The decline in fertility in dairy cows is of international concern. Since 1950, milk production demands have increased while first service rates of conception have decreased. It is unclear why fertility has decreased, however current dairy management practice requires cows to be kept on a tight yearly calving schedule to ensure maximum milk production over the lifetime of the cow. The current postulate suggests that this regime places a high metabolic burden on the cows, which in turn requires the breakdown of tissues such as fat and muscle to provide substrates to meet the increased energy demands of lactation. Immediately after calving, dairy cows enter a state of negative energy balance (EB), as they cannot consume enough energy to sustain lactation. During this period of negative EB, fat is mobilised in the form of non-esterified fatty acids to help supply the body with the extra energy it needs, but fat mobilisation decreases after four weeks while cows remain in a state of negative EB for several more weeks. It is unclear whether or not muscle breakdown occurs and plays a role in the restoration of EB in lactating cows during peak lactation. I hypothesized that the breakdown of muscle does occur in cows during peak lactation, and that it occurs to a greater extent in cows producing higher amounts of milk. Dairy cows from three strains, NZL, NZH and OSH, representing cows with differing milk production abilities (low, intermediate and high, respectively), were studied for 12 weeks postpartum. Blood was drawn at weekly intervals and muscle biopsies taken at -1, 1, 4, 8, and 12 weeks postpartum. Analysis of plasma revealed an increase in the abundance of troponin I-fs (a marker of muscle breakdown) over the period of study, suggesting that breakdown of skeletal muscle was occurring. Real-time polymerase chain reaction analysis showed that expression of the ubiquitin-proteasome (UbP) ligases atrogin-1 (atro1) and muscle ring finger protein 1 (murf1) increased initially, but returned to normal levels by four weeks postpartum. Concentration of mRNA of the lysosomal proteases, cathepsin B, D, H and L, did not change over the period of study. Therefore, the UbP pathway may contribute to the breakdown of muscle detected by troponin I-fs in plasma. Proteins involved in translation initiation were examined by Western blotting. The ratio of phosphorylated over total eIF2alpha and 4E-BP1 remained unchanged throughout the study, indicating that the breakdown of muscle was not a result of decreased protein synthesis. However, there was a greater ratio of phosphorylated to total eIF2alpha in NZL cows compared with NZH and OSH, suggesting that protein synthesis was less overall in NZL cows than other strains. Measurement of myosin heavy chain composition indicated there was no change in the abundance of type I and type IIx muscle fibres and plasma myostatin levels did not change over the period of study. However, the OSH cows had less myostatin in their plasma than the NZL and NZH cows, suggesting that there may be inhibition of muscle growth occurring in this strain. The results of this study suggest that breakdown of muscle could be important in restoring the EB in high-producing dairy cows during peak lactation. Upregulation of the UbP pathway during the first four weeks of lactation may contribute to this muscle breakdown. However, it remains unclear what processes then continue to regulate breakdown of skeletal muscle to maintain the elevated abundance of troponin I-fs in plasma from four to 12 weeks postpartum in lactating dairy cows.

