Neuronatin Promotes SERCA Uncoupling and Its Expression is Inversely Associated with High Fat Diet Induced Weight Gain in vivo

Abstract

Neuronatin (NNAT) is a paternally imprinted gene involved in many aspects of metabolism but the underlying cellular mechanism remains unknown. Its sequence homology with phospholamban (PLN) and sarcolipin (SLN) suggest that NNAT has a putative role in regulating the enzymatic activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) in striated muscles. Both PLN and SLN physically interact and functionally inhibit SERCA by lowering its affinity for Ca2+, but only SLN has the unique ability to uncouple SERCA-mediated Ca2+ transport from ATP hydrolysis in mouse soleus muscles. Uncoupling the SERCA pump increases energy expenditure and may be beneficial in combatting energy imbalances, such as obesity. The objective of this study was to test our hypothesis that like SLN, NNAT would also promote SERCA uncoupling. In addition, we also examined whether NNAT expression would be altered in soleus muscles obtained from C57BL/6 mice fed a high fat diet (HFD). Using human embryonic kidney (HEK) cells co-transfected with SERCA and NNAT cDNA, we measured SERCA coupling ratio using an Indo-1 based fluorometric Ca2+ uptake assay and an enzyme-linked spectrophotometric ATPase assay. NNAT promoted uncoupling of both SERCA1a and SERCA2a isoforms in HEK cells in a dose-dependent manner with SERCA1a showing increased sensitivity when compared to SERCA2a. Western blotting was used to confirm HEK cell transfection as well as to assess NNAT and SERCA content in soleus muscles from mice fed a low (LFD) or HFD (60% kcal) for 12 weeks. A ~40% reduction in SERCA1a and a ~50% increase in SERCA2a protein content was observed in the soleus muscles of mice fed a HFD compared to LFD. Total NNAT content and its levels relative to SERCA (SERCA1a + SERCA2a) were reduced by ~40% and ~45%, respectively in HFD mice compared to LFD mice. Plotting muscle NNAT content (relative to SERCA) against total weight gained after 12 weeks of high fat feeding also revealed a significant inverse relationship (r2=0.49, p=0.01), suggesting that higher NNAT content (relative to SERCA) is related to reduced weight gained from a HFD in vivo. This is the first study to demonstrate NNAT as a SERCA uncoupler and that its expression in muscle is negatively associated with HFD induced weight gain. These findings suggest that NNAT may regulate whole-body metabolism and energy balance via SERCA uncoupling.