim 1 demonstrated that under pathologic conditions such as isolated cerebral hypoxia with systemic normoxia, tissue specific patterns of compensatory responses to the hypoxic stressor exist. Further, this study showed that the differences in lung and brain redox signaling pathways during hypoxia can have different systemic outcomes through modulation of the sympathetic nervous system (SNS). Studies in aim 2 demonstrated a unique model of contractile activity in vitro, which was successful in simulating the cellular adaptations to a single bout of endurance exercise such as greater rate of cytosolic protein synthesis, upregulation of antioxidants and mitochondrial protein markers as well as AMP activated protein kinase (AMPK). Studies in aim 3 demonstrated that H2O2 did not increase mitochondrial biogenesis. Further, increasing Nrf2 activation maintained the rate of mitochondrial protein synthesis during H2O2 treatment, while treatment with the exogenous antioxidant failed to restore the H2O2 induced decreases in mitochondrial biogenesis during H2O2 treatment. Collectively, we have used different models of oxidative stress in vitro and in vivo to evaluate some of the mechanisms involved in cell adaptations responses. Findings from these experiments provide insight into understanding the role of redox signaling in pathologic and non-pathologic circumstances and can help future therapeutic recommendations for battling the consequences of oxidative stress on health.Item Open Access Effect of treatment with a Nrf2 activator on in vivo proteostasis in mice(Colorado State University. Libraries, 2019) Valenti, Zackary J., author; Hamilton, Karyn L., advisor; Lark, Daniel S., committee member; Gentile, Christoper L., committee memberAging is characterized by progressive declines in cellular function, often resulting from oxidative stress. Redox homeostasis is perturbed when the production of reactive oxygen species (ROS) exceeds the capacity of antioxidant defenses to eliminate ROS. Chronic imbalances in ROS production and clearance can lead to disruptions in proteostasis by causing unrepairable damage to proteins. Nuclear factor erythroid-derived 2-like 2 (Nrf2) exerts transcriptional regulation over endogenous antioxidant defenses by regulating the transcription of antioxidant enzymes and a myriad of cytoprotective proteins. Nrf2 activation has received attention as a therapeutic intervention to preserve cellular function. Our lab has characterized the treatment effects of Protandim and PB125, phytochemical compounds known to activate Nrf2. The collective findings from our group using both in vitro and in vivo experiments suggest that both compounds are effective for improving proteostasis; however, compared to Protandim, PB125 is more efficacious for sustained Nrf2 activation due to its ability to inhibit mechanisms of the Nrf2 shutdown pathway. We speculate that PB125 may have additional benefits on mechanism of proteostasis in vivo; thus, the purpose of the present study was to examine the effects of two doses of PB125 supplementation on proteostasis. We randomly assigned 51 male C57BL6/J mice aged 15-16 months to diets with 0 ppm (CON), 100 ppm (LOW), or 300 ppm (HIGH) doses of PB125 n=18/group during a 5-week feeding study. Mice were isotopically labeled with 8% deuterium oxide (D2O) administered in the drinking water to simultaneously measure protein and DNA synthesis rates in mitochondrial (mito), cytosolic (cyto), and mixed (mixed) subcellular fractions of heart, liver, and skeletal muscle tissues. We hypothesized that mice treated with PB125 would have enhanced proteostasis outcomes; however, our results indicate that PB125 supplementation did not affect mechanisms of proteostasis. No significant differences were found in protein or DNA synthesis rates between treatment groups, and our secondary measures further support that PB125 did not affect the proteostatic network, as there were no significant differences observed in Nrf2-regulated protein expression or protein aggregation. From our data we were able to confirm that oral administration of PB125 is safe; however, further in vivo investigations are warranted in order to confirm the role of PB125 in modulating mechanisms of proteostasis.Item Open Access Evaluation of a novel phytochemical Nrf2 activator on cytoprotective gene expression and proteostasis in vivo(Colorado State University. Libraries, 2017) Laurin, Jaime L., author; Miller, Benjamin F., advisor; Hamilton, Karyn