Androgen endocrine physiology is responsible for many processes in the body, from bone metabolism to skeletal muscle maintenance. Testosterone is one of the most potent androgens produced, with decades of research highlighting its androgenic-anabolic effect on skeletal muscle growth. The research has been further driven through the emergence of pharmaceutical derivates (steroids) of testosterone and their use in interventions for muscle wasting associated with disease and advancing age (sarcopenia). However, the molecular regulation of testosterone and the influence it has on cellular processes in promoting muscle development, hypertrophy and satellite cell activation (proliferation, differentiation) remain poorly understood. In summary, this thesis has highlighted the impact of testosterone in rescuing an aged phenotype in myoblast cells (PD cells) displaying prior reductions in regeneration and hypertrophy vs. relevant ‘un-aged’ controls (Sharples et al., 2011, Sharples et al., 2012). Importantly, the results provide evidence towards the mechanisms for testosterone induced hypertrophy, where in the presence of testosterone and a functional androgen receptor (shown via AR antagonism/ flutamide administration) testosterone is fundamental in regulating appropriate myoD, myogenin and myostatin expression in the control of myotube differentiation and hypertrophy. Increases in androgen receptor protein levels in PD myoblasts under a testosterone stimulus highlight the intrinsic responsiveness of aged myoblasts to a hypertrophic stimulus which may somewhat explain the use of testosterone as an effective clinical treatment in muscle wasting disease. Finally, myotube hypertrophy occurred in both aged and un-aged myoblasts with testosterone administration even in the presence of an IGF-I receptor inhibitor (Picropodophyllin) suggesting a limited role of the IGF-IR and associated signaling in testosterones mediated increases in differentiation and hypertrophy. Testosterone did appear however to increase Akt phosphorylation in cells that show a prior impaired regenerative capacity (PD cells), even in the presence-IGF-IR inhibitor suggesting that testosterone can directly activate Akt independently of upstream IGF-IR a novel finding that is presented in chapter 5. This interaction maybe mediated by myostatin, where increases in myostatin has been linked with directly inhibiting Akt (Dubois et al., 2014, Léger et al., 2008, Morissette et al., 2009), and in the present study we see an increase in myostatin mRNA and corresponding decrease in phospho-Akt when AR’s (via flutamide) action is inhibited and the protein level of AR is low (chapter 5). Furthermore in chapter 6 we further showed that inhibition of PI3K/AKT using LY294002 was sufficient to remove the prior rescuing of differentiation and hypertrophy in aged cells after testosterone administration. This was coupled with reductions in myostatin and increases in mTOR with testosterone administration. Overall AR seems to be the most fundamental pathway in testosterones induction of myotube differentiation and hypertrophy with important roles for myogenin, Akt, mTOR and myostatin in mediating these processes.

