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Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures a

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Dong Fu , Jennifer Lippincott-Schwartz & Irwin M Arias a

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Faculty of Pharmacy; University of Sydney; NSW, Australia

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Eunice Kennedy Shriver National Institute of Child Health and Human Development; National Institutes of Health; Bethesda, MD USA Published online: 27 Sep 2013.

To cite this article: Dong Fu, Jennifer Lippincott-Schwartz & Irwin M Arias (2013) Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures, Autophagy, 9:12, 2154-2155, DOI: 10.4161/ auto.26167 To link to this article: http://dx.doi.org/10.4161/auto.26167

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Autophagic Punctum

Autophagy 9:12, 2154–2155; December 2013; © 2013 Landes Bioscience

Dong Fu,1 Jennifer Lippincott-Schwartz,2,* and Irwin M Arias2,* Faculty of Pharmacy; University of Sydney; NSW, Australia; 2Eunice Kennedy Shriver National Institute of Child Health and Human Development; National Institutes of Health; Bethesda, MD USA

Downloaded by [University of New Hampshire] at 06:59 17 March 2015

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Keywords: polarization, mitochondrial fusion, autophagy, oxidative phosphorylation, lipid droplets Submitted: 07/26/2013 Revised: 08/15/2013 Accepted: 08/15/2013 http://dx.doi.org/10.4161/auto.26167 *Correspondence to: Jennifer Lippincott-Schwartz; Email: [email protected]; Irwin M Arias; Email: [email protected] Punctum to: Fu D, Mitra K, Sengupta P, Jarnik M, Lippincott-Schwartz J, Arias IM. Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization. Proc Natl Acad Sci U S A 2013; 110:7288–93; PMID:23589864; http://dx.doi. org/10.1073/pnas.1304285110

reshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the events underlying this repolarization process, focusing on how hepatocytes restore ATP synthesis and resupply biosynthetic precursors after the stress of being isolated from liver. We found that soon after being plated in collagen sandwich cultures, hepatocytes converted their mitochondria into highly fused networks. This occurred through a combination of upregulation of mitochondrial fusion proteins and downregulation of a mitochondrial fission protein. Mitochondria also became more active for oxidative phosphorylation, leading to overall increased ATP levels within cells. We further observed that autophagy was upregulated in the repolarizing hepatocytes. Boosted autophagy levels likely served to recycle cellular precursors, supplying building blocks for repolarization. Repolarizing hepatocytes also extensively degraded lipid droplets, whose fatty acids provide precursors for β-oxidation to fuel oxidative phosphorylation in mitochondria. Thus, through coordination of mitochondrial fusion, autophagy, and lipid droplet consumption, depolarized hepatocytes are able to boost ATP synthesis and biosynthetic precursors to efficiently repolarize in collagen sandwich cultures. The polarized morphology of hepatocytes is essential for many liver functions, including secretion of bile acids and excretion of exogenous substrates. Many

2154 Autophagy

energy-consuming cellular processes are required to form and maintain this polarized state. These include: assembly of tight junctions between adjacent cellular membranes; selective protein trafficking to apical or basolateral plasma membrane domains; and creation of contractile structural scaffolds of actin and myosin. Hepatocytes rapidly lose their polarized morphology under stress conditions, such as after isolation from rat liver. Depolarization under these conditions coincides with internalization of membrane proteins, such as the bile salt export pump (ABCB11), P-glycoprotein (ABCB1) and likely other transporters/ receptors required for accessing external nutrients. How depolarized hepatocytes survive these conditions to ultimately repolarize has been unclear. We examined whether mitochondria and autophagy play an important role in hepatocyte repolarization after isolation from liver. We reasoned that mitochondria and autophagy could help mediate hepatocyte repolarization by serving to restore energy and biosynthetic precursor levels. To address this possibility, we examined the metabolic status of repolarizing hepatocytes in collagen sandwich systems, which have previously been used to study repolarization of hepatocytes isolated from liver. Hepatocytes were freshly isolated from liver and then placed in the collagen sandwich system to monitor their repolarization. Within 5–6 days of culturing, the hepatocytes undergo repolarization to generate bile canaliculi. Repolarization coincides with a significant rise in cellular ATP