L., advisor; Johnson, Sarah A., committee memberAging is associated with increases in oxidative stress. Redox imbalance occurs when chronic production of reactive oxygen species (ROS) exceeds the capacity of antioxidant enzymes to eliminate ROS. Chronic levels of intracellular ROS can compromise proteostasis by causing irreversible damage to proteins. The transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) mediates the cellular endogenous antioxidant defense system by regulating antioxidant enzymes that are cytoprotective against ROS. The phytochemical dietary supplement Protandim activates Nrf2. Previous work from our lab has demonstrated that Protandim treatment can improve proteostasis in skeletal muscle in vivo. Recently, we have begun to characterize a second-generation Nrf2 activator dietary supplement (PB125) that can inhibit components of the Nrf2 shutdown pathway, potentially allowing Nrf2 to stay transcriptionally active for longer. Therefore, we speculated that PB125 might have additional benefits on proteostatic processes. The purpose of the present study was to examine in vivo the effects of three doses (low, medium, high) of PB125 supplementation on Nrf2 activation and proteostasis. We assigned sixty male CB6F1 mice aged 10-11 months to diets containing low, medium, or high doses of PB125 in a 5 week feeding study. Mice were isotopically labeled with 8% deuterium oxide (D2O) administered in the drinking water to simultaneously measure protein and DNA fractional synthesis rates in liver, heart, and skeletal muscle. We assessed Nrf2 activation through analysis of gene expression profiles via Affymetrix GeneChip microarray. Proteostatic mechanisms increased in the liver mitochondrial fraction in the low treatment group. There were no differences in proteostatic mechanisms in heart. In the skeletal muscle mixed fraction, there was a reduction in proteostatic mechanisms in the medium treatment group. In the medium treatment group, there was also upregulation of Nrf2-dependent cytoprotective genes (Akr1c19, Akr1d1, Gpx2, Gclm, Fthl17b) as detected by microarray analysis. From our data we were able to conclude that all three doses were safe, and that 100 ppm was effective at activating Nrf2. In addition, there was an indication of increased proteostatic processes in the liver, but not heart or skeletal muscle, perhaps due to the healthy status of the mice.Item Open Access Lipids and oxidative stress as mediators of endothelial pathophysiology in obesity(Colorado State University. Libraries, 2011) Donovan, Elise Laura, author; Miller, Benjamin F., advisor; Hamilton, Karyn L., advisor; Hickey, Matthew S., committee member; Frye, Melinda A., committee memberBecause obesity is a well established independent risk factor for diabetes and coronary artery disease (CAD), it is important to identify factors associated with obesity that are responsible for disease progression and interventions to decrease risk of developing obesity associated co-morbidities. Two of the many mediators of obesity associated risk for diabetes and CAD are oxidative stress and oxidized phospholipids, which have been implicated in vascular disease initiation and progression through endothelial cell activation, macrophage recruitment and advanced plaque rupture. Plasma platelet activating factor acetylhydrolase (Lp-PLA2) is an enzyme that circulates bound to LDL cholesterol and degrades platelet activating factor (PAF), a potent inducer of the platelet coagulation cascade and thrombosis. In addition, Lp-PLA2 degrades oxidized phospholipids to lysophospholipid products and fatty acids that may also induce inflammatory changes in multiple cell types, including vascular endothelial cells. Exogenous antioxidant supplementation has been examined as a means of decreasing vascular oxidative stress. However, data show that exogenous antioxidant supplementation has little or no effect on CVD outcomes, and in some cases it may increase mortality. A novel approach to protecting cells from oxidative stress is to increase cellular endogenous antioxidant defenses. NF-E2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) promoter region of many genes including phase II antioxidant enzymes. Protandim is a combination of phytochemicals that is thought to induce Nrf2 stabilization and translocation to the nucleus, with subsequent increases in phase II antioxidant enzymes and protection against oxidative stress. The overall objectives of the three studies we performed