The effects of sarcolipin over-expression in mouse skeletal muscle on metabolic activity.
Bottom Line: The mechanism proposed is uncoupling of the sarcoplasmic reticulum calcium pump.Sarcolipin levels were so low that it is unlikely that knocking out sarcolipin would have a measurable effect on thermogenesis by SERCA.In addition, overexpression of neither wild type nor FLAG-tagged variants of mouse sarcolipin in transgenic mice had any major significant effects on body mass, energy expenditure, even when mice were fed on a high fat diet.
Affiliation: Centre for Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK.Show MeSH
Mentions: An examination of the sarcolipin levels in sarcoplasmic reticulum from total skeletal muscle of FVBN mice confirmed the finding of Vangheluwe et al.  that sarcolipin was below the level of detection in mouse skeletal muscle using western blotting techniques (Fig. 2A; lane 2) though as little as 1 ng of synthetic sarcolipin was detectable in this blot (lane 5). By contrast, the western blot procedure was able to detect sarcolipin in the SR of transgenic FVBN mice over-expressing this protein (lane 1). Sarcolipin knockout studies have been reported using C57BL6 mice [5,6,10], so the level of sarcolipin in this strain of mice was also examined. Sarcolipin was undetected by western blotting in homogenates of combined limb skeletal muscle taken from this mouse strain (Fig. 2B, lane 2), but FLAG-tagged sarcolipin and sarcolipin were readily detectable in muscle homogenates of transgenic FVBN mice over-expressing these proteins (lanes 1 and 3 respectively). The FLAG-tagged sarcolipin migrated under SDS–PAGE with an apparent Mr of around 6000. Previously, studies have shown that sarcolipin can be detected in soleus muscle from C57/BL6 mice [5,8] however the study by Bombardier et al.  clearly shows that the sarcolipin content of 100 μg of soleus is far less, by some considerable margin, than that contained by 1 μg of atrial tissue. Given that limb skeletal muscles such as white gastrocnemius, extensor digitorum longus and quadriceps contain little or no sarcolipin, presumably sarcolipin is diluted to such an extent that it is difficult to detect when C57/BL6 limb muscle is combined as in Fig. 2B. Soleus appears to be one of the limb skeletal muscles containing the highest levels of sarcolipin in mouse [8,9,16]. In this study the level of sarcolipin in 120 μg of homogenised soleus from 30 week old male FVBN mice was not detectable (Fig. 2D, lane 4) and was certainly less than 1 ng sarcolipin, which is just detectable (lane 1). Using the value for SERCA in soleus homogenates determined by western blotting of 21 μg SERCA/mg protein (data nor shown), which is comparable with the value from  of 16 μg SERCA/mg protein, the sarcolipin level in soleus is <0.012 mol sarcolipin/mol SERCA. Although Bombardier et al.  were able to demonstrate the presence of sarcolipin in wild type soleus this was close to the limits of detection, requiring an enhancement of the image. As previously described , the high fat diet did increase the level of sarcolipin in mouse soleus muscle. In Fig. 2D (lane 3) 120 μg of homogenised soleus from 30-week-old male FVBN mice fed on a high fat diet (lane 3) contained around 2 ng sarcolipin (2 ng sarcolipin standard in lane 2), which translates to 0.023 mol sarcolipin/mol SERCA.
Affiliation: Centre for Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK.