Inflammation is a central feature of various metabolic diseases including obesity and type-II diabetes. For this study, we hypothesized postprandial metabolism following an acute, high fat (HF) meal to be impaired in mice pre-injected with an inflammatory agonist. To this end, C57BL/6J mice were injected with saline or lipopolysaccharide (LPS, 1μg/kgbw) following an overnight fast and gavaged 2hr post-injection with water or a HF meal in liquid form (5kcal; 21.4%SF, 40.8%UF, 27.1%CHO, 10.7%PRO). Blood and muscle samples taken 3hr post-gavage underwent ex vivo analysis. Overall, results demonstrated a metabolic response to a HF meal that was blocked in the presence of LPS. Metabolic flexibility, though unchanged following the HF meal alone, was reduced following the HF meal in the presence of LPS. Additionally, state-4 uncoupled mitochondrial respiration, which was increased following the HF meal, was also reduced following the HF meal in the presence of LPS. Similar near-significant trends were also observed with total palmitate oxidation. Although no independent response to a HF meal or LPS exposure was observed, a unique interaction between treatments significantly diminished ADP dependent, state-3 and maximal respiration. These effects do not appear to be dependent on the production of reactive oxygen species (ROS) since neither the HF meal nor LPS exposure resulted in increased production of ROS. In conclusion, these results demonstrate that acute activation of inflammatory pathways results in alterations in metabolic response to a HF meal in skeletal muscle from mice, although the mechanism underlying these effects is not yet understood. / Master of Science

Skeletal muscle is highly involved in macronutrient metabolism. To maintain proper energy metabolism and physiology, skeletal muscle must adapt to nutrient supply. Thus, diet macronutrient composition is an important modulator of skeletal muscle metabolism. Evidence from rodent and human models show high-fat diets contribute to impaired insulin signaling, as well as decreased fatty acid and glucose oxidation. Utilizing proteomic analysis of metabolic proteins in humans may lead to the mechanism behind skeletal muscle adaption to macronutrient composition, potentially providing the groundwork for characterizing the etiology of high-fat feeding induced metabolic disease. The objective of this study was to compare the substrate oxidation patterns and the levels of metabolic proteins in the fasted skeletal muscle of lean, healthy males that either increased fatty acid oxidation in response to the high-fat diet, termed responders, or males that decreased fatty acid oxidation, termed non-responders. We employed a controlled feeding study design, where the participants served as their own controls. Following a 2-week control diet (30% fat, 55% carbohydrate and 15% protein), participants came to the lab fasted overnight and a muscle biopsy was taken from their vastus lateralis muscle. Participants were then placed on a 5-day high-fat diet (50% fat [45% saturated fat], 35% carbohydrate, and 15% protein). Following this diet, participants again came to the lab fasted overnight and another muscle biopsy was taken from their vastus lateralis muscle. Both the control and the high-fat diets were isocaloric to habitual diets. Muscle from the biopsies were utilized for substrate metabolism measures and mass spectrometry. We did not observe any significant differences in glucose oxidation between responders and non-responders, prior to or following the high-fat diet. Our proteomic analysis identified 81 proteins and protein subunits involved in substrate metabolism but only 6 were differentially regulated by the high-fat diet. Independent of the high-fat diet, compared to non-responders, responders contained an overall higher content of protein subunits belonging to Complex I and ATP synthase. The findings from this study suggest that adaption to high-fat feeding is individual specific and proteomic changes alone cannot explain high-fat feeding induced metabolic changes. / Ph. D. / Skeletal muscle is highly involved in macronutrient metabolism, which consist of the breakdown and utilization of glucose and fatty acids, thus making the foods we ingest a major modulator of skeletal muscle metabolism. Over the last few decades, Americans have increased their ingestion of foods high in saturated fats, which has coincided with the increased prevalence of obesity and type 2 diabetes. Further, evidence suggests these metabolic diseases are associated with the skeletal muscle’s inability to switch between the utilization of glucose and fatty acid in response to nutrient supply. Analyzing metabolic protein content in humans may lead to the mechanism behind skeletal muscle adaption to macronutrient composition, potentially leading to the cause behind the development of high-fat feeding induced metabolic disease. The objective of our controlled feeding study was to compare the macronutrient metabolism and the content of metabolic proteins in the fasted skeletal muscle of healthy males that either increased fatty acid utilization in response to a high-fat diet, termed responders, or males that decreased fatty acid utilization, termed non-responders. Following a 2-week control diet (30% fat, 55% carbohydrate and 15% protein), participants came to the lab fasted overnight and a biopsy was taken from their thigh muscle called the vastus lateralis. Participants then began a 5-day high-fat diet (50% fat [45% saturated fat], 35% carbohydrate, and 15% protein). Following this diet, participants came to the lab fasted overnight and another biopsy was taken from their vastus lateralis muscle. Both the control and the high-fat diets were isocaloric to habitual diets. The muscle samples were used to analyze macronutrient metabolism and identify metabolic protein content. We did not observe differences in glucose utilization between responders and non-responders, prior to or following the high-fat diet. We identified 81 metabolic proteins and protein subunits but only 6 were differentially regulated by the high-fat diet. Independent of diet, responders contained higher levels of subunits from 2 proteins involved in cell energy production, Complex I and ATP synthase. Our findings suggest that adaption to high-fat feeding is individual specific and protein content changes alone cannot explain high-fat feeding induced metabolic changes.