Altas doses de esteróides anabólicos androgênicos (EAA) são utilizadas, sem indicação terapêutica, por indivíduos que visam aumentara força muscular ou melhorar a aparência física. Entretanto, os efeitos benéficos destas substâncias sobre o desempenho atlético são questionáveis, e sabe-se que esta prática é acompanhada de muitos efeitos colaterais. Este estudo teve por objetivo analisar as alterações histológicas e morfométricas das fibras do músculo sóleo e melhora no desempenho de ratos submetidos a um programa de treinamento físico, associado ou não à administração do esteróide anabólito decanoato de nandrolona. Os animais foram divididos em quatro grupos: sedentário + veículo (SC); treinado + veículo (TC); sedentário + EAA (SE); treinado + EAA (TE). Os animais foram tratados, por 4 semanas, com veículo (propilenoglicol, 0,2 mL/Kg) ou decanoato de nandrolona (Deca-Durabolin, 5 mg/Kg), i.m., 1 x/semana. O treinamento físico foi realizado através de saltos em meio líquido (4 séries, 10 repetições, 60 segundos de intervalo entre as séries), 50-80% do peso corporal, 3 dias/semana, 4 semanas. Antes e após o treinamento os animais foram submetidos a um teste de fadiga. Após o sacrifício, o músculo sóleo direito foi retirado. Cortes do terço médio desse músculo foram feitos em micrótomo criostato e corados pela técnica HE. Os animais submetidos ao treinamento físico (TC) e (TE) apresentaram fibras musculares com maior área, quando comparados com os animais controle (SC e SE). Não foram observadas diferenças no desempenho entre os grupos. Os resultados sugerem que o treinamento físico produz hipertrofia de forma semelhante, tanto no grupo que recebeu EAA quanto no que recebeu óleo mineral. No entanto, o grupo TE apresentou sinais de maior degeneração. / High doses of anabolic androgenic steroids (AAS) are used without therapeutic indication in order to increase the muscle power or improve the physical appearance. However the beneficial effects of AAS on the athletic performance are still questionable and it is well established that AAS abuse is associated with many detrimental side-effects. The objective of this study was to analyze the histological and morphometrical alterations of the fibers from the soleum muscle and performance improvement of rats submitted to a physical training, associated not to the administration of the anabolic steroid decanoate of nandrolone. Male Wistar rats were divided into four groups: sedentary + vehicle (SC), trained + vehicle (TC), sedentary + AAS (SE) and trained + AAS (TE). The animals were treated for 4 weeks with vehicle (propyleneglycol, 0.2 mL/Kg) or nandrolone decanoate (Deca-Durabolin, 5 mg/Kg), i.m., 1x/week. Training was performed by jumping into water (4 sets, 10 repetitions, 60 seconds rest), 50-80% body weight-load, 3 days/week, 4 weeks). Before and after training the animals were submitted a test of fatigue. After sacrifice, the right soleum muscle was removed. Third middle cuts of this muscle were made in microtome cryostat and stained through HE technique. The animals submitted to physical training (TC) and (TE) presented muscle fibers with bigger area when compared to the ones from the control group (SC and SE). Performance improvement of all experimental groups was not altered. The results suggest that physical training produces muscle hypertrophy similarly, not only in the group that received AAS, but also in the one that received mineral oil. However, the group TE presented signs of greater muscle degeneration.

Left Ventricular Hypertrophy (LVH) is common in Type 2 Diabetes (T2DM) and despite optimal treatment of blood pressure can still persist. We know LVH is a cardiovascular (CV) risk factor in its own right and contributes to high CV event rates in patients with T2DM. Apart from hypertension, other factors contribute to the development of LVH in patients with T2DM, in particular oxidative stress (OS) has been implicated in LVH development. Allopurinol is a potent anti-oxidant, acting by blocking the enzyme Xanthine Oxidase, and has been previously shown to reduce vascular OS. Therefore the main aim of this thesis was to investigate whether allopurinol regresses LVH in patients with T2DM. The trial design was a randomised, double blind, placebo controlled study in 66 patients with T2DM with echocardiographic evidence of LVH. Allopurinol 600mg/day or placebo was given for nine months over the study period. The primary outcome was reduction in left ventricular mass (LVM) as calculated by cardiac magnetic resonance imaging (CMR) at baseline and at nine months follow-up. The secondary end-points were change in flow mediated dilatation (FMD) and augmentation index (AIx). Allopurinol significantly reduced absolute LVM (-2.65 ± 5.91g and placebo group +1.21 ± 5.10g (p=0.012)) and LVM indexed to body surface area (-1.32 ± 2.84g/m2 and placebo group +0.65 ± 3.07g/m2 (p=0.017)). When analysis was made of high and low baseline LVM then the effects of allopurinol were exaggerated in the high LVM mass group. No significant change was seen in either FMD or AIx. This thesis shows that allopurinol regresses LVM in patients with T2DM and LVH and controlled blood pressure. Regressing LVH has been shown previously to improve CV mortality and morbidity. Therefore allopurinol may become a useful therapy to reduce CV events in T2DM patients with LVH.

