Naresh C. Bal

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

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 Ca2+-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 Ca2+ 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 Ca2+, 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.

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

Background: Sarcolipin (SLN), a regulator of SR Ca2+ ATPase (SERCA) in muscle, can promote the uncoupling of SERCA from Ca2+ transport and increase heat production. Results: Overexpression of SLN in muscle increases energy expenditure and provides resistance against diet-induced obesity. Conclusion: SLN plays a role in whole-body metabolism. Significance: SLN can serve as novel target to increase energy expenditure in muscle. Sarcolipin (SLN) is a novel regulator of sarcoplasmic reticulum Ca2+ ATPase (SERCA) in muscle. SLN binding to SERCA uncouples Ca2+ 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 (SlnOE) mice to explore energy metabolism by pair feeding (fixed calories) and high-fat diet feeding (ad libitum). Our results show that, upon pair feeding, SlnOE mice lost weight compared with the WT, but Sln−/− mice gained weight. Interestingly, when fed with a high-fat diet, SlnOE 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 SlnOE mice. There was also an increase in both mitochondrial number and size in SlnOE 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.

The role of skeletal-muscle-based thermogenic mechanisms in vertebrate endothermy.

Thermogenesis is one of the most important homeostatic mechanisms that evolved during vertebrate evolution. Despite its importance for the survival of the organism, the mechanistic details behind various thermogenic processes remain incompletely understood. Although heat production from muscle has long been recognized as a thermogenic mechanism, whether muscle can produce heat independently of contraction remains controversial. Studies in birds and mammals suggest that skeletal muscle can be an important site of non‐shivering thermogenesis (NST) and can be recruited during cold adaptation, although unequivocal evidence is lacking. Much research on thermogenesis during the last two decades has been focused on brown adipose tissue (BAT). These studies clearly implicate BAT as an important site of NST in mammals, in particular in newborns and rodents. However, BAT is either absent, as in birds and pigs, or is only a minor component, as in adult large mammals including humans, bringing into question the BAT‐centric view of thermogenesis. This review focuses on the evolution and emergence of various thermogenic mechanisms in vertebrates from fish to man. A careful analysis of the existing data reveals that muscle was the earliest facultative thermogenic organ to emerge in vertebrates, long before the appearance of BAT in eutherian mammals. Additionally, these studies suggest that muscle‐based thermogenesis is the dominant mechanism of heat production in many species including birds, marsupials, and certain mammals where BAT‐mediated thermogenesis is absent or limited. We discuss the relevance of our recent findings showing that uncoupling of sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) by sarcolipin (SLN), resulting in futile cycling and increased heat production, could be the basis for NST in skeletal muscle. The overall goal of this review is to highlight the role of skeletal muscle as a thermogenic organ and provide a balanced view of thermogenesis in vertebrates.

Sarcolipin Protein Interaction with Sarco(endo)plasmic Reticulum Ca2+ATPase (SERCA) Is Distinct from Phospholamban Protein, and Only Sarcolipin Can Promote Uncoupling of the SERCA Pump

Background: Sarcolipin and phospholamban, the regulators of SERCA, are differentially expressed in muscle. Results: Only sarcolipin binds to SERCA in the presence of Ca2+ and interacts with SERCA throughout the kinetic cycle. Conclusion: Sarcolipin alone promotes uncoupling of the SERCA pump leading to increased heat production. Significance: Sarcolipin-mediated regulation of SERCA plays an important role in muscle-based thermogenesis. Sarco(endo)plasmic reticulum Ca2+ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547–554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575–1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the Vmax of SERCA-mediated Ca2+ uptake but not the pump affinity for Ca2+; 2) SLN can bind to SERCA in the presence of high Ca2+, but PLB can only interact to the ATP-bound Ca2+-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca2+ causes uncoupling of the SERCA pump and increased heat production.

IKKα and alternative NF-κB regulate PGC-1β to promote oxidative muscle metabolism

Alternative NF-κB signaling modulates the activity of PGC-1β to promote oxidative metabolism in skeletal muscle.

