The effect of SERCA2 activation on liver metabolism during acute lipotoxicity in vivo

Abstract

Non‐alcoholic fatty liver disease (NAFLD) is the most common form of liver disease affecting about 30% of the US population. The prognosis for simple liver steatosis is relatively benign; however, chronic inflammation and progressive fibrosis may lead to nonalcoholic steatohepatitis (NASH) and later to cirrhosis and hepatocellular carcinoma. There is currently no established treatment to stop progression from NAFLD to NASH. It was previously shown (Egnatchik RA et al. Mol Metab. 2014; 3 (5): 544–53) that lipotoxic concentrations of fatty acids alter endoplasmic reticulum (ER) calcium stores and induce mitochondrial dysfunction, characterized by elevated citric acid cycle (CAC) flux and reactive oxygen species accumulation, leading to apoptosis. Furthermore, the function of SERCA2 (sarco/endoplasmic reticulum Ca2+‐ATPase), the main protein in hepatocytes that is responsible for transporting Ca2+ from cytoplasm to ER, is impaired in obese mice (Fu S et al. Nature. 2011; 473 (7348): 528–31). We thus hypothesize that restoration of SERCA2 activity might improve mitochondrial function in hepatocytes during acute lipotoxicity.To test this hypothesis, we developed a protocol to investigate the acute impact of fatty acids on liver energy metabolism. Conscious, unrestrained, overnight‐fasted, 12‐week‐old male C57Bl/6J mice were given continuous, intravenous infusions of either saline (control) or 20% lard oil (as described in Stein DT et al. J Clin Invest. 1997; 100 (2): 398–403) for 5 hrs to induce hepatic lipotoxicity. A third group of animals was given lard oil in combination with single intraperitoneal injection of small molecular allosteric activator CDN1163 (50 mg/kg) one week prior to study to activate SERCA2 (Kang S et al. J Biol Chem. 2016; 291 (10): 5185–98). Animals were simultaneously administered a primed, continuous infusion of [6,6‐2H2] glucose and sodium [13C3]lactate intravenously for 4 and 2 hrs, respectively, to assess in vivo liver metabolic fluxes. 13C and 2H‐enrichment of plasma glucose and liver alanine, glutamate, lactate and urea obtained at the end of the study were measured by GC‐MS and used to regress liver metabolic fluxes using a flexible modeling platform (INCA).Our analysis showed that by the end of the experiment both plasma and liver free fatty acids were significantly elevated after lard oil infusion, whereas liver triglycerides and plasma glucose were not affected, regardless of CDN1163 treatment. In contrast, CDN1163 significantly increased alanine aminotransferase and aspartate transaminase content in liver, and restored basal insulin level, which was significantly downregulated in the presence of lard oil alone. Analysis of liver metabolic fluxes revealed that lard oil increased fatty acid oxidation flux. On the other hand, lard oil coupled with CDN1163 led to further upregulation of fatty acid oxidation flux, but at the same time hepatocytes were characterized by increased endogenous glucose production, elevated pyruvate cycling and upregulated CAC activity. These data suggest that SERCA2 activation increases fatty acid oxidation in mitochondria, but at the same time proportionally increases other hepatic fluxes, which might restore energy balance and protect liver during acute lipotoxicity.Support or Funding InformationThis research was supported by NIH grant R01 DK106348.