1 / Sarah Elizabeth Galvez

Aging is associated with attenuated blood flow and vasodilator responses during rhythmic exercise. Older adults also demonstrate attenuated blood flow and vasodilator responses following single skeletal muscle contractions (contraction-induced rapid onset vasodilation, ROV) within the forearm. These age-associated attenuations within the forearm have been demonstrated to be a result of endothelial and neural mechanisms. The objective of this research was to examine: 1) whether age-associated attenuations within the forearm are from mechanical factors; 2) whether age-associated attentions in ROV are present within the leg, as well as explore potential mechanisms for these age-associated attenuations in ROV; 3) examine whether aging is associated with a slower rate of adjustment in vasodilation (vasodilator kinetics) during rhythmic exercise preceding steady-state exercise; and 4) examine approaches to ameliorate age-related attenuations in blood flow and vasodilation within the leg across the entire exercise transient (onset to steady-state). The novel findings of this research are that 1) age-associated attenuations in ROV within the forearm are independent of mechanical factors; 2) older adults demonstrate attenuated ROV responses within the leg; 3) age-related attenuations in ROV within the leg are not explained by enhanced sympathetic adrenergic vasoconstriction; 4) older adults exhibit prolonged vasodilator kinetics preceding steady-state exercise; and 5) when examined in a cross-sectional design chronic exercise training improves ROV, vasodilator kinetics, as well as steady-state blood flow and vasodilator responses in older adults; 6) acute supplementation with dietary nitrate fails to exert any effect on blood flow and vasodilator responses during any domain of exercise. Collectively, this work establishes that aging is associated with reductions in blood flow and vasodilation across the entire exercise transient (onset to steady-state) within the leg, which is offset by chronic exercise training. Mechanistically, the current data suggests that mechanical and sympathetic factors do not explain age-related reductions in ROV in the arm and leg, respectively. Furthermore, acute supplementation of dietary nitrate does not impact leg blood flow and vasodilator responses in older adults during any domain of the exercise transient.

Recovery after prolonged or high-intensity exercise is characterised by a substantial increase in adipose tissue lipolysis, resulting in elevated rates of plasma-derived fat oxidation. Despite the large increase in circulating fatty acids (FAs) after exercise, only a small fraction of this is taken up by exercised muscle in the lower extremities. Indeed, the predominant fate of non-oxidised FAs derived from post-exercise lipolysis is reesteriflcation hi the liver. During recovery from endurance exercise, a number of changes also occur hi skeletal muscle that allow for a high metabolic priority towards glycogen resynthesis. Reducing muscle glycogen during exercise potentiates these effects, however the cellular and molecular mechanisms regulating substrate oxidation following exercise remain poorly defined. The broad arm of this thesis was to examine the regulation of fat metabolism during recovery from glycogen-lowering exercise hi the presence of altered fat and glucose availability. In study I, eight endurance-trained males completed a bout of exhaustive exercise followed by ingestion of carbohydrate (CHO)-rich meals (64-70% of energy from CHO) at 1, 4, and 7 h of recovery. Duplicate muscle biopsies were obtained at exhaustion and 3, 6 and 18 h of recovery. Despite the large intake of CHO during recovery (491 ± 28 g or 6.8 + 0.3 g • kg-1), respiratory exchange ratio values of 0.77 to 0.84 indicated a greater reliance on fat as an oxidative fuel. Intramuscular triacylglycerol (IMTG) content remained unchanged in the presence of elevated glucose and insulin levels during recovery , suggesting IMTG has a negligible role in contributing to the enhanced fat oxidation after exhaustive exercise. It appears that the partitioning of exogenous glucose towards glycogen resynthesis is of high metabolic priority during immediate post-exercise recovery, supported by the trend towards reduced pyruvate dehydrogenase (PDH) activity and increased fat oxidation. The effect of altering plasma FA availability during post-exercise recovery was examined in study II. Eight endurance-trained males performed three trials consisting of glycogen-lowering exercise, followed by infusion of either saline (CON), saline + nicotinic acid (NA) (LFA) or Intralipid and heparin (HFA). Muscle biopsies were obtained at the end of exercise (0 h) and at 3 and 6 h in recovery. Altering the availability of plasma FAs during recovery induced changes in whole-body fat oxidation that were unrelated to differences in skeletal muscle malonyl-CoA. Furthermore, fat oxidation and acetyl-CoA carboxylase (ACC) phosphorylation appear to be dissociated after exercise, suggesting mechanisms other than phosphorylation-mediated changes in ACC activity have an important role in regulating malonyl-CoA and fat metabolism in human skeletal muscle after exercise. Alternative mechanisms include citrate and long-chain fatty acyl-CoA mediated changes in ACC activity, or differences in malonyl-CoA decarboxylase (MCD) activity. Reducing plasma FA concentrations with NA attenuated the post-exercise increase in MCD and pyruvate dehydrogenase kinase 4 (PDK4) gene expression, suggesting that FAs and/or other factors induced by NA are involved hi the regulation of these genes. Despite marked changes hi plasma FA availability, no significant changes in IMTG concentration were detected, providing further evidence that plasma-derived FAs are the preferential fuel source contributing to the enhanced fat oxidation post-exercise during recovery. To further examine the effect of substrate availability after exercise, Study III investigated the regulation of fat metabolism during a 6 h recovery period with or without glucose infusion. Enhanced glucose availability significantly increased CHO oxidation compared with the fasted state, although no differences in whole-body fat oxidation were apparent. Consistent with the similar rates of fat metabolism, no difference hi AMPK or ACCβ phosphorylation were observed between trials. In addition, no significant treatment or time effects for IMTG concentration were detected during recovery. The large exercise-induced PDK4 gene expression was attenuated when plasma FAs were reduced during glucose infusion, supporting the hypothesis that PDK4 is responsive to sustained changes in lipid availability and/or changes in plasma insulin. Furthermore, the possibility exists that the suppression of PDK4 mRNA also reduced PDK activity and thus maintained PDH activity to account for the higher rates of CHO oxidation observed during glucose infusion compared with the control trial.