Uncoupling Protein 1 and Sarcolipin Are Required to Maintain Optimal Thermogenesis, and Loss of Both Systems Compromises Survival of Mice under Cold Stress

Background: The mechanisms underlying UCP1-independent thermogenesis are not well understood. Results: Loss of both SLN and UCP1 results in compromised thermogenic ability and severe sensitivity to acute cold. Conclusion: Sarcolipin-mediated thermogenesis is required for optimal thermogenesis and is up-regulated in the absence of UCP1. Significance: Sarcolipin is a crucial contributor to thermogenesis and energy expenditure. The importance of brown adipose tissue as a site of nonshivering thermogenesis has been well documented. Emerging studies suggest that skeletal muscle is also an important site of thermogenesis especially when brown adipose tissue function is lacking. We recently showed that sarcolipin (SLN), an uncoupler of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump, could contribute to heat production in skeletal muscle. In this study, we sought to understand how loss of UCP1 or SLN is compensated during cold exposure and whether they are both necessary for thermogenesis. Toward this goal, we generated a UCP1;SLN double knock-out (DKO) mouse model and challenged the single and DKO mice to acute and long-term cold exposures. Results from this study show that there is up-regulation of SLN expression in UCP1-KO mice, and loss of SLN is compensated by increased expression of UCP1 and browning of white adipose tissue. We found that the DKO mice were viable when reared at thermoneutrality. When challenged to acute cold, the DKO were extremely cold-sensitive and became hypothermic. Paradoxically, the DKO mice were able to survive gradual cold challenge, but these mice lost significant weight and depleted their fat stores, despite having higher caloric intake. These studies suggest that UCP1 and SLN are required to maintain optimal thermogenesis and that loss of both systems compromises survival of mice under cold stress.

Sarcolipin trumps β-adrenergic receptor signaling as the favored mechanism for muscle-based diet-induced thermogenesis

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.—Bombardier, E., Smith, I. C., Gamu, D., Fajardo, V. A., Vigna, C., Sayer, R. A., Gupta, S. C., Bal, N. C., Periasamy, M., Tupling, A. R., Sarcolipin trumps β‐adrenergic receptor signaling as the favored mechanism for muscle‐based diet‐induced thermogenesis. FASEB J. 27, 3871–3878 (2013). www.fasebj.org

The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport.

Background: Both phospholamban (PLB) and sarcolipin (SLN) regulate SERCA activity, however, only SLN uncouples SERCA. Results: The N and C termini of SLN, or the N terminus and transmembrane region of PLB, confer protein-specific function. Conclusion: SLN N terminus plays a role in dynamic interaction and uncoupling of SERCA. Significance: SERCA uncoupling by SLN increases heat production implicating SLN-SERCA interaction in muscle thermogenesis. The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is responsible for intracellular Ca2+ homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca2+-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca2+ transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues 2ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.

The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering

Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2D307H protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca2+ buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca2+. These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca2+ activation.

Increased Reliance on Muscle-based Thermogenesis upon Acute Minimization of Brown Adipose Tissue Function.

Skeletal muscle has been suggested as a site of nonshivering thermogenesis (NST) besides brown adipose tissue (BAT). Studies in birds, which do not contain BAT, have demonstrated the importance of skeletal muscle-based NST. However, muscle-based NST in mammals remains poorly characterized. We recently reported that sarco/endoplasmic reticulum Ca2+ cycling and that its regulation by SLN can be the basis for muscle NST. Because of the dominant role of BAT-mediated thermogenesis in rodents, the role of muscle-based NST is less obvious. In this study, we investigated whether muscle will become an important site of NST when BAT function is conditionally minimized in mice. We surgically removed interscapular BAT (iBAT, which constitutes ∼70% of total BAT) and exposed the mice to prolonged cold (4 °C) for 9 days. The iBAT-ablated mice were able to maintain optimal body temperature (∼35–37 °C) during the entire period of cold exposure. After 4 days in the cold, both sham controls and iBAT-ablated mice stopped shivering and resumed routine physical activity, indicating that they are cold-adapted. The iBAT-ablated mice showed higher oxygen consumption and decreased body weight and fat mass, suggesting an increased energy cost of cold adaptation. The skeletal muscles in these mice underwent extensive remodeling of both the sarcoplasmic reticulum and mitochondria, including alteration in the expression of key components of Ca2+ handling and mitochondrial metabolism. These changes, along with increased sarcolipin expression, provide evidence for the recruitment of NST in skeletal muscle. These studies collectively suggest that skeletal muscle becomes the major site of NST when BAT activity is minimized.