Sarcolipin (SLN) regulates muscle-based nonshivering thermogenesis and is up-regulated with high-fat feeding (HFF). To investigate whether other muscle-based thermogenic systems compensate for a lack of Sln and to firmly establish SLN as a mediator of diet-induced thermogenesis (DIT), we measured muscle and whole-body energy expenditure in chow- and high-fat-fed Sln(-/-) and wild-type (WT) mice. Following HFF, resting muscle metabolic rate (VO2, μl/g/s) was increased similarly in WT (0.28±0.02 vs. 0.31±0.03) and Sln(-/-) (0.23±0.03 vs. 0.35±0.02) mice due to increased sympathetic nervous system activation in Sln(-/-) mice; however, whole-body metabolic rate (VO2, ml/kg/h) was lower in Sln(-/-) compared with WT mice following HFF but only during periods when the mice were active in their cages (WT, 2894±87 vs. Sln(-/-), 2708±61). Treatment with the β-adrenergic receptor (β-AR) antagonist propranolol during HFF completely prevented muscle-based DIT in Sln(-/-) mice; however, it had no effect in WT mice, resulting in greater differences in whole-body metabolic rate and diet-induced weight gain. Our results suggest that β-AR signaling partially compensates for a lack of SLN to activate muscle-based DIT, but SLN is the primary and more effective mediator.

The main purpose of this study was to directly quantify the relative contribution of Ca2+ cycling to resting metabolic rate in mouse fast (extensor digitorum longus, EDL) and slow (soleus) twitch skeletal muscle. Resting oxygen consumption of isolated muscles (VO2, µL/g wet weight/s) measured polarographically at 30°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.022) than in EDL (0.261 ± 0.020). In order to quantify the specific contribution of Ca2+ cycling to resting metabolic rate, the concentration of MgCl2 in the bath was increased to 10 mM to block Ca2+ release through the ryanodine receptor, thus eliminating a major source of Ca2+ leak from the sarcoplasmic reticulum (SR), and thereby indirectly inhibiting the activity of the sarco(endo) plasmic reticulum Ca2+-ATPases (SERCAs). The relative (%) reduction in muscle VO2 in response to 10 mM MgCl2 was similar between soleus (48.0±3.7) and EDL (42.4±3.2). Using a different approach, we attempted to directly inhibit SERCA ATPase activity in stretched EDL and soleus muscles (1.42x optimum length) using the specific SERCA inhibitor cyclopiazonic acid (CPA, up to 160 µM), but were unsuccessful in removing the energetic cost of Ca2+ cycling in resting isolated muscles. The results of the MgCl2 experiments indicate that ATP consumption by SERCAs is responsible for 40–50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30°C, or 12–15% of whole body resting VO2. Thus, SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.

a b s t r a c tThe purpose of this study was to examine the effects of sarcolipin (SLN) on sarco(endo) plasmic reticulum Ca 2+ -ATPase (SERCA pump) energetics in vivo and resting skeletal muscle metabolic rate. Using SLN knockout (Sln À/À ) mice we show that SLN ablation increases the transport stoichiometry of SERCA pumps (Ca 2+ uptake/Ca 2+ -ATPase activity) and decreases the relative contribution of SERCA pumps to resting oxygen consumption (VO 2 ) in soleus without affecting soleus or whole body VO 2 . These data suggest that the lower energy requirements for Ca 2+ cycling in resting skeletal muscle of Sln À/À mice do not impact significantly either skeletal muscle or whole body metabolic rate.

To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (ϩdF/dt). Maximum relaxation rates (ϪdF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or ϩdF/dt; however, forcefrequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus forcefrequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (ϪdF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca 2ϩ regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca 2ϩ -ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates. knockout mouse; muscle contractility; isolated skeletal muscle; Ca 2ϩ pump SARCO (ENDO) PLASMIC RETICULUM (SER) Ca 2ϩ -ATPases (SERCAs) are ubiquitously expressed, integral membrane proteins that transport Ca 2ϩ ions from the cytosol to the lumen of the SER. In the heart, a 52 amino acid transmembrane protein, phospholamban (PLN), interacts physically with SERCA2a, lowering the apparent Ca 2ϩ affinity of the PLN-SERCA2a complex (19,30). The inhibited complex is disrupted by phosphorylation of PLN or by elevation of cytosolic Ca 2ϩ , leading to the reversal of SERCA2a inhibition. Sarcolipin (SLN), a 31 amino acid protein, shares substantial identity with PLN in both primary sequence and gene structure (25, 35) and, like PLN, is an effective inhibitor of SERCA molecules (1-3, 16, 24). SLN was originally identified as a proteolipid that copurified with SERCA1a in rabbit fast-twitch skeletal muscle (20). Subsequently, SLN was found to be expressed highly in both human and rabbit fast-twitch skeletal muscle and to a lesser extent in slow-twitch and cardiac muscle, based on mRNA levels (25). SLN expression in the heart is largely restricted to the atrium (22), whereas PLN is highly expressed in the ventricle and not in the atrium ...

Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) by reducing their apparent affinity for Ca2+. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca2+ affinity of SERCA (K Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.