were to 1) globally analyze obesity associated lipid and oxidative stress using lipidomics techniques 2) determine the effects of identified obesity associated oxidative and lipid stress on the vascular endothelium 3) determine whether Protandim treatment could protect vascular endothelial cells from an oxidative challenge, and 4) characterize Lp-PLA2 in human adipose and skeletal muscle. Experiment 1 tested the hypothesis that oxidized phospholipids would be greater in morbidly obese gastric bypass patients compared to lean surgical controls, and that global lipid profiles would differ between groups. To test this hypothesis we performed a combined targeted and global lipidomic analysis of plasma lipids from morbidly obese gastric bypass patients and lean controls. We identified a group of ether-linked lipids that were greater in obese subjects compared to lean, and further examined whether a representative lipid from this group induced pathophysiological phenotypic changes in vascular endothelial cells. Experiment 2 tested the hypothesis that Protandim would protect human coronary artery endothelial cells (HCAEC) against an oxidative challenge by increasing phase II antioxidant enzymes in a Nrf2 dependent manner. To do this we performed a series of in vitro experiments treating HCAEC with Protandim and determined that Protandim induced Nrf2 nuclear localization, increased phase II antioxidant enzyme expression, and protected cells from undergoing apoptosis in response to an oxidative challenge. Silencing Nrf2 prior to the oxidative challenge inhibited the Protandim induced protection. Experiment 3 tested the hypothesis that Lp-PLA2 would be detectable in human adipose tissue and that Lp-PLA2 would be greater in adipose from morbidly obese gastric bypass patients compared to lean. In addition, we examined whether adipose Lp-PLA2 may be related to circulating Lp-PLA2 activity, inflammation, and glucose intolerance. We have identified ether-linked lipids that are elevated in obese subjects compared to lean. We found that Lp-PLA2 is expressed in human adipose for the first time, adipose Lp-PLA2 is co-localized with macrophages, and report relationships between Lp-PLA2 and indices of glucose homeostasis and inflammation. Lastly, we found that Protandim protects endothelial cells from an oxidative challenge in a Nrf2 dependent manner. Collectively, these data provide insight into the oxidative and lipid stress milieu that occurs in obese subjects.Item Open Access Nrf2 activation but not vitamin C treatment promotes proteostatic maintenance during an oxidative challenge(Colorado State University. Libraries, 2016) Ehrlicher, Sarah E., author; Miller, Benjamin F., advisor; Hamilton, Karyn L., advisor; Melby, Christopher, committee memberImproved proteostasis may be a mechanism of stress resistance, and it is likely that the increased protein turnover with exercise training contributes to adaptation to stress. Exogenous antioxidant treatments such as vitamin C (VitC) target the detrimental effects of reactive oxygen species (ROS), but may simultaneously prevent the beneficial redox signaling associated with exercise. A possible alternative strategy to prevent oxidative damage while permitting redox-sensitive signaling is to increase endogenous antioxidants. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) increases the transcription of endogenous antioxidants by binding to the antioxidant response element in the promoter region of target genes. Protandim (Pro, LifeVantage), a combination of five phytochemicals, activates Nrf2 by increasing its translocation to the nucleus. We hypothesized that, compared to VitC, treatment with the Nrf2 activator Pro would not blunt ROS induced proteostatic maintenance. To mimic ROS signaling, C2C12 myoblasts were treated with H2O2. Treatment occurred alone or in combination with either VitC or Pro. Deuterium oxide labeling was used to measure protein synthesis in the mitochondrial and cytosolic cell fractions after 2, 4, 8, and 12 hours of treatment. Simultaneously cell proliferation was measured by deuterium incorporation into DNA. Compared to the untreated control, H2O2 alone increased DNA synthesis but did not increase mitochondrial protein synthesis, resulting in decreased proteostasis. Compared to H2O2 alone, Pro decreased protein synthesis in both cytosolic and mitochondrial fractions. However, Pro also decreased DNA synthesis. This resulted in a greater protein to DNA ratio suggesting