Myostatin is a negative regulator of muscle growth. When it is not present or non-functional double-muscling occurs, the primary characteristic of this phenotype being an increase in muscle mass. Another characteristic of double-muscling is an increased proportion of type IIB muscle fibres, which rely on glycolysis as their primary energy source, as opposed to type IIA and type I fibres which rely on oxidative phosphorylation. This switch in muscle metabolism directly impacts on the mitochondria, as mitochondria from glycolytic muscle fibres have been shown to have differences in metabolic activity. The increased proportion of glycolytic muscle fibres present in myostatin knockout animals provides a unique model to investigate alterations in muscle fibre type metabolism. The mouse model of myostatin knockout utilised during this study was generated by genetic deletion of exon three of the myostatin gene. Verification of this knockout was attempted by western blot analysis, but only the latency associated protein (LAP) was detected. Interestingly, the LAP was barely detectable in the knockout muscle suggesting deletion of exon three affects binding of anti-myostatin antibodies to the LAP, as that part of the gene is not deleted. A comparison of the basal mitochondrial stress levels was made, also by western blot analysis. The knockout mitochondria showed no change in levels of heat shock protein 60 or superoxide dismutase 2, indicating that they are not being subjected to any increased stress due to the myostatin knockout phenotype. A comparative proteomics approach was used to detect changes in the mitochondrial proteome of myostatin knockout gastrocnemius muscle to gain clues to how mitochondria from glycolytic muscle fibres differ from those present in oxidative fibres. This was undertaken using two-dimensional electrophoresis (2-DE), in-gel tryptic digests and peptide mass fingerprinting by mass spectrometry. A 2-DE gel protein loading of 220 g was shown to give the best protein spot resolution and the most crucial step in the loading process was found to be the laying of the immobilized pH gradient, which had to be performed very carefully to obtain a consistent loading pattern. This study resolved only around 160 protein spots out of the estimated 1,000 to 2,000 proteins present in the mitochondria. Modulation of six proteins was seen at a plt0.1 level, but were unable to be identified using the current methodology. More abundant mitochondrial proteins were able to be identified, but showed no significant modulation. Malate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase, which were identified during this study, have been reported to have decreased activity in mitochondria from glycolytic muscle fibres. This study suggests that the change in activity observed by other researchers is due to inhibition of these enzymes in the glycolytic fibres or activation in the oxidative fibres.

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.

Increased circulating levels of glucocorticoids observed in many catabolic conditions play a major role in the induction of muscle atrophy. Indeed, inhibition of glucocorticoid action by glucocorticoid receptor antagonist attenuates and, in some cases, abolishes muscle atrophy. Circulating and tissue levels of IGF-I, a growth factor that stimulates the development of muscle mass, are frequently reduced in response to glucocorticoids. This decline could therefore trigger muscle atrophy in catabolic conditions. Indeed, systemic administration of IGF-I prevents glucocorticoid-induced muscle atrophy. However, use of systemic IGF-I administration is limited by its hypoglycemic and cardiac hypertrophic actions. Moreover, local IGF-I seems to play a more important role in the regulation of muscle mass than systemic IGF-I. Therefore, to limit loss of muscle mass observed in catabolic states, IGF-I administration must mimic as close as possible the autocrine production of IGF-I. The aim of this thesis was to investigate whether the restoration of IGF-I muscle content could reverse muscle atrophy induced by glucocorticoids. In this work we have tested the hypothesis that the local decrease in muscle IGF-I content might be responsible for the muscular atrophy induced by glucocorticoids. In our work, we have demonstrated that localized overexpression of IGF-I by gene electrotransfer prevents muscle atrophy in glucocorticoid-treated rats. High rate of fiber transfection and long term gene expression were obtained by combining multiple injection sites of DNA with electroporation. Human IGF-I gene electrotransfer using this optimised protocol resulted in increased muscle IGF-I mRNA and protein levels together with prevention of loss of skeletal muscle mass. Furthermore, alterations in the Akt/GSK-3â/â-catenin signaling pathway caused by glucocorticoids were prevented by local IGF-I gene overexpression. Finally, muscle overexpression of caAkt, dnGSK-3b and ÄNb-catenin was sufficient to mimic the anti-atrophic effect of IGF-I supporting the role of this signalling pathway in muscle atrophy caused by glucocorticoids. Taken together, our results show, for the first time in vivo, the role of the IGF-I/Akt/GSK-3b/b-catenin pathway in the skeletal muscle atrophy caused by glucocorticoids. In conclusion, our work highlights the crucial role of decreased muscle IGF-I in glucocorticoid-induced muscle atrophy. Indeed, the data presented in this thesis support the fact that the atrophic action of glucocorticoids is in part due to the downregulation of IGF-I, leading to the inhibition of its signalling pathways while restoration of muscle IGF-I levels is able to counteract totally muscle atrophy.

Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.

