Introduction
The opinion review by Campbell and Dicke questioned if sarcolipin (SLN) is involved
in adaptive thermogenesis especially cold adaptation, while agreeing to SLN-mediated
heat production (Campbell and Dicke, 2018). Several interesting questions were raised
concerning the data implicating SLN in muscle nonshivering thermogenesis (NST). Here,
we intend to clarify to the research community that SLN-mediated heat production and
adaptive thermogenesis are inseparable. This is because heat production by SLN-SERCA
interaction is a biochemical mechanism, whose physiological implication is adaptive
thermogenesis. Recent studies from several groups have identified SLN as an important
regulator of SERCA pump (Smith et al., 2002; Tupling et al., 2002; Asahi et al., 2003;
MacLennan et al., 2003; Vangheluwe et al., 2005; Mall et al., 2006; Morita et al.,
2008; Gorski et al., 2013, 2017; Toyoshima et al., 2013; Winther et al., 2013; Gamu
et al., 2014, 2015; Montigny et al., 2014; Fajardo et al., 2015). X-ray Crystallographic
studies notably from two groups Toyoshima and Nissen have provided the molecular details
of SLN binding to SERCA, inside the TM groove (Toyoshima et al., 2013; Winther et
al., 2013). Studies using genetically altered mouse models provided convincing data
that SLN is important for muscle thermogenesis and metabolism (Bombardier et al.,
2013; Sahoo et al., 2013; Maurya et al., 2015; Rowland et al., 2015a; Bal et al.,
2017). Although the molecular details of SLN function in muscle physiology continues
to evolve, there is critical evidence that SLN is a key regulator muscle NST.
SLN is an uncoupler of SERCA pump leading to increased heat production
SERCA is a Ca2+ ion transport pump in muscle and its activity is regulated by small
peptides including phospholamban (PLB-52 aa) and sarcolipin (SLN-31 aa), whose interaction
with SERCA alters the dynamics of Ca2+ cycling (Odermatt et al., 1998; Maclennan,
2004; Bhupathy et al., 2007; Traaseth et al., 2008; Periasamy et al., 2017). SLN expression
is muscle specific, and its expression level is highly regulated during muscle development
and disease states (Vangheluwe et al., 2005; Babu et al., 2007; Pant et al., 2015).
PLB is known as an affinity modulator of SERCA pump for Ca2+. Interestingly PLB binds
to SERCA only to the Ca2+-free E2-state, whereas SLN can bind to Ca2+-bound SERCA
and can remain bound during the Ca2+-transport cycle. The presence of SLN decreases
the Vmax of Ca2+-uptake but not the amount of ATP hydrolyzed (Sahoo et al., 2013,
2015; Shaikh et al., 2016). Anthony Lee and his colleagues were the first to suggest
that SLN binding to SERCA could promote uncoupling of SERCA-mediated ATP hydrolysis
from Ca2+ transport (Smith et al., 2002; Mall et al., 2006). They further showed that
Ca2+ accumulation in the vesicles decreased, but the heat released by SERCA increased
in the presence of SLN. Based on this, they suggested that SLN binding to SERCA prevents
release of Ca2+ into the lumen and promotes slippage of Ca2+ back to the cytosol.
The efficiency of SLN uncoupling of SERCA may depend on several factors including
a) the ratio of SLN to SERCA; b) cytosolic Ca2+-concentration; c) SR luminal Ca2+-load;
d) ATP availability in the local milieu and e) other factors yet to be defined. A
recent study by Nowack et al provides strong evidence that SLN-to-SERCA ratio is utilized
by pigs to recruit skeletal muscle thermogenesis independent of shivering. Using muscle
SR vesicles expressing high SLN, we determined that the efficiency of SLN uncoupling
is not 100% but amounts to no more than 30% of SERCA which suggests while SLN uncoupling
function does not interfere with normal muscle function since SERCA pump is in excess.
Considering the fact that muscle forms more than 40% of the body weight of all endothermic
vertebrates, even a small fraction of heat coming from SLN-mediated uncoupling of
SERCA function can have a significant impact on thermoregulation.