maintenance of proteostasis. VitC with H2O2 increased DNA synthesis and decreased proteostasis, similar to H2O2 treatment alone. From these data, it appears that although treatment with exogenous antioxidants increases proliferation, activation of Nrf2 maintains mitochondrial protein synthesis despite a reduction in proliferation. Further study into the role of Nrf2 in improving mitochondrial proteostasis to promote stress resistance is warranted.Item Open Access Oxidative and energetic stress: regulation of Nrf2 and mitochondrial biogenesis for slowed aging interventions(Colorado State University. Libraries, 2013) Bruns, Danielle Reuland, author; Hamilton, Karyn L., advisor; Miller, Benjamin F., advisor; Wilusz, Carol J., committee member; Pagliassotti, Michael J., committee memberThe following dissertation describes a series of experiments with the overall aim to understand the cellular energetic and oxidative stresses associated with aging, and to investigate treatments which may attenuate these stresses and promote healthspan. The specific aims of the four sets of experiments were 1) to determine if treatment with the phytochemicals in Protandim activates Nrf2 and 2) the mechanisms by which this activation occurs; 3) to assess sexually dimorphic Nrf2 signaling across three rodent models of longevity; and 4) to determine if mitochondrial related proteins are preferentially translated under energetic stress. In Experiments #1 and #2, we found that phytochemicals activate Nrf2 and protect cells against oxidant stress. None of the mechanisms we investigated appear to be responsible for phytochemical-induced Nrf2 activation, and continued investigations must be undertaken to identify how Protandim robustly induces Nrf2 nuclear accumulation. In Experiment #3, we found that Nrf2 signaling was not consistently upregulated in tissues from long-lived models compared to controls, but we did elucidate important sex differences, with female mice generally displaying greater Nrf2 signaling than male mice. We believe this finding, in the context of sexual dimorphism in aging, warrants future investigations into Nrf2, stress resistance, and longevity between males and females. In Experiment #4, we found that mitochondrial proteins were preferentially translated upon pharmaceutical energetic stress, and that this selective translation occurred in the vicinity of the mitochondria. Our results indicate activation of Nrf2 protects cells against oxidant stress, and may be a therapeutic target for cardiovascular diseases and other age-related diseases. Further, we assess selective translation of mitochondrial proteins during energetic stress as a means of understanding how energetic stress mimetics selectively facilitate the translation of key mitochondrial proteins. Taken together, these studies provide the basis for future work aimed at attenuating diseases with oxidant stress and mitochondrial dysfunction components.Item Open Access Protein synthesis in slowed aging: insights into shared characteristics of long-lived mouse models(Colorado State University. Libraries, 2014) Drake, Joshua Chadwick, author; Miller, Benjamin F., advisor; Hamilton, Karyn L., advisor; Wilusz, Carol J., committee member; Hickey, Matthew S., committee memberThe following dissertation describes a series of experiments with the overall aim to understand the role that changes in protein synthesis have in slowed aging. The specific aims of the three sets of experiments were 1) to determine if chronic administration of the mTORC1 inhibitor rapamycin to mice increases proteostatic mechanisms in skeletal muscle, heart, and liver; 2) to determine if an underdeveloped anterior pituitary, caused by deletion of the Pit-1 gene in mice, increases proteostatic mechanisms in skeletal muscle, heart, and liver of long-lived Snell dwarf mice; 3) and to determine if transient nutrient restriction during the suckling period in mice (i.e. crowded litter), increases proteostatic mechanisms in skeletal muscle, heart, and liver later in life. In Experiment #1 we found that mitochondrial proteins were preferentially synthesized in skeletal muscle and that global protein synthesis in the heart was maintained despite reduced cellular proliferation and mTORC1 activity in mice fed rapamycin compared to normal diet controls. Originally we determined that these data were indicative of an improved somatic maintenance of skeletal muscle mitochondria and the heart proteome. Since we could not account for