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.

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.

No description available.

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.

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.

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.

No description available.

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.

No description available.

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.