The structural features involved in SLN binding and uncoupling of SERCA
The detailed mechanism of SLN uncoupling is an evolving area of research, but many
structural features of SLN/SERCA interaction were identified by protein cross-linking
and using SERCA/SLN co-crystals. The X-ray crystals showed that SLN binds to the SERCA
TM groove formed by TMs 2, 6, and 9 (Sahoo et al., 2015; Shaikh et al., 2016) however,
these studies could not localize the N-terminus of SLN. Previous studies have shown
that the C-terminal residues of SLN (Tyr29 and Tyr31), are important for regulation
of SERCA (Odermatt et al., 1998) and or SLN localization and interaction with SERCA
(Butler et al., 2011). Employing protein chimeras of SLN and PLB, we determined the
function of individual domains in uncoupling. We found addition of SLN C-terminus
to PLB can increase PLB binding affinity to SERCA but does not promote uncoupling
of SERCA (Sahoo et al., 2015). Recent studies from our laboratory suggest that the
N-terminus of SLN is important for SLN function (Sahoo et al., 2015). The short N-terminus
varies across species and 2ERSTQE sequence in rodents change to 2ERSTRE in rabbit
and to 2GINTRE in primates. It contains a conserved Thr5 residue that has been proposed
to be a site of Phosphorylation (Bhupathy et al., 2009). Using mutagenesis and chimeric
proteins made between SLN and PLB, we have shown that SLN requires its N-terminus
for its uncoupling function. Deletion of the N-terminal residues (MERSTQ) caused SLN
to constitutively bind to the SERCA groove but did not possess the uncoupling ability.
Recently, Autry et al speculated that the negatively charged Glutamate residues are
critical for the uncoupling function (Autry et al., 2016). This suggests that mere
occupation of the groove is insufficient for uncoupling function but requires the
dynamic interaction between the N-terminus, the TM, and the C-terminus of SLN with
SERCA for its regulation.
SLN plays a role in both shivering and non-shivering thermogenesis
The opinion review questioned if SLN is recruited during adaptive thermogenesis. While
skeletal muscle has been known to generate heat through shivering, the role of NST
in muscle has not been fully appreciated until recently. Studies in birds and mammals
suggest that cold adaptation involves upregulation of SR proteins in addition to metabolic
remodeling of the muscle (Rowland et al., 2015a; Bal et al., 2016, 2017). Both shivering
and NST rely on SR Ca2+-cycling, so invariably recruits SLN and SERCA, therefore SLN-dependent
heat production is a component of both shivering and NST mechanisms. It is interesting
to point out that during shivering the level of Ca2+-leak from the SR is very high
which would recruit myosin-ATPase as well. Further, as discussed earlier high cytosolic
Ca2+-concentration reduce SLN interaction/uncoupling of SERCA, so SLN function during
shivering might be an important but minor. During prolonged cold adaptation animals
switch from shivering to NST mechanisms; which is activated through an increase in
cytosolic Ca2+ either via RYR1-mediated Ca2+-leak or Ca2+-entry through channels in
the plasma membrane. It is known that cold adapted mice including UCP1 knockout (UCP1-KO)
do not shiver when challenged with cold which argues that NST in muscle is primarily
responsible for heat production. This idea is further supported by two independent
observations made recently. First, mice with surgical ablation of interscapular BAT
acclimated to cold significantly upregulate SLN expression in their skeletal muscles
(Bal et al., 2016). Similarly, SLN is upregulated in skeletal muscles of UCP1-KO mice,
even when exposed to mild cold (Bal et al., 2017). Despite these progress, there are
several gaps in our knowledge including how SLN-based NST is activated, whether this
involves neurohormonal signaling to trigger Ca2+-entry through plasma membrane channels
and/or release from the SR. At this time the major mechanism appears to be RYR1-mediated
Ca2+-leak that can trigger muscle heat production during cold adaptation (Dumonteil
et al., 1993, 1995; Aydin et al., 2008; Bal et al., 2016).