An acute bout of resistance exercise (RE) and dietary protein consumption stimulate muscle protein synthesis (MPS). This anabolic effect is believed to be attenuated with resistance exercise training (RET), however, the mechanism for this plateau" is unknown. In addition, the ideal timing for protein consumption to optimize MPS is not well characterized. The central hypothesis of this research is that RE stimulates cumulative (measured over 24-36 h) MPS in rats and humans. Study one determined whether an acute bout of RE in rats enhances MPS when assessed with the traditional flooding dose (~ 25 min) and 2H2O (4 and 24 h measurements); thus a comparison of the two methodologies was made. An acute session of RE did not result in an elevation in MPS when quantified by either the flooding dose or 2H2O over 4 and 24 h (methods compared qualitatively). Therefore, an acute bout of RE in rats does not appear to be anabolic and adaptation resulting from multiple bouts is likely necessary. Study two determined if RET in rats results in attenuation in MPS (plateau effect) 16 h following the final RE session (peak anabolic window) and if it is due to an increase in 4E-BP1 (a key regulator of mRNA translation initiation) activity; or if the timing in anabolism changes, which could be detected with a cumulative assessment (2H2O). MPS at 16 h was unchanged following RE training. Consistent with this finding, there were no differences in 4E-BP1 activity. Conversely, cumulative MPS was significantly increased with RET, suggesting a temporal shift in anabolism. Study three determined if dietary protein consumed immediately following RE augments cumulative (24 h) MPS in young adult human males when energy and macronutrients are controlled. RE and post-RE protein had no effect on mixed MPS; however, myofibrillar MPS was significantly increased with RE suggesting specific changes within a heterogeneous protein pool. Collectively, these are the first studies to assess changes in cumulative MPS with RE in rats and humans. The long term goals of this research are to understand muscle protein anabolism in "free-living" mammals and the mechanisms that regulate this process.

The first objective of my work was to use the ank/ank (progressive ankylosis) mutant mice, which have a deficiency in inorganic pyrophosphate transport, to address the role of Ank in joint ankylosis. I observed the presence of hypertrophic chondrocytes in the uncalcified ank/ank mice articular cartilage. This novel phenotype is likely due to a dysregulation of chondrocyte maturation as these chondrocytes expressed hypertrophic chondrocyte markers (collagen type X and tissue non-specific alkaline phosphatase). I also showed by immunohistochemical staining that beta-catenin expression was upregulated and localized in the nuclei of articular ank/ank chondrocytes, suggesting activation of Wnt/beta-catenin signaling in these chondrocytes. The second objective was to use ank/ank mice as an informative model for understanding ankylosis mechanisms in human ankylosing spondylitis (AS) patients, as WNT/beta-catenin signaling plays an important role in ankylosis in AS patients. We attempted rescue of joint ankylosis in ank/ank mice by gene transfer of noggin, an antagonist of BMP signaling. Paradoxically, noggin-treated ank/ank mice had accelerated ankylosis, as evidenced by joint pathology and IHC staining of beta-catenin showed more intense signals in the spinal chondrocytes of the treated mice. As noggin and sclerostin (an antagonist of beta-catenin signaling) form a mutually inhibitory complex, we hypothesize that the formation of this complex results in relieving suppression of both beta-catenin and BMP signaling, leading to more severe ankylosis in ank/ank mice. By quantitative molecular imaging, I have demonstrated that ankylosis in these mutant mice developed simultaneously in distal and axial joint, instead of being a centripetal process. In summary, I have made three original observations in the ank/ank mice: the hypertrophic chondrocyte phenotype; activation of beta-catenin signaling and the simultaneous development of ankylosis in distal and axial joints. These mutant mice serve as valuable model for pre-clinical studies which enable modeling and testing of novel anti-ankylosis treatments.