changes to other energetic processes (e.g. metabolism), we reasoned that our data was more consistent with proteostasis, a component of somatic maintenance. In Experiment #2 we developed a novel method for assessing proteostasis and determined that Snell dwarf mice had an increase in proteostatic mechanisms across sub-cellular fractions within skeletal muscle and heart compared control mice, despite differential rates of protein synthesis in the face of decreased mTORC1. Together with our previous investigations into rapamycin fed and caloric restriction models of long-life we concluded that increased proteostatic mechanisms may be a shared characteristic of models of slowed aging. In Experiment #3 we demonstrate that the crowded litter mouse transitions from growth to maintenance as it ages. Furthermore, in the crowded litter mouse, we demonstrate that proteostasis is not dependent upon decreased mTORC1. Our results indicate that decreased mTORC1 does not necessarily correlate to decreases in protein synthesis across all sub-cellular fractions. Discerning which proteins and the mechanism(s) of how specific proteins can be preferentially synthesized despite decreases in protein synthesis in other fractions and decreased mTORC1, may give further insight into characteristics of slowed aging. Further, we demonstrate that increases in proteostasic mechanisms are a shared characteristic of multiple unique models of slowed aging and therefore, provides a basis for future work aimed at slowing the aging process.Item Embargo Targeting proteostatic maintenance and mitochondrial function with phytochemical compounds in models of brain and skeletal muscle aging(Colorado State University. Libraries, 2024) Walsh, Maureen Ann, author; Hamilton, Karyn L., advisor; Fling, Brett W., committee member; Moreno, Julie A., committee member; LaRocca, Thomas J., committee member; Santangelo, Kelly S., committee memberThere is a growing population of older adults (>65+ years) worldwide that is projected to increase in coming decades, presenting both a challenge and an opportunity. Specifically, age is the number one risk factor for chronic diseases like sarcopenia, the loss of muscle mass and function, and neurodegenerative diseases such as Alzheimer's Disease. The twelve hallmarks of aging are a collection of cellular changes that drive the aging process. Two highly interconnected hallmarks of aging that drive the development and progression of sarcopenia and neurodegeneration are loss of proteostasis (protein homeostasis) and mitochondrial dysfunction. While progress has been made in understanding the etiology of chronic diseases, treatments for age-related chronic diseases affecting skeletal muscle and the brain are lacking. One reason for the lack of effective treatments in humans is the absence of preclinical animal models that recapitulate human aging. However, our group previously identified the Hartley guinea pig as a novel model of brain and skeletal muscle aging. We then treated these guinea pigs with a phytochemical compound to delay the onset and/or slow the progression of brain and skeletal muscle aging. Through the experiments in this dissertation, I observed that: 1.) phytochemical compounds, branded as Protandim, can improve mechanisms of proteostasis independent of changes in mitochondrial respiration in muscle precursor cells; 2.) the phytochemical compound, branded as PB125, can improve mechanisms of skeletal muscle proteostasis in the Hartley guinea pig; 3.) PB125 can also decrease neuroinflammation in the Hartley guinea pig; and 4.) despite the lack of declines in hippocampal mitochondrial respiration with age, Hartley guinea pigs exhibit decreased mitochondrial efficiency. Collectively, this dissertation builds on prior work suggesting that the Hartley guinea pig is a valuable model to test preclinical interventions.Item Open Access Targeting skeletal muscle mitochondrial function with a Nrf2 activator in a novel model of musculoskeletal decline(Colorado State University. Libraries, 2020) Musci, Robert Vincent, author; Hamilton, Karyn L., advisor; Hickey, Matthew S., committee member; Lark, Daniel S., committee member; Santangelo, Kelly S., committee memberThis dissertation describes a series of three experiments with an overall objective to understand how targeting mitochondrial function with a phytochemical Nrf2 activator can prevent the onset of or mitigate the progression of mitochondrial dysfunction and sarcopenia in a novel model