The Role of Sarcolipin in Calcium Handling and Obesity
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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.