Changes in intracellular Ca2+ ([Ca2+]f) and high-energy phosphates are known to induce adaptive changes in skeletal muscle during endurance exercising training, including mitochondrial biogenesis. Levels of [Ca2+]f are regulated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) which are further regulated by sarcolipin (SLN), through a reduction in the apparent affinity of SERCAs for Ca2+. Furthermore, SLN reduces the efficiency of Ca2+ transport by SERCAs supporting a thermogenic role for SLN in skeletal muscle. Thus, it is possible SLN ablation could reduce Ca2+ and metabolic signaling during exercise training and attenuate increases in mitochondrial content. To investigate the potential role of SLN in the exercise-induced adaptive response of skeletal muscle, mice devoid of SLN (SLNKO) underwent endurance training for 8 weeks and were compared to WT controls. Maximal oxygen uptake (V̇O2 max) was measured with an exercise stress test while mitochondrial content was assessed through measurement of protein expression and maximal enzyme activities of several mitochondrial enzymes in soleus and extensor digitorum longus (EDL) muscles, which express high and low levels of SLN, respectively. All data were analyzed using a two-way analysis of variance (ANOVA) and student t-tests were conducted on enzyme data. V̇O2 max was found to not be significantly altered with exercise training in either genotype. Exercise training significantly increased the contents of adenine nucleotide translocase (ANT), cytochrome-c (cyt-c) and cytochrome-c oxidase subunit IV (COXIV) in soleus independent of genotype. Likewise, exercise training significantly increased cyt-c and COXIV expression (P<0.04), while increases in ANT expression were not significant (P=0.13) in the EDL. Two-way ANOVAs of mitochondrial enzymes in soleus revealed an interaction existed for succinate dehydrogenase (SDH) where its activity was increased only in the SLNKO mice (P<0.02). In comparison, exercise training significantly elevated activities of cytochrome c oxidase (COX) and citrate synthase (CS) activities (P<0.02) but not β-hydroxyacyl-CoA dehydrogenase (β-HAD; P=0.08), independent of genotype. Upon closer examination using student t-tests, it was determined that exercise training induced greater increases in COX and CS activity in SLNKO compared to WT controls (P<0.02), similar to and consistent with SDH data. In EDL, only SDH activity increased following exercise training, an effect that was independent of genotype. In conclusion, these data suggest that SLN ablation does not attenuate exercise-induced mitochondrial adaptations and may increase mitochondrial enzyme adaptations to exercise training in slow-twitch muscle. Further examination of the effects of SLN on Ca2+ and metabolic signaling may provide mechanisms explaining the results of this thesis.

Polyunsaturated fatty acids (PUFA) affect key cellular and physiological processes in the body ranging from cell signalling to inflammation. Compositional, dietary refinement and bioassay studies have shown strong associations between the PUFA composition of skeletal muscle with various contractile properties as well as the development of obesity and insulin resistance. The incorporation of PUFAs into rat soleus (slow-twitch oxidative), red gastrocnemius (fast-twitch oxidative), and white gastrocnemius (fast-twitch glycolytic) muscle were examined using stable isotope-labelled fatty acids. Two separate tracer studies were conducted. In the first study, four groups of rats were orally dosed with one of three isotopes of 18:2n-6; 13C18-18:2n-6 ethyl ester, 13C18- 18:2n-6 nonesterified fatty acid or 2H5-18:2n-6 ethyl ester and a control group received the vehicle only (olive oil). Animals were sacrificed 8 hours post dosing and soleus, red and white gastrocnemius muscles were collected for lipid analysis. In the second study, rats were orally administered a single dose of a mixture of 4 isotopes (13C18-18:2n-6, 2H5-18:3n-3, 13C16-16:0, and 2H2-18:1n-9) or vehicle only (olive oil) as a control. Groups of animals were sacrificed at 8, 24, and 48 h after dosing and four muscle types (heart, soleus, red and white gastrocnemius) were collected and analyzed for isotopic signal of these fatty acids and their corresponding desaturation and/or elongation products. Soleus accumulated significantly higher concentrations of labelled 18:2n-6, 18:3n-3 and most of n-6 fatty acids derived from 18:2n-6 followed by red gastrocnemius and white gastrocnemius. Heart muscle accumulated 20:5n-3 and 22:6n-3 increasingly over time while skeletal muscle accumulation was variable across muscle types. Labelled 20:5n-3 was detected in red and white gastrocnemius at 8 and 24 h with levels declining by 48 h while no 20:5n-3 was detected in soleus at anytime. Labelled 22:6n-3 was not detected in white gastrocnemius, but 22:6n-3 appeared to be increasing in red gastrocnemius over time. Soleus demonstrated a large accumulation of 22:6n-3 at 8 h with no detectable levels at 48 h. In conclusion we were able to demonstrate that the distribution and metabolism of various PUFAs differ in muscle types with distinct fibre type composition.

