Deletion of Sarco(endo)plasmic Reticulum Ca2+-ATPase Regulatory Proteins: Impacts on Energy Metabolism and Obesity
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The sarco(endo)plasmic reticulum (SR) Ca2+-ATPase (SERCA) is responsible for maintaining low cytosolic [Ca2+] through the ATP-dependent pumping of Ca2+ ions from the cytosol into the SR lumen. SERCA activity has the potential to be a critical regulator of body mass and adiposity given that it is estimated to contribute upwards of 20% of daily energy expenditure (Smith et al. 2013. PLoS One. 8: e68924). Two well-characterized regulators of SERCAs are the homologous proteins sarcolipin (SLN) and phospholamban (PLN), which reduce its Ca2+ affinity/maximal activity either on their own or through a ternary super-inhibitory complex (Asahi et al. 2002. J Biol Chem. 277: 26725-26728). Our group has shown that SLN uncouples SERCA function within oxidative skeletal muscle (Bombardier et al. 2013. FEBS Lett. 587: 1687-1692), and mice lacking SLN (Sln-/-) are susceptible to diet-induced obesity (Bombardier et al. 2013. FASEB J. 27: 3871-3878). However, it remains unclear whether skeletal muscle PLN has a similar role in regulating SERCA efficiency and diet-induced thermogenesis. Furthermore, double knock-out (DKO) mice for both SLN and PLN display a cardiac phenotype distinct from either single KO model (Shanmugam et al. 2011. Cardiovasc Res. 89: 353-361), suggesting the combined action of SLN and PLN, possibly even the super-inhibitory complex, is integral for regulating cardiac Ca2+-handling. Given that both SERCA regulators are expressed within oxidative muscle, and even within the same muscle fibre (Fajardo et al. 2013. PLoS One. 8: e84304), potential exists for their combined role in the regulation of SERCA efficiency within skeletal muscle, and consequently whole-body metabolism. The two major objectives of this thesis were to: 1) determine whether skeletal muscle PLN reduces SERCA efficiency and is involved in diet-induced thermogenesis, and 2) characterize the impact of dual SLN/PLN ablation on skeletal muscle SERCA efficiency, whole-body metabolism and susceptibility to obesity. To address objective 1) we utilized Pln-/- and wild-type (WT) littermates, and to address objective 2) we utilized DKO (Sln-/-/Pln-/-) and WT (Sln+/+/Pln+/+) control mice. We hypothesized that 1) PLN would uncouple SERCA function and Pln-/- mice would develop an obesogenic phenotype, and 2) DKO mice would have improved SERCA efficiency within skeletal muscle and as a result be susceptible to an obesogenic phenotype. We chose to focus our examination using the soleus (SOL), as this muscle endogenously expresses PLN. Pln-/- mice showed no changes in histological variables or SR protein expression within SOL, including SLN. While PLN ablation increased maximal Ca2+-ATPase activity ~33% (P < 0.01), SERCA pumping efficiency was similar to that of WT littermates, suggesting that PLN does not uncouple Ca2+-uptake from ATP hydrolysis in oxidative muscle. Not surprisingly then, whole-body metabolic rate, metabolic efficiency, glucose tolerance, fat pad mass and adiposity were comparable between WT and Pln-/- littermates following 8 weeks of high-fat feeding (42% kcal from fat). This lack of an obesogenic phenotype in Pln-/- was not the result of compensation from other known mechanisms of diet-induced thermogenesis, namely skeletal muscle SLN or brown adipose tissue (BAT) uncoupling protein (UCP)-1 expression. Furthermore, cumulative food consumption was also similar between Pln-/- mice and WT littermates. Interestingly, SOL PLN expression of high-fat fed WT mice was reduced ~60% (P < 0.05) compared to chow-fed controls. Furthermore, there was a tendency (P = 0.07) for high-fat feeding to increase the non-inhibitory phosphorylated form of PLN, together suggesting that the physical interaction of PLN with SERCA is reduced by caloric surfeit. These results indicate that, unlike SLN, skeletal muscle PLN does not increase the energy demand of SERCA to pump Ca2+ and is not involved in adaptive diet-induced thermogenesis. We next examined Ca2+-handling within the SOL of DKO mice. To our surprise, maximal Ca2+-ATPase activity tended (P = 0.06) to be lower in DKO SOL. However, we observed a fast-to-slow fibre type shift within