SLN is recruited in diet induced adaptive thermogenesis
Compared to rodents, in large adult mammals including rabbits, dogs and humans, BAT-mediated
thermogenesis is negligent and SLN levels are much more abundant (Babu et al., 2007;
Rowland et al., 2015b). To mimic SLN expression found in large mammals, we overexpressed
SLN using skeletal α-actin gene promoter (Maurya et al., 2015). Sln over-expression
(Sln-OE) did not affect muscle function but increased BMR. When pair-fed, Sln-OE mice
showed a higher rate of oxygen consumption and lost its fat mass, whereas Sln-KO mice
consumed less oxygen and gained fat mass. We further showed that Sln-OE mice were
resistant to high fat diet induced obesity and were protected from lipotoxicity in
muscle, suggesting higher SLN leads to enhanced energy expenditure through increased
mitochondrial biogenesis (Maurya and Periasamy, 2015; Maurya et al., 2015; Sopariwala
et al., 2015). A recent study by Maurya et al, has demonstrated that SLN-mediated
Ca2+-signaling promote mitochondrial health and muscle metabolism (Maurya et al.,
2018). On the other hand, a manuscript published by Butler et al. shows that SLN could
not be detected in rodent muscle and they were unable to increase SLN expression through
transgenesis in muscle (Butler et al., 2015). We believe that there might be technical
issues with SLN detection since this protein is highly hydrophobic and of very small
size (~3.5 kDa) and the use different mouse strain could also contribute to this discrepancy
since our Sln-OE mouse model is in C57Bl6/j background. It has been shown that cold
intolerance of UCP1-KO varies significantly between different mouse strains (Hofmann
et al., 2001).
SLN based muscle NST can compensate for the loss of BAT function in mice
Recent studies also highlighted that in rodents muscle based thermogenesis is recruited
in addition to BAT during cold adaptation (Monemdjou et al., 2000; Anunciado-Koza
et al., 2008, 2011; Bruton et al., 2010; Shabalina et al., 2010; Rowland et al., 2015a;
Bal et al., 2016, 2017; Stanford et al., 2018). Barbara Cannon's group showed that
cold adaptation of UCP1-KO mice affect skeletal muscle metabolism (Aydin et al., 2008),
but argued that this is primarily due to muscle shivering. Interestingly, several
groups have found that Ca2+-handling in the skeletal muscle is altered in cold adapted
UCP1-KO mice. We further investigated the importance of muscle NST in UCP1-KO mice
by exposing them to different regimens of cold challenge. We found that skeletal muscles
from gradually cold adapted UCP1-KO mice upregulated SLN expression, become redder
with increased mitochondrial content, succinate dehydrogenase (SDH) activity as opposed
to SLN–KO mice. Interestingly, SLN knockout mice adapted to cold showed significant
upregulation of UCP1 and higher mitochondrial content in BAT suggesting that SLN-KO
mice increasingly rely on BAT-based NST to compensate for decreased muscle thermogenesis.
Using double knockout (DKO) mice for SLN and UCP1, we studied their ability to adapt
to cold. These DKO mice were unable to withstand acute cold (4°C) exposure, while
UCP1-KO exhibit only 30% cold sensitivity (Rowland et al., 2015a). Further, we do
not believe shivering is the sole mechanism for the survival of UCP1-KO mice during
cold adaptation. To gain insight into this question we have performed cold adaptation
studies on UCP1-KO mice employing gradual decrease (2.0°C/day) and acute decrease
(5°C/day) of ambient temperature of mice reared at 29°C. During this cold exposure,
the UCP1-KO show visible shivering only for a short period. When acutely shifted from
29 to 16°C and from 16 to 4°C, mice showed visible shivering for up to 2 days but
recognizable shivering decreases and stops by 4th day. Based on these studies we suggest
that, subsequent to an initial phase of shivering, SLN-based NST becomes the primary
mechanism of thermogenesis as continuous shivering will lead to muscle damage.
SLN might be the major determinant of muscle thermogenesis in birds and BAT-Deficient
mammals
From the studies described here, it is evident that SLN plays a very important role
in thermogenesis, both in shivering and muscle NST. Campbell and Dicke rightly pointed
out that all our studies have been in the mouse model. We agree that we have not studied
large animal models and/or avian species. However, we want to emphasize that we have
compared SLN protein expression in mice vs. large animals (like rabbit, dog), and
humans (Babu et al., 2007; Rowland et al., 2015b). SLN is highly abundant in all the
muscles of larger mammals including human and SLN has a better intracellular milieu
for its physiological function in oxidative fibers [discussed by Nowack et al. (2017)].
