The role of hepatocyte Na⁺/H⁺ exchanger 1 (NHE1) in non-alcoholic fatty liver disease (NAFLD) in vitro and in vivo

Non-alcoholic fatty liver disease (NAFLD) affects up to one third of the global population and constitutes a major cause of liver-related mortality. It is the hepatic manifestation of obesity, diabetes type II and the metabolic syndrome. Thus, NAFLD demands intense investigation to enable treatment and prevention of disease progression. Na+/H+ exchanger 1 (NHE1) was previously implicated in the development of hepatic steatosis and fibrogenesis. Thus, the focus of the present study was to investigate the effects of NHE1 knockout (KO) in hepatocytes on steatosis, cellular stress, fibrosis, and mitochondrial function in vitro and in vivo as well as characterizing the Ca2+/calmodulin (CaM)-mediated interaction and regulation of NHE1.

In Paper I, we employed biophysical and cellular techniques to gain structural and functional insights into Ca2+-mediated regulation of NHE1, with Ca2+ being a dysregulated signaling pathway in NAFLD. We showed that CaM dynamically interacts with NHE1 tuned by protein concentrations, Ca2+ availability, and phosphorylation, and that CaM affects dimerization of NHE1. Although the interaction was important for activation of NHE1 in cells, a rise in free cytosolic Ca2+ or mutations to the high affinity CaM binding site in NHE1 did not influence proximity of NHE1-CaM in cells. Furthermore, in Paper II, we studied the effect of a palmitic acid (PA)-induced NAFLD-like phenotype in hepatocytes in vitro on NHE1 activity. While PAtreatment caused lipid buildup, it did not impact NHE1 activity. Instead, we investigated if NHE1 KO in PA-treated hepatocytes conferred protective effects on steatosis, lipotoxicity, inflammatory signaling and hepatic stellate cell (HSC) activation. We showed that NHE1 protein content decreased, and that NHE1 KO protected against apoptosis and activation of the NAFLD-associated MAP kinase p38. Treatment of HSCs with hepatocyte conditioned medium showed that loss of hepatocyte NHE1 affected HSC protein expression. To evaluate these results in vivo, we knocked out NHE1 in hepatocytes in mice and fed them a methioninecholine-deficient diet to induce NAFLD-associated liver damage (Paper III). Microscopy and western blot analyses showed that hepatic steatosis, inflammatory signaling and macrophage accumulation and fibrosis were unaffected by hepatocyte NHE1 KO. Yet, respirometric analysis of reactive oxygen species (ROS) emission suggested a slight dependency on NHE1 for basal mitochondrial function. Overall, these data indicate that hepatocyte NHE1 KO does not ameliorate NAFLD-associated hepatic injury in vivo.

Collectively, the work presented shows that, firstly, although CaM interaction affects cellular NHE1 activity, NAFLD-like conditions in hepatocytes do not significantly impact NHE1 activity. And, secondly, despite protective effects in vitro, hepatocyte NHE1 KO does not severely alter NAFLD-associated liver damage in vivo. These novel insights require adjustments of current knowledge regarding the role of NHE1 in NAFLD.