DKO SOL, along with hypertrophy of type I and IIA fibres (P < 0.05). Correspondingly, SERCA2a protein expression was elevated (P < 0.05), while SERCA1a was reduced (P < 0.05) in DKO SOL. Thus, the reduction of maximal SERCA function in DKO mice likely reflects an overall reduction of total SERCA density resulting from the fibre type shift of these animals. Despite these changes, SERCA efficiency tended (P = 0.08) to be higher in DKO SOL, consistent with the absence of SLN, and suggests a lower energy demand of SERCA to pump Ca2+ in these mice. Interestingly, 24-hr whole-body metabolic rate (ml O2/kg body mass/hr) was ~4% higher (P < 0.05) in DKO mice, although they were also more (P < 0.05) spontaneously active during metabolic measurements. Unlike hypothesized, DKO mice were in fact protected against diet-induced obesity compared to WT control animals as noted by their lower dietary mass gain, smaller subcutaneous/visceral fat pad mass, and lower adiposity index when fed both a chow or HFD for 8 weeks (P < 0.05). Protection of DKO mice against obesity was unrelated to energy intake, as cumulative food consumption was similar to that of WT control mice. Interestingly, weekly metabolic efficiency was lower (P < 0.05) in DKO mice across 8 weeks of chow feeding and between weeks 1 to 5 of the HFD, suggesting greater energy expenditure of these animals. Although HFD-fed DKO mice were more spontaneously active during metabolic measurements both pre- and post-HFD (P < 0.05), whole-body energy expenditure was greater (P < 0.05) post-HFD in DKO mice, even during states of physical inactivity; therefore, greater cage activity can only partly explain their lean phenotype. Although SERCA isoforms were altered within DKO SOL in both chow- and HFD-fed animals as described above, no genotype differences were observed in the expression of proteins involved in SR Ca2+ release or storage, regardless of diet. PLN protein expression was again reduced in WT SOL by ~55% (P < 0.05) in response to the HFD, in addition to increasing its non-inhibitory phosphorylated form (P < 0.05). Furthermore, while BAT UCP-1 protein expression of WT and DKO mice was increased ~25% (P < 0.01) in response to the HFD, no genotype differences in UCP-1 expression existed. Thus, protection against obesity was not the result of any change in skeletal muscle or BAT proteins measured in response to dual SLN/PLN ablation. This thesis revealed several novel findings. First, we show that physiological levels of PLN protein within oxidative skeletal muscle do not uncouple SERCA function. Not surprisingly then, Pln-/- animals were not susceptible to an excessively obese phenotype when given a “Westernized” HFD, which is distinctly different from that previously shown for Sln-/- mice. Secondly, we showed that PLN protein expression is responsive to calorie surfeit. Specifically, high-fat feeding reduced PLN’s interaction with SERCA by both decreasing its expression and increasing its non-inhibitory phosphorylated form. These data are in line with a growing body of literature suggesting that SLN and PLN serve distinct physiological roles within skeletal muscle. We conclude that, unlike SLN, PLN is not involved in skeletal muscle adaptive diet-induced thermogenesis. Thirdly, we show that the combined regulation of SERCA by SLN and PLN are required for the regulation of muscle fibre-type and size as noted by the fast-to-slow fibre-type shift and hypertrophy of type I and IIA fibres with DKO SOL. Lastly, despite the inability to activate SLN-mediated thermogenesis, DKO animals were surprisingly protected against obesity. While not examined here, the muscular phenotype of DKO animals is consistent with the activation of cytosolic Ca2+-signaling proteins. Given that in vivo SR Ca2+ load is likely to be higher in DKO skeletal muscle, this may result in a greater SR gradient favoring SR Ca2+ leak and subsequent activation of cytosolic Ca2+-signaling proteins. Furthermore, a futile cycle of SR Ca2+-leak and re-uptake may explain, in part, hypermetabolic phenotype and protection against obesity of DKO mice.
Cite this version of the work
Daniel Gamu (2017). Deletion of Sarco(endo)plasmic Reticulum Ca2+-ATPase Regulatory Proteins: Impacts on Energy Metabolism and Obesity. UWSpace. http://hdl.handle.net/10012/12671