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Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures

Downloaded by [University of New Hampshire] at 06:59 17 March 2015

levels. As mitochondria are efficient ATP generators through oxidative phosphorylation (OXPHOS), we tested whether the rise in ATP levels during hepatocyte repolarization came from mitochondrial OXPHOS. Supporting such a role, treatment with oligomycin to inhibit OXPHOS in the first few days of cultures prevents polarization. Oligomycin treatment also leads to decreased overall cellular ATP levels. By contrast, treatment with 2-deoxyglucose to inhibit glycolysis neither inhibits repolarization nor affects cellular ATP levels. Only after hepatocytes became fully polarized does inhibiting glycolysis lead to reduced cellular ATP levels. Thus, the increased energy requirement for hepatocyte repolarization is primarily supplied by OXPHOS. In this way, the large and urgent demand for energy production required to initiate repolarization is met. The shift to OXPHOS by repolarizing cells coincides with mitochondria becoming morphologically highly fused. This involves upregulation of the mitochondrial fusion proteins OPA1, MFN1, and MFN2, and downregulation of the mitochondrial fission protein DNM1L/DRP1. Because changes in abundance of the mitochondrial fusion/fission proteins correlate with the shift to OXPHOS, tubulated mitochondria could play a role in the metabolic shift. Moreover, the tubular mitochondria could be additionally important for protecting mitochondria from mitophagy, which is reduced when

mitochondria are highly fused. More mitochondria would thereby be available to drive OXPHOS during repolarization. To test what nutrient source is used to fuel OXPHOS during repolarization, we blocked fatty acid β-oxidation within mitochondria using the drug etomoxir. We found that hepatocyte repolarization is inhibited. This suggested that repolarization pathways utilize β-oxidation of fatty acids to drive OXPHOS. Because lipid droplets disappear in repolarizing hepatocytes, the lipid droplets likely provide the fatty acid precursors required for β-oxidation. Supporting this, lipid droplets are abundant in the early cultures when hepatocytes are under stress and not polarized. Disappearance of these lipid droplets correlates with increasing ATP levels during polarization. Autophagy is upregulated throughout hepatocyte repolarization and appears to be required for it because inhibiting autophagy impairs polarization. One obvious role for autophagy during repolarization is to recycle endogenous substrates for new protein synthesis. Additionally, we found that autophagy contributes to boosting ATP levels during hepatocyte repolarization since inhibiting autophagy lowers cellular ATP levels. Autophagy’s role in ATP production during repolarization could be for breaking down proteins and membranes, which would allow amino acids and lipids to be imported into mitochondria to help fuel OXPHOS.

Overall, our results reveal that mitochondria and autophagy coordinate to supply essential bioenergetics and biosynthetic precursors during hepatocyte repolarization after the stress of cellular isolation. To produce sufficient energy and nutrients to undergo such repolarization, hepatocytes increase autophagy and shift their metabolism from glycolysis to OXPHOS. The shift to OXPHOS allows the cells to be more efficient energy producers since OXPHOS produces fifteen times more ATP than glycolysis. The increased levels of autophagy in the repolarizing hepatocytes provide nutrients by degradation and reutilization of internal macromolecules. Together, the shift in metabolism allows depolarized hepatocytes to rely on internal stores of nutrients to survive and undergo repolarization under these conditions. These findings have implications for other stresses, regeneration and pathological conditions that affect hepatocytes and other epithelial cells. Loss of polarization is likely a common consequence in liver failure associated with hepatitis, drug-induced liver injury and liver cancer. Mitochondrial fusion, OXPHOS, autophagy, lipid droplets consumption or β-oxidation could be potential treatment targets. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

www.landesbioscience.com Autophagy 2155

©2013 Landes Bioscience. Do not distribute.

Autophagic Punctum

Autophagic Punctum

Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures.

Freshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the ...
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