of musculoskeletal aging. The specific aims of the three experiments were to 1) characterize the age-related changes in skeletal muscle in Dunkin-Hartley guinea pigs; 2) assess the effect of Nrf2 activator treatment on skeletal muscle energetics by measuring mitochondrial function; and 3) determine how Nrf2 activator treatment influences components of skeletal muscle proteostasis. Dunkin-Hartley guinea pigs exhibit several characteristics reflective of human musculoskeletal aging including a decline in the proportion of type II muscle fibers, a shift towards a smaller myofiber size distribution, and a decline in muscle density in the gastrocnemius, as well as a decline in protein synthesis in both the soleus and gastrocnemius. In the second experiment, Nrf2 activator treatment improved mitochondrial respiration in both 5- and 15-month-old male and female guinea pigs. Moreover, Nrf2 activator treatment attenuated the age-related decline in mitochondrial respiration. In the third experiment, Nrf2 activator treatment attenuated the age-related decline in protein synthesis in Dunkin-Hartley guinea pigs. Altogether, these data demonstrate 1) Dunkin-Hartley guinea pigs experience age-related changes in skeletal muscle consistent with the aged musculoskeletal phenotype in humans 2) this phytochemical Nrf2 activator can improve mitochondrial function and 3) targeting mitochondrial dysfunction is an efficacious intervention to mitigate age-related declines in components of proteostasis in skeletal muscle and improve overall musculoskeletal function.Item Open Access The evaluation of myofiber remodeling and skeletal muscle inflammaging using a novel guinea pig model(Colorado State University. Libraries, 2020) Walsh, Maureen Ann, author; Hamilton, Karyn L., advisor; Lark, Daniel S., committee member; Reiser, Raoul F., II, committee member; Santangelo, Kelly S., committee memberApproximately 40% of total body mass is accounted for by the musculoskeletal system and thus, when it becomes dysfunctional it strongly influences whole body function. Sarcopenia is one facet of musculoskeletal aging and contributes to other age-related chronic diseases. Aging is a major risk factor for osteoarthritis, which is characterized by a concomitant loss of skeletal muscle, further contributing to decreased mobility. Age-related increases in low-grade inflammation and oxidative stress, referred to as the "inflammaging" phenotype, is common to both osteoarthritis and sarcopenia. While we have begun to understand the underlying pathology of sarcopenia, treatments are still lacking. One barrier to progress in identifying treatments is lack of a preclinical model that recapitulates the human skeletal muscle aging phenotype. Dunkin Hartley guinea pigs rapidly and spontaneously develop primary osteoarthritis beginning at about 4 months of age. The purpose of these studies was to determine if the Dunkin Hartley guinea pig can serve as a model to understand human skeletal muscle aging. Thus, we speculate that the Dunkin Hartley guinea pig may also be a valuable model of myofiber remodeling and skeletal muscle inflammaging. We compared skeletal muscle myofiber properties of the gastrocnemius and soleus from 5, 9, and 15-month Dunkin Hartley guinea pigs. We also compared these changes to a strain of guinea pig, Strain 13, that does not develop knee osteoarthritis at an early age. Additionally, in a second study, we assessed markers of skeletal muscle inflammation, oxidatively modified proteins, and redox signaling in 5 and 15-month Dunkin Hartley guinea pigs. The Dunkin Hartley guinea pig showed evidence of skeletal muscle aging including declines in gastrocnemius density and a shift in myofiber size distribution to encompass a greater percentage of smaller myofibers in both the gastrocnemius and soleus. Male Dunkin Hartley guinea pigs experience a trend to decrease Nrf2 protein content from 5 to 15-months implying altered redox signaling, while female Dunkin Hartley guinea pigs experienced a significant increase from 5 to 15-months. Skeletal muscle myofiber remodeling, a component of musculoskeletal aging, influences both muscle function and quality of life. Based on these analyses, Dunkin Hartley guinea pigs appear to be a potentially valuable model of musculoskeletal aging.Item Open Access Treatment with rapamycin or rapamycin in combination with metformin contributes to mechanisms of mitochondrial proteostasis in vivo and in vitro(Colorado State University. Libraries, 