The first objective of my work was to use the ank/ank (progressive ankylosis) mutant mice, which have a deficiency in inorganic pyrophosphate transport, to address the role of Ank in joint ankylosis. I observed the presence of hypertrophic chondrocytes in the uncalcified ank/ank mice articular cartilage. This novel phenotype is likely due to a dysregulation of chondrocyte maturation as these chondrocytes expressed hypertrophic chondrocyte markers (collagen type X and tissue non-specific alkaline phosphatase). I also showed by immunohistochemical staining that beta-catenin expression was upregulated and localized in the nuclei of articular ank/ank chondrocytes, suggesting activation of Wnt/beta-catenin signaling in these chondrocytes. The second objective was to use ank/ank mice as an informative model for understanding ankylosis mechanisms in human ankylosing spondylitis (AS) patients, as WNT/beta-catenin signaling plays an important role in ankylosis in AS patients. We attempted rescue of joint ankylosis in ank/ank mice by gene transfer of noggin, an antagonist of BMP signaling. Paradoxically, noggin-treated ank/ank mice had accelerated ankylosis, as evidenced by joint pathology and IHC staining of beta-catenin showed more intense signals in the spinal chondrocytes of the treated mice. As noggin and sclerostin (an antagonist of beta-catenin signaling) form a mutually inhibitory complex, we hypothesize that the formation of this complex results in relieving suppression of both beta-catenin and BMP signaling, leading to more severe ankylosis in ank/ank mice. By quantitative molecular imaging, I have demonstrated that ankylosis in these mutant mice developed simultaneously in distal and axial joint, instead of being a centripetal process. In summary, I have made three original observations in the ank/ank mice: the hypertrophic chondrocyte phenotype; activation of beta-catenin signaling and the simultaneous development of ankylosis in distal and axial joints. These mutant mice serve as valuable model for pre-clinical studies which enable modeling and testing of novel anti-ankylosis treatments.

An acute bout of resistance exercise (RE) and dietary protein consumption stimulate muscle protein synthesis (MPS). This anabolic effect is believed to be attenuated with resistance exercise training (RET), however, the mechanism for this plateau" is unknown. In addition, the ideal timing for protein consumption to optimize MPS is not well characterized. The central hypothesis of this research is that RE stimulates cumulative (measured over 24-36 h) MPS in rats and humans. Study one determined whether an acute bout of RE in rats enhances MPS when assessed with the traditional flooding dose (~ 25 min) and 2H2O (4 and 24 h measurements); thus a comparison of the two methodologies was made. An acute session of RE did not result in an elevation in MPS when quantified by either the flooding dose or 2H2O over 4 and 24 h (methods compared qualitatively). Therefore, an acute bout of RE in rats does not appear to be anabolic and adaptation resulting from multiple bouts is likely necessary. Study two determined if RET in rats results in attenuation in MPS (plateau effect) 16 h following the final RE session (peak anabolic window) and if it is due to an increase in 4E-BP1 (a key regulator of mRNA translation initiation) activity; or if the timing in anabolism changes, which could be detected with a cumulative assessment (2H2O). MPS at 16 h was unchanged following RE training. Consistent with this finding, there were no differences in 4E-BP1 activity. Conversely, cumulative MPS was significantly increased with RET, suggesting a temporal shift in anabolism. Study three determined if dietary protein consumed immediately following RE augments cumulative (24 h) MPS in young adult human males when energy and macronutrients are controlled. RE and post-RE protein had no effect on mixed MPS; however, myofibrillar MPS was significantly increased with RE suggesting specific changes within a heterogeneous protein pool. Collectively, these are the first studies to assess changes in cumulative MPS with RE in rats and humans. The long term goals of this research are to understand muscle protein anabolism in "free-living" mammals and the mechanisms that regulate this process.