Polyunsaturated fatty acids (PUFA) affect key cellular and physiological processes in the body ranging from cell signalling to inflammation. Compositional, dietary refinement and bioassay studies have shown strong associations between the PUFA composition of skeletal muscle with various contractile properties as well as the development of obesity and insulin resistance. The incorporation of PUFAs into rat soleus (slow-twitch oxidative), red gastrocnemius (fast-twitch oxidative), and white gastrocnemius (fast-twitch glycolytic) muscle were examined using stable isotope-labelled fatty acids. Two separate tracer studies were conducted. In the first study, four groups of rats were orally dosed with one of three isotopes of 18:2n-6; 13C18-18:2n-6 ethyl ester, 13C18- 18:2n-6 nonesterified fatty acid or 2H5-18:2n-6 ethyl ester and a control group received the vehicle only (olive oil). Animals were sacrificed 8 hours post dosing and soleus, red and white gastrocnemius muscles were collected for lipid analysis. In the second study, rats were orally administered a single dose of a mixture of 4 isotopes (13C18-18:2n-6, 2H5-18:3n-3, 13C16-16:0, and 2H2-18:1n-9) or vehicle only (olive oil) as a control. Groups of animals were sacrificed at 8, 24, and 48 h after dosing and four muscle types (heart, soleus, red and white gastrocnemius) were collected and analyzed for isotopic signal of these fatty acids and their corresponding desaturation and/or elongation products. Soleus accumulated significantly higher concentrations of labelled 18:2n-6, 18:3n-3 and most of n-6 fatty acids derived from 18:2n-6 followed by red gastrocnemius and white gastrocnemius. Heart muscle accumulated 20:5n-3 and 22:6n-3 increasingly over time while skeletal muscle accumulation was variable across muscle types. Labelled 20:5n-3 was detected in red and white gastrocnemius at 8 and 24 h with levels declining by 48 h while no 20:5n-3 was detected in soleus at anytime. Labelled 22:6n-3 was not detected in white gastrocnemius, but 22:6n-3 appeared to be increasing in red gastrocnemius over time. Soleus demonstrated a large accumulation of 22:6n-3 at 8 h with no detectable levels at 48 h. In conclusion we were able to demonstrate that the distribution and metabolism of various PUFAs differ in muscle types with distinct fibre type composition.

