This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.

Type II diabetes mellitus (T2DM) has been associated with the onset of diet-induced obesity, which is currently on the rise worldwide. T2DM is typically characterized by insulin resistance in peripheral tissues such as adipose tissue, liver, and skeletal muscle. In skeletal muscle it is widely accepted that the defective insulin action is due to the inability of the cell to sufficiently activate the insulin signalling pathway and promote systemic glucose uptake. The sarcolipin-null (KO) mouse is a potential novel model for diet-induced obesity and diabetes. KO mice become significantly more obese and display a greater glucose intolerance than wildtype (WT) mice following an 8-week high-fat diet (HFD; 42% calories from fat) but the underlying mechanisms are still unknown. In this study the role of defective skeletal muscle insulin signalling in the development of the impaired glucose tolerance in KO mice was investigated. It was hypothesized that the HFD fed KO mice would exhibit greater reductions in IRS1 tyr628 and Akt ser473 phosphorylation (i.e. decreased activation of the insulin signalling pathway) than controls. Furthermore, it was believed that KO mice would display increased phosphorylation of IRS1 ser307, which is commonly associated with insulin resistance. At 16-weeks of age KO mice and littermates were subdivided into two groups and placed on either a HFD (n=30) or chow diet (n=24) for an 8-week period. Changes in body weight, glucose tolerance, and insulin tolerance were assessed pre- and post-diet period. Following the completion of the diet intervention mice were treated with an intraperitoneal injection of insulin (0.75U/kg) or vehicle solution and sacrificed for tissue collection. Epididymal/inguinal and retroperitoneal fat pads were removed for assessment of whole body adiposity. Whole gastrocnemius muscle was excised and homogenized for Western blot analysis of several key proteins of the insulin signalling cascade. Following completion of the HFD KO mice (48.6 ± 1.6 g) weighed significantly more than HFD fed wildtype (WT) mice (41.5 ± 1.6 g), and all chow fed mice (KO: 36.8 ± 1.5 g; WT: 35.2 ± 1.2 g; p<0.001). Glucose tolerance testing showed that KO mice exhibited significantly greater glucose intolerance compared to control mice post-HFD (p<0.001). Insulin tolerance testing, however, revealed no change in insulin sensitivity in KO or WT mice post-HFD (p>0.05). The HFD fed KO mice (0.73 ± 0.06 g) had an elevated retroperitoneal fat pad weight than HFD fed WT (0.49 ± 0.05 g) and all chow fed mice (KO: 0.28 ± 0.04 g; WT: 0.24 ± 0.04 g; p<0.01). Western blot analysis revealed a similar reduction in insulin receptor substrate-1 (IRS1) tyr628 phosphorylation in both KO and WT mice following the HFD (Con WT: 2.82 ± 0.69; Con KO: 3.06 ± 0.73; HFD WT: 1.71 ± 0.28; HFD KO: 1.28 ± 0.11 fold increase over non-insulin stimulated mice; p<0.02). IRS1 ser307 phosphorylation was elevated in both genotypes post-HFD (HFD WT: 2.97 ± 1.19; HFD KO: 2.17 ± 0.59 fold increase over standard chow fed control mice; p<0.03). Insulin treatment did not stimulate phosphorylation of Akt ser473 in KO or WT mice regardless of diet (p>0.05). In summary there was no difference between KO and WT mice in skeletal muscle insulin sensitivity as assessed by the phosphorylation of insulin signalling intermediates. An increase in IRS1 ser307 phosphorylation appears to be the primary mechanism for the reduced activation of IRS1 following the HFD in both KO and WT mice. However, the results from the current investigation did not support the notion that impaired skeletal muscle insulin signalling is responsible for the more pronounced diet-induced glucose intolerance observed in KO mice. Future studies investigating the viability of skeletal muscle GLUT4 translocation and glucose uptake as well as the glucose-induced insulin secretion of pancreatic β-cells following consumption of a HFD would help elucidate the mechanism of glucose intolerance in KO mice.