It has been recently demonstrated that the cytokine interleukin (IL)-6 is unique among the so called

<p>Glucose metabolism in insulin-sensitive tissues such as skeletal muscle and adipose tissue is tightly regulated by external stimuli. Metabolic changes in these tissues have direct effects on whole body metabolism. Such metabolic changes can be induced or influenced by adrenergic stimulation.</p><p>In L6 skeletal muscle cells, we have seen that the β2-adrenergic receptor increases glycogen synthesis to the same extent as insulin. The β2-adrenergically mediated effect is independent of cyclic AMP but dependent on PI3K.</p><p>In brown adipocytes, our data suggest that signaling from the β-adrenergic receptors consists of an acute cyclic AMP effect that is rapidly desensitized and then a prolonged signal involving PI3K.</p><p>In skeletal muscle cells in culture, we have shown that DPI (a NADPH oxidase inhibitor) increases glucose uptake through a signaling pathway independent of NADPH oxidase and insulin signaling. DPI instead inhibits complex 1 in the mitochondrial respiratory chain, which lowers ATP levels. This activates AMPK, an activator of glucose uptake.</p><p>Furthermore, we have developed a model system for ordered fusion of skeletal muscle cells in culture. In this system, differentiating skeletal muscle cells can be studied separately. This system is optimal for microscopy techniques and easily adaptable for micromanipulations. We have seen that the myogenic factor MyoD can have different expression of the protein in different nuclei within the same myotube. This system could be used with advantage for intracellular signaling and metabolic studies.</p>