This research investigated the changes in body adipocyte size and density as well as parameters relating to adipogenesis in growing cobia, Rachycentron canadum. The cobia from a same batch were sampled 5 , 7 , 9 , 13 , 17 , 21 , 26 and 31 weeks after hatching, Liver activities of fatty acid synthase, (FAS), mailc enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PDH) as well as serum total triacylglycerol (TG), total cholesterol (TC), phospholipid (PL), non-esterified fatty acid (NEFA) were assayed. Adipocyte abundance and size spectrum in liver, ventral muscle and dorsal muscle were determined. The cobia attained an average weight of 13.6 ¡Ó 0.5 g at week 5, and were cultured in nursery outdoor ponds until an average weight 87.5 ¡Ó 3.8 g when moved to open ocean cages. They grew to an average weight 1690.8 ¡Ó 106.6 g at week 31. The crude lipid content in liver, ventral and dorsal muscle increased significantly with fish age, and were 36.1 ¡Ó 3.2 g, 14.8 ¡Ó 0.2g, 4.6 ¡Ó 0.0 g at week 31. No significant change was found in hepatic FAS activity. Hepatic ME activity increased significantly after the fish were stocked in the cages. Whilr hepatic ME activity reduced with age, TG and PL were both increased significantly with age. While adipocyte diameter in liver showed a decreasing trend as the fish grew its abundance increased significantly at week 13 and maintained between 3070¡ã3356 x 104 cell/g tissue during week 17-31. Similarly, adipocyte abundance in ventral muscle increased significantly at week 13, and maintained between 255¡ã269 x 104 cell/g tissue afterwards. There was no significant change in size of ventral muscle adipocyte as fish grew. These results indicate that the adipocyte hyperplasia occurred during week 7- 13, when body weight was 100- 400 g. Adipocyte hypertrophy was observed when the fish reached week 31, there was a sign of adipocyte hypertrophy in liver when the fish were at the last phase of nursery outdoor ponds before being moved to the offshore cages.

published_or_final_version / Community Medicine / Master / Master of Philosophy

Objective: Heme oxygenase-1 (HO-1) has been well established as a cytoprotective molecule, and has been shown to exert cardioprotective effects in both hypertension and cardiac hypertrophy. However, the precise mechanism of the cardioprotective effect of HO-1 has yet to be fully elucidated. The natriuretic peptide system (NPS) is also a key player in cardiovascular homeostasis and tissue dynamics, and has also been shown to be cardioprotective in a variety of pathologic conditions. This study examined the effect of high dietary salt treatment in genetic models of HO-1, and assessed the expression of the NPS in the left ventricle (LV), in order to gain insight into the relationship between varying levels of HO-1 expression with the development of cardiac hypertrophy and the expression of the NPS. Methods: Age-matched 12-week old male HO-1 knockout (HO-1-/-) and HO-1 cardiomyocyte-specific transgenic overexpressing (HO-1Tg) mice were treated with either normal salt (NS; 0.8%) or high salt (HS; 8.0%) chow for 5 weeks. LV mRNA expression was determined using quantitative real-time RT-PCR. Results: HO-1-/- mice fed HS diet had significantly higher left ventricle-to-body weight ratio (LV/BW) compared to HO-1+/+ mice fed NS diet. HO-1-/- mice had significantly reduced expression of the NPS compared to controls, and these mice did not exhibit a salt-induced increase in ANP expression. HS treatment had no effect on LV/BW in HO-1Tg mice compared to controls. HO-1Tg mice had significantly higher ANP and BNP expression compared to controls. Conclusions: The presence of HO-1 is required for normal salt-induced changes in the local cardiac NPS. HO-1 ablation resulted in significantly lower mRNA expression of the NPS, whereas HO-1 overexpression resulted in higher mRNA expression of the NPS. These data indicate that the detrimental effect of reduced HO-1 expression and the cardioprotective effect of increased HO-1 expression may be due, in part, to altered expression of the NPS. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2009-10-20 09:15:20.541