Sarcolipin Is a Key Determinant of the Basal Metabolic Rate, and Its Overexpression Enhances Energy Expenditure and Resistance against Diet-induced Obesity

Abstract

Sarcolipin (SLN) is a novel regulator of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) in muscle. SLN binding to SERCA uncouples Ca(2+) transport from ATP hydrolysis. By this mechanism, SLN promotes the futile cycling of SERCA, contributing to muscle heat production. We recently showed that SLN plays an important role in cold- and diet-induced thermogenesis. However, the detailed mechanism of how SLN regulates muscle metabolism remains unclear. In this study, we used both SLN knockout (Sln(-/-)) and skeletal muscle-specific SLN overexpression (Sln(OE)) mice to explore energy metabolism by pair feeding (fixed calories) and high-fat diet feeding (ad libitum). Our results show that, upon pair feeding, Sln(OE) mice lost weight compared with the WT, but Sln(-/-) mice gained weight. Interestingly, when fed with a high-fat diet, Sln(OE) mice consumed more calories but gained less weight and maintained a normal metabolic profile in comparison with WT and Sln(-/-) mice. We found that oxygen consumption and fatty acid oxidation were increased markedly in Sln(OE) mice. There was also an increase in both mitochondrial number and size in Sln(OE) muscle, together with increased expression of peroxisome proliferator-activated receptor δ (PPARδ) and PPAR γ coactivator 1 α (PGC1α), key transcriptional activators of mitochondrial biogenesis and enzymes involved in oxidative metabolism. These results, taken together, establish an important role for SLN in muscle metabolism and energy expenditure. On the basis of these data we propose that SLN is a novel target for enhancing whole-body energy expenditure.

Keywords: Energy Expenditure; Mitochondria; Obesity; Oxidative Metabolism; SERCA; Sarcolipin; Sarcoplasmic Reticulum (SR); Skeletal Muscle; Skeletal Muscle Metabolism.

FIGURE 1.

Pair feeding of Sln^OE mice results in a significant loss in body weight and fat mass. A, whole-body VO₂ in Sln^−/−, WT, and Sln^OE mice. B, body before and after pair feeding (n = 7). C, caloric efficiency shown as change in body weight per gram of diet consumed. D, mass of total WAT after pair feeding. E, H&E staining of sections of WAT. F, SDH staining of TA muscle. *, p < 0.05; #, p < 0.001.

FIGURE 2.

Sln^OE mice show resistance to high-fat diet-induced obesity. A, weight gain during 12 weeks of feeding (n = 15). B, net weight after 12 weeks of feeding. C, caloric intake is significantly higher in Sln^OE mice. D, caloric efficiency (weight gain per kilocalorie consumed). E, intraperitoneal glucose tolerance test. F, fasting blood cholesterol level. G, triglyceride levels. H, nonesterified fatty acids (NEFA) levels. I, mass of total body white fat. J, MRI images of WT and Sln^OE mice (fat depots are shown in red). K, H&E staining of WAT (top panel) and brown adipose tissue (BAT) sections (bottom panel). L, H&E staining (top panel) and osmium tetraoxide (tet) staining (bottom panel) of liver sections. *, p < 0.05; **, p < 0.01; #, p < 0.001.

FIGURE 3.

Sln^OE mice display increased oxygen consumption, energy expenditure, and fat utilization. A and B, whole-body VO₂. C, RER (VCO₂/VO₂). D, 24-h whole-body energy expenditure (EE). E, regression-adjusted 24-h energy expenditure. F, overexpression of SLN increases muscle oxygen consumption. Isolated soleus muscle from Sln^OE mice show increased oxygen consumption in the resting state and when stimulated with electrical stimulation ± caffeine (3 mm), as measured by a TIOX tissue bath system (n = 4). *, p < 0.05; **, p < 0.01; #, p < 0.001.

FIGURE 4.

Overexpression of SLN increases mitochondrial content. A, representative transmission electron micrographs of TA muscles from HFD-fed WT, Sln^OE, and Sln^−/− mice. B, electron micrographs of extensor digitorum longus muscle. C, average mitochondrial numbers in TA and extensor digitorum longus muscle (n = 5). D, higher magnification electron micrographs showing much larger mitochondria with elaborate cristae in Sln^OE muscle. E, mitochondrial DNA quantification (the mitochondrial gene Nd1 was normalized to the nuclear gene Ppia). *, p < 0.05; **, p < 0.01.

FIGURE 5.

Overexpression of SLN increases oxidative capacity and transcriptional activators of mitochondrial biogenesis. A, expression pattern of mitochondrial electron transport chain (ETC) genes. B, Western blot showing expression of different subunits of electron transport chain proteins. C, SDH staining of TA muscle of HFD-fed WT and Sln^OE mice. D, gene expression of enzymes involved in fatty acid transporters and oxidation. E, protein level of CPT1-M and lipoprotein lipase (LPL) in various skeletal muscles (gastrocnemius (Gastro), quadriceps (Quad), and TA). F, mRNA expression level of genes involved mitochondrial biogenesis. Tfam, mitochondrial transcription factor A. G, PPARδ and PGC1α protein expression levels in various muscles. H, gene expression of calcium signaling mediators. CamKII, Ca²⁺/calmodulin-dependent protein kinase II. I, calcineurin activity in the whole homogenates of the indicated muscles. J, phosphorylated (pNFAT) and total NFAT protein levels. K, densitometric analysis of pNFAT/NFAT. *, p < 0.05; **, p < 0.01; #, p < 0.001.

FIGURE 6.

Formoterol treatment increases energy expenditure in Sln^OE but not in Sln^−/− mice. A and B, oxygen consumption before and after treatment with formoterol, a β₂-adrenergic receptor-selective agonist (n = 8). C, percentage change in fatty acid utilization after formoterol administration. *, p = 0.05; **, p < 0.01; ns, not significant.

FIGURE 7.

Proposed mechanism to show how SLN/SERCA interaction affects muscle metabolism. SERCA uses ATP hydrolysis to actively transport Ca²⁺ from the cytosol into the sarcoplasmic reticulum lumen. SLN binding to SERCA causes uncoupling of Ca²⁺ transport from ATP hydrolysis. This leads to futile cycling of the pump and increased ATP hydrolysis/heat production, thereby creating energy demand. At the same time, uncoupling of SERCA prolongs the cytosolic Ca²⁺ transient, thereby activating the mitochondrial oxidative metabolism and ATP synthesis (i) and Ca²⁺-dependent signaling pathways promoting mitochondrial biogenesis (ii). Therefore, SLN plays a dual role. It creates an energy demand and signals mitochondria to increase ATP production. The SLN-mediated increase in metabolism can also be recruited by SNS stimulation.