The cholinergic system drives muscle contraction and plays a central role in the formation, maintenance, and repair of mammalian neuromuscular junctions (NMJs) and skeletal muscles. Because of these essential actions, much effort has been devoted to identifying primary and auxiliary modulatory components of the cholinergic system at NMJs and throughout skeletal muscles. Here, I asked if Lynx1, a GPI-anchored protein shown to modulate nAChRs in the brain, is present and affects the activity of nAChRs at NMJs. Molecular and cellular analysis revealed that Lynx1 levels increase in skeletal muscles, specifically at NMJs, during development. Its expression pattern also closely mirrors changes in cholinergic transmission in vivo and in vitro. As expected, I found by co-immunoprecipitation that Lynx1 interacts with muscle nAChRs and using electrophysiology, I show that Lynx1 desensitizes nAChRs to ACh at NMJs. These findings demonstrate that Lynx1 regulates the cholinergic system at NMJs, suggesting roles for this gene in developing and adult NMJs. To determine the role of Lynx1 at NMJs, I examined Lynx1 knockout mice at different ages. While deletion of Lynx1 has no discernable effect on developing NMJs, its absence increases the incidence of NMJs with age-related morphological features, such as fragmentation and denervation, in young adult and middle-aged mice. Loss of Lynx1 also increases the number of slow-type muscle fibers in young and middle-aged mice, another hallmark of aging. Along with these morphological changes, deletion of Lynx1 affects expression of genes associated with NMJ stability, myogenesis, and muscle atrophy in young adult and middle-aged mice. Not surprisingly, the loss of Lynx1 reduces the density and stability of nAChRs at NMJs. Because of these findings, I surmised that loss of Lynx1 would adversely affect NMJs under other physiological stressors. However, I found the opposite as the loss of Lynx1 augments the capacity of NMJs to repair damages during exercise, following injury to motor axons, and during the initial symptomatic stage of amyotrophic lateral sclerosis (ALS). Since Lynx1 modulates the activity of nAChRs, these contrasting findings likely represent the positive and negative effects of heightened cholinergic transmission on aging compared to injury and disease-afflicted NMJs. / Doctor of Philosophy / During normal aging and in neuromuscular diseases, such as amyotrophic lateral sclerosis (ALS), voluntary movement becomes compromised. This is largely due to deterioration of the synapse between motor neurons and skeletal muscles, called the neuromuscular junction (NMJ), which is responsible for voluntary movement. Signaling at the NMJ is driven by cholinergic transmission, which when dysregulated can directly result in degeneration of the NMJ, similar to that seen in both aging and ALS. Thus, it is critical to maintain proper cholinergic transmission for preservation of the NMJ. For the first time, I have characterized the role of an endogenous protein, Lynx1, in modulating cholinergic transmission at the NMJ. Lynx1 functions to dampen cholinergic activity to prevent muscles from becoming overwhelmed and fatigued. The work outlined in this dissertation proposes Lynx1 as a novel therapeutic candidate for preventing neuromuscular degeneration in conditions associated with dysregulated cholinergic transmission, such as ALS.

No

Skeletal muscle atrophy is a common, debilitating consequence of muscle disuse, malnutrition, critical illness, musculoskeletal conditions, neurological disease, cancer, and organ failure. Despite its prevalence, little is known about the molecular pathogenesis of this devastating condition due in large part to an incomplete understanding of the molecular mechanisms that drive the atrophy process. In previous studies, we identified the transcription factor ATF4 as a critical mediator of skeletal muscle atrophy. We found that ATF4 is necessary and sufficient for skeletal muscle atrophy during limb immobilization. However, ATF4 mKO mice were only partially protected from skeletal muscle atrophy during limb immobilization, indicating the existence of another pro-atrophy factor that acts independently of the ATF4 pathway. Using mouse models, we identify p53 as this ATF4-independent factor. We show that skeletal muscle atrophy increases p53 expression in skeletal muscle fibers. In addition, overexpression of p53 causes skeletal muscle atrophy. Further, p53 mKO mice are partially resistant to muscle atrophy during limb immobilization. Taken together, these data indicate that like ATF4, p53 is sufficient and required for skeletal muscle atrophy during limb immobilization. Importantly, overexpression of p53 induces muscle atrophy in the absence of ATF4, whereas ATF4-mediated muscle atrophy does not require p53. Furthermore, overexpression of p53 and ATF4 induces greater muscle atrophy than p53 or ATF4 alone. Moreover, skeletal muscle lacking both p53 and ATF4 is more resistant to skeletal muscle atrophy than muscle lacking either p53 or ATF4 alone. Taken together, these data indicate that p53 and ATF4 mediate distinct and additive mechanisms to skeletal muscle atrophy. However, the precise mechanism by which p53 and ATF4 cause skeletal muscle atrophy remained unclear. Using genome-wide expression arrays, we identify p21 as a skeletal muscle mRNA that is highly induced by p53 and ATF4 during limb immobilization. Further, overexpression of p21 causes skeletal muscle atrophy. In addition, p21 is required for muscle atrophy due to limb immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as critical and complementary mediators of skeletal muscle atrophy during limb immobilization, and discover p21 as an essential downstream mediator of the p53 and ATF4 pathways.