Sarcolipin (SLN), a small molecular weight, hydrophobic protein found in skeletal muscle, is a known regulator of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pumps. Earlier in vitro reconstitution experiments have shown that SLN uncouples ATP hydrolysis from Ca2+ transport by the SERCA pumps and increases the amount of heat released per mol of ATP hydrolyzed by inducing an increased rate of “slippage” during the reaction cycle of SERCA pumps. In order to determine whether SLN causes slippage of SERCA activity by uncoupling ATP hydrolysis from Ca2+ transport under more physiological conditions, comparisons were made between skeletal muscle Ca2+ ATPase activity and Ca2+ uptake in homogenates from soleus muscle of wild-type (WT) and Sln-null (KO) mice under conditions in which a Ca2+ gradient was preserved across the sarcoplasmic reticulum (SR) vesicles. Ca2+ ATPase activity, measured in the absence of the Ca2+ ionophore, A23187, was 15-25% lower in KO muscles, compared with WT, consistent with the proposal that SLN increases “slippage” and reduces the extent of back-inhibition of the Ca2+ ATPase. Ca2+ uptake, measured in homogenates without oxalate, was not different (p>0.05) in SR vesicles from WT and KO mice, indicating that the calculated Ca2+ transport efficiency (coupling ratio) in KO mice was increased by about 20% (P<0.04). The basal oxygen consumption (VO2) of soleus muscles isolated from WT and KO mice and the contribution of energy utilized by SERCA was also compared. Surprisingly, basal VO2 was not lower in the soleus of KO mice, but the contribution of energy utilized by SERCA pumps was about 7% lower (P<0.0001). It was also found that uncoupling protein 3 (UCP-3) was expressed at a higher (P<0.03) concentration in soleus muscle of KO compared to WT. Thus UCP-3 could, potentially, provide compensation, resulting in higher basal VO2 in KO mice than expected. These data show that at physiological SLN:SERCA ratios, SLN uncouples ATP hydrolysis from SR Ca2+ uptake in skeletal muscle, resulting in a lower contribution of Ca2+ handling to basal VO2. Thus, SLN is a key regulator of both ATP utilization in Ca2+ handling and of overall energy metabolism in skeletal muscle. To further examine the role of SLN in adaptive thermogenesis, obesity and glucose intolerance, KO and WT mice were placed on a high fat diet (HFD; 42% of kcal derived from fat) for an eight week period. Whole body metabolism, weight gain, glucose tolerance and insulin tolerance were measured before and after the HFD. Fat pads, liver, pancreas, hindlimb muscles and plasma samples were collected from standard chow fed control and HFD WT and KO mice. KO mice gained more weight (P<0.05) and became more obese (P<0.05) than WT mice after consuming the HFD. The comprehensive laboratory animal monitoring system (CLAMS) revealed no differences in whole body metabolic rate (ml O¬2/kg/hr) between KO and WT mice pre diet; however, daily metabolic rate was lower (P<0.05) in KO mice compared with WT mice after the HFD which may explain the increased obesity in KO mice. Western blotting analyses revealed SLN protein content to be 3.8 fold higher (P<0.05) in WT soleus post HFD compared to control. Phospholamban (PLN), a homologue of SLN, was found to be 2.1 fold higher (P<0.05) in brown adipose tissue (BAT) in both WT and KO mice post HFD. Protein contents of other Ca2+ handling proteins (SERCA1a, SERCA2a, PLN and calsequestrin) within fast (white gastrocnemius) and slow (soleus) twitch muscle were not different between KO and WT mice following the HFD. Collectively, these results suggest that PLN and SLN could play a role in adaptive diet-induced thermogenesis. On the other hand, compared with chow fed control mice, the metabolic cost of Ca2+ handling in soleus muscle was significantly reduced post HFD in both WT and KO mice, although to a greater extent (P<0.05) in KO mice than WT mice. Moreover, there were no differences in resting energy expenditure of soleus muscles between WT and KO mice following the HFD. These observations can be accounted for by diet-induced increases in sympathetic nervous system activity in KO mice and other adaptive responses leading to increased energy expenditure of soleus in both WT and KO mice. Therefore, differences in whole body metabolic rate and obesity between high fat fed WT and KO mice do not appear to be due to adaptive thermogenesis mechanisms in skeletal muscle involving SLN. Interestingly, soleus and EDL muscle weights increased proportionately to body weight in high fat fed WT mice but not KO mice. Therefore, lower lean body tissue mass may explain the lower whole body metabolic rate and increased susceptibility to obesity in KO mice compared with WT mice. With increased obesity, KO mice became extremely glucose intolerant (P<0.05) post HFD compared to WT mice who also demonstrated glucose intolerance (P<0.05) compared to the pre-HFD values. Surprisingly, the insulin tolerance test responses were not different between KO and WT mice post HFD suggesting that KO mice did not develop greater whole body insulin resistance despite being more obese than WT mice. Blood serum analysis showed that non-esterified fatty acids (NEFA) and LDL cholesterol levels were also increased more (P<0.05) in KO mice compared to the WT mice post HFD. Overall, it is concluded that SLNs impact on Ca2+ handling influences not only ATP consumption by SERCA pumps in resting soleus muscle via uncoupling of ATP hydrolysis from SR Ca2+ uptake but also blunts the negative effect of high fat feeding by increasing resistance to diet-induced obesity and glucose intolerance in mice through mechanisms which are currently unidentified.

