Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals

Abstract

The role of skeletal muscle in nonshivering thermogenesis (NST) is not well understood. Here we show that sarcolipin (Sln), a newly identified regulator of the sarco/endoplasmic reticulum Ca(2+)-ATPase (Serca) pump, is necessary for muscle-based thermogenesis. When challenged to acute cold (4 °C), Sln(-/-) mice were not able to maintain their core body temperature (37 °C) and developed hypothermia. Surgical ablation of brown adipose tissue and functional knockdown of Ucp1 allowed us to highlight the role of muscle in NST. Overexpression of Sln in the Sln-null background fully restored muscle-based thermogenesis, suggesting that Sln is the basis for Serca-mediated heat production. We show that ryanodine receptor 1 (Ryr1)-mediated Ca(2+) leak is an important mechanism for Serca-activated heat generation. Here we present data to suggest that Sln can continue to interact with Serca in the presence of Ca(2+), which can promote uncoupling of the Serca pump and cause futile cycling. We further show that loss of Sln predisposes mice to diet-induced obesity, which suggests that Sln-mediated NST is recruited during metabolic overload. These data collectively suggest that SLN is an important mediator of muscle thermogenesis and whole-body energy metabolism.

Figure 1

Sln^−/− mice are not able to maintain optimal core temperature (~37 °C) and develop hypothermia when challenged with acute cold. (a) Infrared imaging of surface body heat in WT and Sln^−/− mice with or without iBAT at 22 °C and 4 °C. (b) Core body temperature after acute cold exposure in WT and Sln^−/− mice, with and without iBAT. n = 22 (WT + iBAT), n = 23 (WT – iBAT), n = 22 (Sln^−/− + iBAT), n = 27 (Sln^−/− – iBAT). (c) Percentage of mice reaching ERC. (d,e) Average drop in core temperature (Tc) during the first 2 h of 4 °C challenge in WT and Sln^−/− mice first acclimatized to 22 °C (d) or 30 °C (e). (f,g) Oxygen consumption averaged over the first two hours of acute cold exposure in WT and Sln^−/− mice first acclimatized to 22 °C (f) or 30 °C (g). *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant as analyzed by Student’s t test or one-way analysis of variance (ANOVA). All data are means ± s.e.m.

Figure 2

Reintroduction of Sln in Sln^−/− mice completely restores thermogenesis, and Sln is necessary for muscle-based NST. (a) Transgenic overexpression of Sln in Sln^−/−/OE mice. R, red gastrocnemius; S, soleus; D, diaphragm; E, extensor digitorum longus; T, tibialis anterior; G, gastrocnemius; Q, quadriceps; Casq1, skeletal calsequestrin. (b) Core body temperature during acute cold exposure after Sln overexpression in the Sln^−/− (n = 8) background. (c–e) Core body temperature during acute cold exposure in WT mice (c) and in Sln^−/− mice (d,e) after curare treatment. (f) Physical activity of the indicated mouse strains with and without curare treatment. **P < 0.01, ***P < 0.001 by Student’s t test. All data are means ± s.e.m.

Figure 3

Molecular basis of Sln-mediated thermogenesis. (a,b) Core body temperature after acute cold exposure and after dantrolene treatment in WT mice (a) and Sln^−/− mice (b) with or without iBAT. n = 8 (WT + iBAT), n = 8 (WT – iBAT), n = 11 (Sln^−/− + iBAT). (c) Alignment of the mouse Sln and Plb protein sequences. The amino acid residues Asp30 in Plb and Glu7 in Sln were mutated to cysteine for crosslinking experiments with Serca1a. (d) Western blot analysis showing Sln crosslinking to Serca1a in the presence of Ca²⁺ (0 –100 μM). (e) Western blot showing crosslinking of Plb with Serca1a in the presence of Ca²⁺ (0 – 100 μM). All data are means ± s.e.m.

Figure 4

Sln^−/− mice are prone to develop obesity when fed HFD. (a) Increase in body weight during 12 weeks of HFD feeding (n = 8 mice per group). (b) Sln^−/− mice fed HFD become obese compared to WT mice. (c) MRI of the fat distribution in representative mice fed on HFD. (d) Determination of the fat content of the mice in a. WAT, white adipose tissue. (e) Representative histological sections of intraperitoneal WAT, BAT and liver stained with H&E and osmium tetroxide (osmium tet) (seen as black dots). Scale bar, 25 μm. (f) Representative western blots of soleus and diaphragm muscle from chow-fed and HFD-fed WT and Sln^−/− mice. n = 4. (g) Glucose tolerance test. *P < 0.05, **P < 0.01 for the comparison of WT and Sln^−/− HFD-fed mice by one-way ANOVA. All data are means ± s.e.m.