Neuronatin promotes SERCA uncoupling and its expression is altered in skeletal muscles of high‐fat diet‐fed mice

Abstract

Neuronatin (NNAT) is a transmembrane protein in the endoplasmic reticulum involved in metabolic regulation. It shares sequence homology with sarcolipin (SLN), which negatively regulates the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) that maintains energy homeostasis in muscles. Here, we examined whether NNAT could uncouple the Ca2+ transport activity of SERCA from ATP hydrolysis, similarly to SLN. NNAT significantly reduced Ca2+ uptake without altering SERCA activity, ultimately lowering the apparent coupling ratio of SERCA. This effect of NNAT was reversed by the adenylyl cyclase activator forskolin. Furthermore, soleus muscles from high fat diet (HFD)‐fed mice showed a significant downregulation in NNAT content compared with chow‐fed mice, whereas an upregulation in NNAT content was observed in fast‐twitch muscles from HFD‐ versus chow‐ fed mice. Therefore, NNAT is a SERCA uncoupler in cells and may function in adaptive thermogenesis.

Characterizing SERCA Function in Murine Skeletal Muscles after 35–37 Days of Spaceflight

“…Accompanying the changes in SERCA protein content, three SERCA regulators—PLN, SLN, and NNAT—were also investigated. PLN and NNAT are both highly conserved proteins primarily found in oxidative skeletal muscle [ 22 , 24 , 37 ]. PLN allosterically binds to SERCA and reduces its affinity for calcium [ 22 , 37 ], while NNAT has been shown to inhibit Ca 2+ uptake and promote SERCA uncoupling in vitro [ 24 ].…”

“…PLN and NNAT are both highly conserved proteins primarily found in oxidative skeletal muscle [ 22 , 24 , 37 ]. PLN allosterically binds to SERCA and reduces its affinity for calcium [ 22 , 37 ], while NNAT has been shown to inhibit Ca 2+ uptake and promote SERCA uncoupling in vitro [ 24 ]. In response to spaceflight, significant reductions in both PLN and NNAT protein content were seen, and thus we believe may not contribute to the impairments in SERCA function observed in the postural soleus.…”

“…In response to spaceflight, significant reductions in both PLN and NNAT protein content were seen, and thus we believe may not contribute to the impairments in SERCA function observed in the postural soleus. Further, since both PLN and NNAT are found primarily in oxidative skeletal muscle [ 22 , 24 , 37 ], the reduction in both of their protein contents is not entirely surprising given the fast fiber type shift. Conversely, we found that SLN was significantly upregulated in the soleus in response to spaceflight, which again was, to some extent, expected.…”

“…Rates of Ca 2+ uptake in the muscle homogenates were measured using the Indo-1 Ca 2+ fluorophore as previously described [ 38 , 49 , 55 , 56 ], but fitted onto a 96-well plate [ 24 ]. Briefly, muscle homogenate was added to reaction buffer (200 mM KCl, 20 mM HEPES, 10 mM NaN 3 , 5 µM TPEN, 15 mM MgCl 2 (pH 7.0)) containing Indo-1 (4 µM final concentration; 57180, Sigma-Aldrich, St. Louis, MO, USA).…”

“…In addition to SERCA isoform content, there are several other factors that can regulate SERCA function. SERCA has two main regulatory proteins in skeletal muscle, phospholamban (PLN) [ 15 , 22 ] and sarcolipin (SLN) [ 15 , 22 ]; however, we have also recently identified another regulator in muscle named neuronatin (NNAT) [ 23 , 24 ]. Each of these regulators work to reduce SERCA-mediated Ca 2+ uptake either through reductions in Ca 2+ affinity or causing Ca 2+ ‘slippage’ back into the myoplasm [ 22 , 25 ].…”