Fatty acid (FA) transport proteins are important regulators of FA uptake at the cell surface and the mitochondria where they are oxidized. Tight regulation of this process is necessary in order to meet metabolic requirements, while preventing excess lipid accumulation. In an obese state, there is an increase in FA uptake and increased storage of lipids in skeletal muscle, including diacylglycerol (DAG) and ceramides, which interfere with insulin-stimulated glucose uptake. Leptin administration has been shown to reduce muscle triacylglycerol accumulation and restore insulin response in obese rodents. However, it is not known whether this is mediated through a redistribution of the FA transport proteins to the cell surface and mitochondria. In addition to hyperglycemia, post-prandial lipidemia is also observed in the obese state, suggesting a resistance to insulin-stimulated FA uptake. The possibility that insulin-stimulated FA transporter translocation is impaired has received little attention. Lastly, while recent studies have demonstrated that the transverse (t)-tubules may be an important site for glucose uptake in muscle, this has not yet been examined with regards to the FA transporters. In the first study of this thesis, the recovery of insulin response with short-term (2 week) chronic leptin administration in high-fat fed rats was associated with a decrease in muscle reactive lipid species (DAG, ceramide) and an increase in markers of oxidative capacity. Contrary to our expectations, this was not mirrored by an alteration in the distribution of FA transport proteins (FAT/CD36 or FABPpm) at the sarcolemma or the two major mitochondrial populations. To gain further insight into FA transporters and their localization at the cell surface, the second study of this thesis analyzed both the sarcolemma and t-tubules (constitute 40 and 60% of the cell surface, respectively). The novel observation was made that the t-tubules contain FA transport proteins (FAT/CD36, FABPpm, FATP1 and FATP4), and that the distribution and response of these transporters to acute metabolic stimuli (insulin and muscle contraction) was unique from that of the sarcolemma. The third study of this thesis characterized the translocation of FA transport proteins in response to insulin in the obese, insulin resistant Zucker rat. FA transport proteins were chronically increased on both membrane fractions in muscle from the obese rats. Furthermore, a blunting of the insulin-induced translocation of FA transporters to both cell surface domains was observed, demonstrating that insulin resistance extends to the movement of FA as well as glucose transport proteins. The t-tubules appear to play an important role regarding substrate uptake. Together the data from this thesis suggests that a chronic elevation in FA transporters at both cell surface domains contributes to lipid accumulation in obese skeletal muscle, and that reduced sensitivity of both FA and glucose transport proteins to translocate in response to insulin may explain the lipidemia and hyperglycemia that often characterizes post-prandial situations in the obese condition. As the prevalence of obesity reaches epidemic proportions, research into the functional role of FA transport proteins in the progression of obesity related pathologies is warranted as we work to further our knowledge of this significant health issue. / Natural Sciences and Engineering Research Council, Canadian Institute of Health Research

This thesis is a series of investigations into the regulation of skeletal muscle mitochondrial metabolism. Novel regulatory mechanisms regarding mitochondrial fatty acid oxidation are continually being identified and alterations in skeletal muscle mitochondrial metabolism have been implicated in the pathogenesis of type II diabetes (T2DM). Therefore, advancing our basic understanding of mitochondrial regulatory processes is required to provide insight into the progression of T2DM. In study one, the utilization of knockout mice for the putative mitochondrial fatty acid transport protein FAT/CD36, showed that mitochondrial FAT/CD36 plays a functional role in mitochondrial long chain fatty acid (LCFA) oxidation. Specifically, FAT/CD36 was found to be located on the outer mitochondrial membrane (OMM) upstream of acyl-CoA synthetase. In study two, it was observed that in rat muscle, malonyl-CoA (M-CoA) inhibition kinetics of carnitine palmitoyltransferase I (CPT-I) display a more physiological IC50 in permeabilzed muscle fibre bundles (PmFB) compared to isolated mitochondria. These data suggest that the cytoskeleton may have a role in regulating M-CoA inhibition. Additionally, a significant effect of LCFA-CoA on M-CoA inhibition kinetics was observed. These data indicate that M-CoA content does not need to decrease to promote an increase in CPT-I flux. Finally, in a model of T2DM (ZDF rat), submaximal ADP-stimulated respiration rates and the content of adenine nucleotide translocase 2 (ANT2) content were depressed compared to lean control animals. Resveratrol treatment in ZDF rats recovered these declines concomitantly with improving insulin-stimulated skeletal muscle glucose uptake and the cellular redox state. A number of novel findings are presented, specifically, 1) a functional role for mitochondrial FAT/CD36 in mitochondrial LCFA oxidation was confirmed and the topology of this protein along the OMM is expanded upon, 2) M-CoA inhibition kinetics of CPT-I were re-evaluated in PmFB and a regulatory role of LCFA-CoA on M-CoA inhibition kinetics is established, and 3) submaximal ADP-stimulated respiration rates and ANT2 content are depressed in the ZDF rat and resveratrol supplementation prevents these decrements.

This thesis investigated the effects of omega-3 supplementation (2.0 g/day EPA + 1.0 g/day DHA) for 12 weeks on human skeletal muscle sarcolemmal and mitochondrial membrane fatty acid (FA) composition and whole body energy expenditure in young healthy males. Supplementation resulted in significant incorporation of EPA and DHA into sarcolemmal and mitochondrial membranes, with an increase in total unsaturation of mitochondrial membranes. The incorporation profile of the sarcolemma and mitochondria differed, with the mitochondria mimicking changes in whole muscle. There were no changes in the protein content of mitochondrial and selected proteins involved in energy metabolism, except for a significant increase in the long form of UCP3. Despite changes in membrane FA compositions, there were no changes in whole body substrate oxidation at rest or during exercise. These data demonstrate that omega-3 supplementation for 12 weeks altered the FA composition of sarcolemmal and mitochondrial membranes in human skeletal muscle.

