Alterations in Cytoskeleton and Mitochondria in the Development and Reversal of Steatosis in Human Hepatocytes

Abstract

Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well-established in non-alcoholic fatty liver disease (NAFLD). Patients can undergo lifestyle changes, especially in earlier NAFLD stages, to reverse disease-induced phenotypes on a gross level. Yet, little is known about whether mitochondrial function and injuries recover upon reversal. We, thus, elucidated this question and interplays between cytoskeletal network and mitochondria in the development and reversal of steatosis. We cultured primary human hepatocytes stably for 2 weeks and used free-fatty-acid (FAA) supplementation to induce steatosis over 7 days and reversed steatosis by FAA withdrawal over the next 7 days. We assessed cytoskeletal and mitochondrial morphologies using immunocytochemistry and confocal microscopy. We evaluated mitochondrial respiration and function via the Seahorse analyzer where we fully optimized reagent dosing specifically for human hepatocytes. During early steatosis, intracellular lipid droplets displaced microtubules altering mitochondrial distribution, and disrupted the F-actin network leading to loss of bile canaliculi in steatotic hepatocytes. Basal mitochondrial respiration, maximum respiratory capacity, and resistance to H2O2-induced cell death also increased as an adaptative response. Upon reversal of steatosis, F-actin and bile canaliculi were restored in hepatocytes. Nevertheless, we observed an increase in elongated mitochondrial branches accompanied by decreases in α-tubulin expression, mitochondrial proton leak, and susceptibility to H2O2-induced cell death. Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, mitochondria, in hepatocytes, were impaired due to early adaptative respiratory increase. Hepatocytes were thus highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.