There is substantial evidence that oxidative stress causes negative outcomes in many cell and tissue types. This is especially true of skeletal muscle, as it is continually subjected to various sources of reactive oxygen species (ROS). Oxidative stress in muscle has been linked to several disease states as well as to the normal aging process. Oxidative stress has also been associated with increased apoptotic signalling. Furthermore, elevated apoptosis is consistently observed in aged skeletal muscle and is thought to be one of the mechanisms of age-related muscle atrophy. Due to its post-mitotic nature, skeletal muscle may be more susceptible to the harmful effects of oxidative stress in light of its limited regenerative capacity. As a protective measure, a sophisticated antioxidant system exists in muscle consisting of both enzymatic (superoxide dismutases (SOD’s), catalase, glutathione peroxidase) and non-enzymatic elements (glutathione: GSH). GSH is a ubiquitously expressed tripeptide essential to maintenance of the redox status of the cell. Its role in skeletal muscle apoptosis, especially in different muscle types, is currently unclear. To elucidate the potential role of GSH in skeletal muscle apoptosis and oxidative stress, L-buthionine-[S,R]-sulfoximine (BSO) was used to deplete GSH in young (34.85 ± 0.68 wks) and old (69.11 ± 3.61 wks) male Sprague-Dawley rats. Thiol levels (GSH, GSSG), ROS production, 4-hydroxy-2-nonenal (4HNE) levels, DNA fragmentation and apoptosis-related protein expression were examined in soleus (SOL) and white gastrocnemius (WG) muscle. BSO led to significant GSH depletion (89% in SOL, 96% in WG) compared to age-matched controls. Catalase upregulation, in the absence of change in SOD levels, was evident as a result of BSO treatment and advancing age in both muscle tissues. BSO treatment also resulted in increased DNA fragmentation in WG and SOL, with elevated ROS production in SOL only; both of these effects were independent of age. Advancing age resulted in elevated caspase activity and Hsp70 protein content, with a concomitant decrease in anti-apoptotic ARC in SOL but not WG. Additionally, ROS production, 4HNE content, DNA fragmentation and ARC levels were all significantly elevated in SOL compared to WG. These data indicate that SOL may be subjected to a state of elevated cellular stress. There is also some evidence that GSH depletion increases DNA fragmentation while age contributes to a degradative loss of glycolytic muscle.

Sarcolipin (SLN) is a small sarcoplasmic reticulum (SR) integral membrane protein that regulates the SR Ca2+-ATPase (SERCA). Previous studies indicate that the functional interaction between SLN and SERCA is thermogenic in nature. Recently, SLN knock-out (SLNKO) mice have been shown to develop excessive obesity and glucose intolerance when placed on a high-fat diet (HFD; 42% kcal derived from fat) relative to wild-type (WT) littermates, implicating SLN in diet-induced obesity. The purpose of this thesis was two-fold: 1) to determine whether an excessively obese phenotype persists when SLNKO mice are given access to voluntary exercise, and 2) to determine if SLN ablation results in a deficit in skeletal muscle oxidative capacity, given the integral role cellular Ca2+ plays in mitochondrial metabolism. Mice were fed either standard chow or a HFD for 8 weeks, and remained sedentary or given access to voluntary running wheels during this period. Glucose tolerance was assessed pre- and post-diet, along with weight gain and adiposity. Skeletal muscle succinate dehydrogenase (SDH), citrate synthase (CS), cytochrome c oxidase (COX), and 3-hydroxyacyl CoA dehydrogenase (ß-HAD) activities were measured in the soleus (SOL) and extensor digitorum longus (EDL) of both chow- and high-fat fed sedentary mice. Both average daily running distance and total exercise volume were not different between WT and SLNKO mice given voluntary running wheels. As before, sedentary SLNKO mice gained more mass following the HFD relative to WT counterparts (P < 0.05); however, no difference in mass gain existed between genotype for voluntary exercising mice on a HFD. Despite this, SLNKO animals were more obese and glucose intolerant following high-fat feeding, regardless of activity status (P < 0.05). Under chow-fed conditions COX activity was higher in the EDL of SLNKO mice (P < 0.05), while no differences in SDH, CS, or ß-HAD existed between genotype in either muscle group. Following the HFD, no changes in mitochondrial enzyme activities within the SOL existed. COX activity in the EDL remained elevated in SLNKO mice post-HFD (P < 0.001), while ß-HAD increased in both WT and SLNKO animals relative to chow-fed controls (P < 0.05). These findings suggest that increasing energy expenditure through voluntary activity cannot compensate for increased basal SERCA Ca2+-pumping efficiency during caloric excess. Additionally, ablation of SLN does not result in a metabolic deficit within skeletal muscle, nor does it limit the adaptive enzymatic response of SLNKO mice to high-fat feeding. Thus, the findings of this study provide further support of the view that SLN’s thermogenic role is the primary mechanism of diet-induced obesity in SLNKO mice.