Lithium Inhibition of GSK3 Uncouples SERCA Transport Efficiency in C2C12 Cells and Alters Energy Expenditure in vivo

Abstract

Adaptive thermogenesis is a cellular process that accelerates energy expenditure while increasing heat production in response to prolonged cold exposure or caloric excess. Enhancing adaptive thermogenesis mechanisms may assist in combatting diet-induced obesity. Skeletal muscle via sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) uncoupling and brown/beige adipose via mitochondrial uncoupling are the two primary sites for adaptive thermogenesis in mammals. Recent evidence has shown that glycogen synthase kinase 3 (GSK3) negatively regulates adipose-based thermogenesis by repressing uncoupling protein-1 expression in brown adipocytes. However, to our knowledge, there have been no studies examining whether GSK3 also negatively regulates muscle-based thermogenesis via SERCA uncoupling. Lithium (LiCl), a known GSK3 inhibitor, has previously been shown to offset diet induced obesity in rodents. Thus, we examined the effect of LiCl treatment on energy expenditure first utilizing a cellular model followed by an in vivo mouse model. To this end, C2C12 cells were treated with 0.5 mM LiCl at the onset of differentiation for 7 days. SERCA coupling ratios were obtained by measuring the rates of Ca2+ uptake using a fluorophore-based assay and the rates of ATP hydrolysis using a spectrophotometric assay at physiological Ca2+ concentrations. Our results show that when treated with LiCl, C2C12 cells had significantly reduced Ca2+ uptake (-59%, p = 0.03) with no change in SERCA activity. This led to a significant reduction in SERCA's coupling ratio (p = 0.04). These results are consistent with our previous findings suggesting that LiCl increases basal respiration and SERCA's energy contribution in C2C12 cells. To support this increase in energy expenditure, our Western blot analyses identified that LiCl treatment inhibited GSK3 and showed increased levels of PGC-1a, the master regulator of mitochondrial biogenesis. To investigate the effects of LiCl on energy expenditure in vivo, adult male C57BL/6J mice were provided 10mg/kg/day LiCl via their drinking water for 6 weeks. We have previously shown that this dose and duration of LiCl significantly inhibits GSK3 in murine skeletal muscle. On the 6thweek of treatment, mice were housed in a Promethion metabolic cage system for a period of 48h to examine daily energy expenditure via indirect calorimetry. Metabolic cage data revealed that LiCl fed mice had significantly increased energy expenditure (kcal/kg/hr; p = 0.008) compared to controls; however, the contribution from muscle-based thermogenesis and SERCA uncoupling still needs to be examined. Altogether, these data suggest that GSK3 inhibition via LiCl treatment augments SERCA energy expenditure in muscle by uncoupling Ca2+ transport from ATP hydrolysis. Future studies will determine whether our in vitro findings translate to the in vivo setting.