2018) Wolff, Christopher Andrew, author; Hamilton, Karyn L., advisor; Miller, Benjamin F., advisor; Hickey, Matthew, committee member; Pagliassotti, Michael, committee memberThis dissertation describes three sets of experiments with an overall objective of understanding how lifespan-extending treatments influence mechanisms of mitochondrial protein homeostasis (proteostasis). The specific aims of the three experiments were to 1) determine how the mTORC1 inhibitor rapamycin (Rap), or the anti-diabetes medication metformin in combination with rapamycin (Met+Rap) influence mitochondrial protein synthesis in young mice, and to determine if there are sex-specific differences in protein synthesis following treatment with Rap or Met+Rap; 2) examine the influence of Met+Rap treatment on protein synthesis in the heart, liver, and skeletal muscle of older mice and to determine if there are sex-specific differences in protein synthesis following Met+Rap treatment in old mice; 3) and, finally, to investigate the regulation of how protein turnover contributes to maintaining proteostasis during Rap and Met+Rap treatment, and the contribution of autophagic and mitophagic flux to protein turnover in cultured skeletal myotubes. In the first experiment, both Rap and Met+Rap treatments lowered mitochondrial protein synthesis in male mice compared to control in skeletal muscle. However, in female mice, only Met+Rap treatment significantly lowered skeletal muscle mitochondrial protein synthesis compared to control. Additionally, both Rap and Met+Rap treatments significantly elevated skeletal muscle mitochondrial protein synthesis in female animals compared to males. However, in the heart and liver tissue, there were no differences in mitochondrial protein synthesis between treatments or sexes. In the second experiment, Met+Rap treatment lowered protein synthesis in all three tissues, but in a fraction-specific manner. independent of sex-differences in old mice. For the third experiment, we measured protein synthesis and protein breakdown in cultured skeletal myotubes treated with Rap and Met+Rap. Rap treatment significantly increased mitochondrial protein:DNA compared to control, while Met+Rap did not. We demonstrate that autophagic flux is a large (29%) contributing process to degradation of mitochondrial proteins. Additionally, mitochondrial fission is not essential for mitochondrial protein degradation. The data from these experiments demonstrate that despite sexually dimorphic effects on lifespan, Rap and Met+Rap treatments both enhance the contribution of protein synthesis to maintaining proteostasis in vivo. Further, we demonstrate that both Rap and Met+Rap treatment increased protein:DNA in cultured skeletal myotubes. In summary, these data demonstrate that Rap and Met+Rap treatments increase proteostatic mechanisms, and further research is required to improve the understanding of how Met+Rap treatment influences lifespan.Item Open Access Upregulation of heme oxygenase-1 and activation of Nrf2 by the phytochemicals in protandim(Colorado State University. Libraries, 2010) Reuland, Danielle Judith, author; Hamilton, Karyn L., advisor; Miller, Benjamin F., committee member; Frye, Melinda A., committee memberIncreased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), with enhanced endogenous antioxidants proposed as a potential mechanism for promoting redox balance. Protandim is a well-defined combination of five widely studied medicinal plants derived from botanical sources [Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)]. The purpose of this study was to determine if treatment of cardiomyocytes with Protandim induces phase II detoxification enzymes, including the endogenous antioxidant heme oxygenase-1 (HO-1), with activation of nuclear factor E2 p45-related factor 2 (Nrf2), and protection from oxidative stress induced apoptosis. In cultured cardiomyocytes, treatment with Protandim was associated with activation of Nrf2 and a significant increase in HO-1. Protandim supplemented cells were protected against hydrogen peroxide-induced apoptosis as assessed by TUNEL (35% apoptotic in untreated vs. 5% apoptotic in Protandim treated). These findings support the use of Protandim as a potential method for upregulation of antioxidant defenses and protection of heart cells against an oxidative challenge. Future studies will focus on optimizing phytochemical induction of Nrf2-mediated antioxidant defenses in relevant in vivo models of CVD.