Skeletal muscle (SM) atrophy complicates many illnesses, diminishing quality of life and increasing disease morbidity, health resource utilization and health care costs. In animal models of muscle atrophy, loss of SM mass results predominantly from ubiquitin-mediated proteolysis and ubiquitin ligases are the key enzymes that catalyze protein ubiquitination. We have previously shown that ubiquitin ligase Nedd4-1 is up-regulated in a rodent model of denervation-induced SM atrophy and the constitutive expression of Nedd4-1 is sufficient to induce myotube atrophy in vitro, suggesting an important role for Nedd4-1 in the regulation of muscle mass. In this study we generate a Nedd4-1 SM specific-knockout mouse and demonstrate that the loss of Nedd4-1 partially protects SM from denervation-induced atrophy confirming a regulatory role for Nedd4-1 in the maintenance of muscle mass in vivo. Nedd4-1 did not signal downstream through its known substrates Notch-1, MTMR4 or FGFR1, suggesting a novel substrate mediates Nedd4-1’s induction of SM atrophy.

In the present study Fourier Transform Infrared Spectroscopy was used to examine the effects of streptozotocin-induced diabetes mellitus on the structural components of slow- and fast-twitch rat skeletal muscles, at molecular level. Diabetes mellitus is a chronic disorder of carbohydrate, fat and protein metabolism, which is characterized by hyperglycemia caused by a defective or deficient insulin secretory response. The effect of diabetes is seen on a variety of tissues leading to important secondary complications such as kidney failure, liver dysfunction, cardiac disorders, etc. Skeletal muscle is one of the major tissues determining carbohydrate and lipid metabolism in the body / therefore, is one of the target tissues of diabetes. The two main types of muscle fibers are type I (slow-twitch) and type II (fast-twitch) fibers / having different structural organization and metabolic features. The FTIR spectra revealed a considerable decrease in lipid and protein content of diabetic skeletal muscles, indicating an increased lipolysis and protein breakdown or decreased protein synthesis. Moreover changes in protein structure and conformation were observed. In diabetes, muscle membrane lipids were more ordered and the amount of unsaturated lipids was decreased possibly due to lipid peroxidation. Diabetes caused a decrease in the content of nucleic acids, especially RNA, and hydrogen bonded phospholipids in the membrane structures of skeletal muscles. In all of the spectral parameters investigated slow-twitch muscle was more severely affected from diabetes. Thus, FTIR spectroscopy appears to be a useful method to evaluate the effect of diabetes on skeletal muscle tissues at molecular level.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 63-74). Also available on the Internet.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 18, 2010). Includes bibliographical references.

Congenital myopathies are a rare and heterogeneous group of diseases. They are primarily characterised by skeletal muscle weakness and disease-specific pathological features. They harshly limit ordinary life and in severe cases, these myopathies are associated with early death of the affected individuals. The congenital myopathies investigated in this thesis are nemaline myopathy and myofibrillar myopathy. These diseases are usually caused by missense mutations in genes encoding myofibrillar proteins, but the exact mechanisms by which the point mutations in these proteins cause the overall weakness remain mysterious. Hence, in this thesis two different nemaline myopathy-causing actin mutations and one myofibrillar myopathy-causing myosin-mutation found in both human patients and mouse models were used to investigate the cascades of molecular and cellular events leading to weakness. I performed a broad range of functional and structural experiments including skinned muscle fibre mechanics, small-angle X-ray scattering as well as immunoblotting and histochemical techniques. Interestingly, according to my results, point mutations in myosin and actin differently modify myosin binding to actin, cross-bridge formation and muscle fibre force production revealing divergent mechanisms, that is, gain versus loss of function (papers I, II and IV). In addition, one point mutation in actin appears to have muscle-specific effects.  The presence of that mutant protein in respiratory muscles, i.e. diaphragm, has indeed more damaging consequences on myofibrillar structure than in limb muscles complexifying the pathophysiological mechanisms (paper II). As numerous atrophic muscle fibres can be seen in congenital myopathies, I also considered this phenomenon as a contributing factor to weakness and characterised the underlying causes in presence of one actin mutation. My results highlighted a direct muscle-specific up-regulation of the ubiquitin-proteasome system (paper III). All together, my research work demonstrates that mutation- and muscle-specific mechanisms trigger the muscle weakness in congenital myopathies. This gives important insights into the pathophysiology of congenital myopathies and will undoubtedly help in designing future therapies.