Based on the expression profile, we do not believe that SLN can have a completely
different physiological role in larger mammals compared to mice. We were unable to
study SLN expression in birds using our antibody as it detects the C-terminal sequence
“RSYQY” that is not conserved in birds (KSYQX). However SLN is highly conserved in
the TM region, suggesting avian SLN bind to the same groove of SERCA. The C-terminus
of avian SLN does not have conserved sequence but the N-terminus has conserved Glutamate
at 2nd and 7th position in sparrow, chicken and pigeon important for uncoupling function
(Sahoo et al., 2015; Autry et al., 2016). It is to be noted that avian SERCA bears
several residue substitutions especially in the cytosolic and luminal loop regions
that SLN can potentially interact as shown in Figure 1. Hence, difference in SLN sequence
as seen in Figure 1, might provide better anchoring leading to increased function
and not loss of function as indicated by the authors. Complete sequence alignment
of SERCA is presented as supplemetal data Data Sheet 1. It shows that transmembrane
helices are highly conserved, which provides the groove for SLN to bind to SERCA.
Figure 1
Alignment of SERCA (A) and SLN (B) sequences. Comparison of SERCA sequences from various
species of birds and mammals shows that the cytosolic residues that may potentially
interact with SLN do not have strict conservation. SLN sequences also show divergence
more on the N-terminal cytosolic and C-terminal luminal side. Like other transmembrane
proteins, transmembrane residues of both the proteins are more strictly conserved.
Therefore, avian SLN may be better suited to uncouple avian SERCA.
Perspectives and conclusions
While shivering is an important mechanism of heat production in birds and mammals,
prolonged shivering would compromise many physiological functions of an animal and
even compromise survival in the wild. In addition mild temperature fluctuations do
not activate shivering but rely primarily on NST mechanisms. It is of interest and
surely significant that SLN is expressed throughout the vertebrate, from fish to man
and might be involved in more fundamental role of local heat production. The role
of SR Ca2+-cycling in muscle thermogenesis has been documented in the “heater organ”:
a modified extraocular muscle found in deep sea fishes (Block and Franzini-Armstrong,
1988; Morrissette et al., 2003) and in malignant hyperthermia, a disease where excessive
Ca2+-leaks from RyR coupled with chronic SERCA Ca2+-cycling leading to pathological
heat production (Gommans et al., 2002; Rossi and Dirksen, 2006). These examples do
suggest that SR can be adapted as a heat producing mechanism. While thermogenesis
is often thought as important for whole body temperature regulation, heat production
in muscle plays an intrinsic role in peak muscle performance and more importantly
survival, especially when faced with a predator–prey situation. True endothermy is
seen only in birds and mammals, yet it relies on BAT in only one taxa, the eutherian
mammals. The other extant mammals, monotremes and marsupials, lack BAT but are competent
endotherms. Birds too lack BAT but manage endothermy at the highest body temperatures
of any, 40°C and more. These all must achieve their endothermy by relying on regulatory
NST sourced in the skeletal muscle. [reviewed by Nowack et al. (2017)]. Uncoupling
mechanisms or futile mechanisms are not entirely new but exist at the expense of increased
energy demand. Regulation of SERCA by small peptides including PLB, SLN, myoregulin
and others have provided new roles for SERCA pump activity in muscle Physiology (Anderson
et al., 2015). We suggest that SLN uncoupling of SERCA activity evolved during vertebrate
evolution to support heat production in muscle important for both muscle performance
and thermogenesis. Although the molecular details how SLN binding promotes uncoupling
of SERCA and recruited under different pathophysiological conditions remain to be
solved, the recent studies provide strong evidence that SLN is important for muscle
thermogenesis.
Author contributions
All authors drafted the manuscript together, critically revised the work and approved
the final version. NB prepared the figures.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.