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

Letao Fan, Aslihan Gokaltun, Sarah Maggipinto, Yoshinori Kitagawa, Jeevendra Martyn, Heidi Yeh, Basak E. Uygun, Martin L. Yarmush, O. Berk Usta

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Background & Aims: Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well established in nonalcoholic 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. Thus, we elucidated this question and interplays between the cytoskeletal network and mitochondria in the development and reversal of steatosis. Methods: We cultured primary human hepatocytes stably for 2 weeks and used free fatty acid supplementation to induce steatosis over 7 days and reversed steatosis by free fatty acid 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, in which we fully optimized reagent dosing specifically for human hepatocytes. Results: 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. Conclusions: Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, the mitochondria in hepatocytes were impaired owing to early adaptative respiratory increase. Hepatocytes thus were highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.

Original languageEnglish (US)
Pages (from-to)243-261
Number of pages19
Issue number2
StatePublished - Jan 2023

Bibliographical note

Funding Information:
Funding This research was supported partially by grants from the National Institutes of Health (5R21GM136002, 1R21GM 141683, 1R21GM140656, and 5R01HL145031) for O.B.U, L.F, A.G and M.L.Y, National Science Foundation ( 1941543 ) for O.B.U, A.G. and M.L.Y., and a Massachusetts General Hospital Executive Committee on Research Interim Support Fund for O. B.U.

Publisher Copyright:
© 2023 The Authors


  • Cytoskeleton
  • Mitochondria
  • Nonalcoholic Fatty Liver Disease
  • Steatosis

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't


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