No description available.

No description available.

Skeletal satellite cells are adult stem cells located among muscle fibers. Proliferation, migration and subsequent differentiation of these cells are critical steps in the repair of muscle injury. We document in this study the roles and mechanisms through which the NAPDH oxidase complex regulates skeletal satellite cell proliferation. The NADPH oxidase subunits Nox2, Nox4, p22phox, p47phox and p67 phox were detected in primary human and murine skeletal muscle satellite cells. In human satellite cells, NADPH oxidase-fusion proteins were localized in the cytosolic and membrane compartments of the cell, except for p47 phox, which was detected in the nucleus. In proliferating subconfluent satellite cells, both Nox2 and Nox4 contributed to O2- production. However, Nox4 expression was significantly attenuated in confluent cells and in differentiated myotubes. Proliferation of satellite cells was significantly reduced by antioxidants (N-acetylcysteine and apocynin), inhibition of p22phox expression using siRNA oligonucleotides, and reduction of Nox4 and p47phox activities with dominant-negative vectors resulted in attenuation of activities of the Erk1/2, PI-3 kinase/AKT and NFkappaB pathways and significant reduction in cyclin D1 levels. We conclude that NADPH oxidase is expressed in skeletal satellite cells and that its activity plays an important role in promoting proliferation of these cells.

Cache Valley virus (CVV) induced malformations have been previously reproduced in ovine fetuses; however, no studies have established the CVV infection sequence of the cells targeted by the virus or the development of the antiviral response of the early, infected fetus that results in viral clearance before development of immunocompetency. To address these questions, ovine fetuses at 35 dg were inoculated in utero with CVV and euthanized at 7, 10, 14, 21 and 28 dpi. On postmortem examination arthrogryposis and oligohydramnios were observed in some infected fetuses. Morphologic studies showed necrosis in the central nervous system (CNS) and skeletal muscle of earlier infected fetuses and hydrocephalus, micromyelia and muscular loss in later infected fetuses. Using immunohistochemistry and in situ hybridization, intense CVV viral antigenic signal was detected in the brain, spinal cord, skeletal muscles and fetal membranes of infected fetuses. Viral signal decreased in targeted and infected tissues with the progression of the infection. To determine specific cell types targeted by CVV in the CNS, indirect immunofluorescence was applied to sections of the CNS using a double labeling technique with antibodies against CVV together with antibodies against neurons, astrocytes and microglia. CVV viral antigen was shown within the cytoplasm of neurons in the brain and spinal cord. No viral signal was observed in microglial cells; however, infected animals had marked microgliosis. The antiviral immune response in immature fetuses infected with CVV was evaluated. Gene expression associated with an innate, immune response was quantified by real-time, quantitative PCR. Upregulated genes in infected fetuses included ISG15, Mx1, Mx2, IL-1, IL-6, TNF-?, TLR-7 and TLR-8. The amount of Mx protein, an interferon stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. ISG15 protein expression was significantly increased in target tissues of infected animals. B lymphocytes and immunoglobulin-positive cells were detected in lymphoid tissues and in the meninges of infected animals. This demonstrated that the infected ovine fetus is able to stimulate an innate and adaptive immune response before immunocompetency that presumably contributes to viral clearance in infected animals.

The inflammatory process is a critical component of the repair and regeneration of skeletal muscle following injury. The influx of innate immune cells following injury is intricate, and temporal nature. Although required for proper repair and regeneration, the inflammatory process has been shown to exacerbate initial damage, prolonging the healing process. Complementary Alternative Medicine (CAM) treatments, such as massage therapy, are a promising substitute for pharmaceutical modulation of the inflammatory response, and recent studies into the efficacy of massage have begun to report the physiological benefits of massage application following injury. Nonetheless, there is a significant lack of sound mechanistic investigations into massage application and its effects on unperturbed tissue. To gain insight to its potential influences on healthy skeletal muscle, massage was applied at three different magnitudes of load in vivo. Using a custom fabricated device for cyclic compressive loading, Wistar rats receiving massage had an increased expression in genes associated with the immune response; a significant change in the macrophage populations within the muscle tissue; and demonstrated a systemic effect marked by the increase of immune cells in the non-massaged limb. Further elucidating the systemic and immunomodulatory effects of massage, Long Evans rats receiving non-constrained eccentric exercise followed by a single 30minute bout of massage, displayed a significant crossover effect just 6 hours post exercise through the modulation of inflammatory cells in the non-massaged limb. Together these investigations suggest that mechanotransductive properties of massage can promote modulation of the immune response absent of pharmaceuticals.