The risk of injury, inflammation, and oxidative stress increases in skeletal muscle with aging. It has been postulated that pro-oxidant signaling, including upregulation of inducible nitric oxide synthase (iNOS) contributes to inflammation, pathology, and aging in the brain, liver and heart. Exercise training reduces the risk of injury and inflammation. The purpose of this study was: 1) to identify the mechanisms that upregulate iNOS, pro-oxidant and pro-inflammatory signaling in skeletal muscle, and 2) to identify the mechanisms by which exercise training reduces pro-oxidant signaling. Protein levels and activity of iNOS were measured in 4 groups of male Fischer-344 rats (5 mo and 24 mo, n=10/group), old-control (OC), old-trained (OT), young-control (YC), and young-trained (YT). Exercise training protocol was 60 min at 15 m/min at 15° incline for 5 d/wk for 12 wk. Both iNOS protein expression and activity were significantly higher in OC compared to YC, but exercise training reversed the elevation of iNOS levels lower than OC in tibialis anterior. Surprisingly, NF-κB DNA binding activity was significantly lower in OC than YC, while increased with exercise training in white and red gastrocnemius in both OT and YT. In contrast, protein expression of p65, a regulatory subunit of NF-κB was significantly greater in OC than YC, while exercise training significantly reduced p65 in OT compared to OC from the white gastrocnemius. These data indicate that regulation of NF-κB activity with aging is post-translational and alterations in iNOS expression may result from alternative NF-κB pathways. As decreased NF-κB activity with aging could result in downstream increase in pro-apoptotic signaling, we tested follow-up hypotheses that aging would increase pro-apoptotic regulator Bax and decrease the anti-apoptotic regulator Bcl-2. Bax increased while Bcl-2 decreased in OC in white gastrocnemius when compared to YC. In contrast, exercise training resulted in a dramatic upregulation of Bcl-2 and downregulation of Bax protein expression in OT when compared to OC. These novel results indicate that alterations in pro-inflammatory and pro-apoptotic signaling occur in skeletal muscle during the aging process. Importantly, our findings strongly support the hypothesis that exercise training reverses age-induced changes in pro-inflammatory and pro-apoptotic signaling.

Glucose metabolism in insulin-sensitive tissues such as skeletal muscle and adipose tissue is tightly regulated by external stimuli. Metabolic changes in these tissues have direct effects on whole body metabolism. Such metabolic changes can be induced or influenced by adrenergic stimulation. In L6 skeletal muscle cells, we have seen that the β2-adrenergic receptor increases glycogen synthesis to the same extent as insulin. The β2-adrenergically mediated effect is independent of cyclic AMP but dependent on PI3K. In brown adipocytes, our data suggest that signaling from the β-adrenergic receptors consists of an acute cyclic AMP effect that is rapidly desensitized and then a prolonged signal involving PI3K. In skeletal muscle cells in culture, we have shown that DPI (a NADPH oxidase inhibitor) increases glucose uptake through a signaling pathway independent of NADPH oxidase and insulin signaling. DPI instead inhibits complex 1 in the mitochondrial respiratory chain, which lowers ATP levels. This activates AMPK, an activator of glucose uptake. Furthermore, we have developed a model system for ordered fusion of skeletal muscle cells in culture. In this system, differentiating skeletal muscle cells can be studied separately. This system is optimal for microscopy techniques and easily adaptable for micromanipulations. We have seen that the myogenic factor MyoD can have different expression of the protein in different nuclei within the same myotube. This system could be used with advantage for intracellular signaling and metabolic studies.

The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.