Loss of mitochondrial ATPase ATAD3A contributes to nonalcoholic fatty liver disease through accumulation of lipids and damaged mitochondria

Abstract

Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and nonalcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced nonalcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells and also that triglyceride levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, triglyceride, and damaged mitochondria in hepatocytes. Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and nonalcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced nonalcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells and also that triglyceride levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, triglyceride, and damaged mitochondria in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in the United States and the second most common indicator for liver transplantation (1Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. et al.Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat. Rev. Gastroenterol. Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (3068) Google Scholar). NAFLD is a condition where excess fat accumulates in the liver, inducing impaired hepatocyte function and inflammation leading to liver injury in the absence of alcohol. NAFLD is characterized by excessive triglyceride (TG) and cholesterol accumulation and constitutes a disease spectrum, with stages ranging from simple steatosis (SS) with excessive fat accumulation in the liver to nonalcoholic steatohepatitis (NASH), where inflammation and fibrosis first appear. NASH can progress further into cirrhosis and hepatocellular carcinoma at end stages (2Huang T.D. Behary J. Zekry A. Non-alcoholic fatty liver disease: a review of epidemiology, risk factors, diagnosis and management.Intern. Med. J. 2020; 50: 1038-1047Crossref PubMed Scopus (92) Google Scholar). Free cholesterol (FC) has been shown to disrupt mitochondrial function and induce hepatocyte death, contributing to NAFLD progression (3Arguello G. Balboa E. Arrese M. Zanlungo S. Recent insights on the role of cholesterol in non-alcoholic fatty liver disease.Biochim. Biophys. Acta. 2015; 1852: 1765-1778Crossref PubMed Scopus (211) Google Scholar). In SS, excessive fats such as cholesterol and TG accumulate in hepatocytes, disrupting the intracellular homeostasis of liver cells, resulting in liver injury. Lipid disruption, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and reactive oxygen species have been shown to contribute to liver-cell damage and promote the progression of SS to NASH with concomitant fibrosis (4Cao S.S. Kaufman R.J. Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease.Antioxid. Redox Signal. 2014; 21: 396-413Crossref PubMed Scopus (871) Google Scholar, 5Pang L. Liu K. Liu D. Lv F. Zang Y. Xie F. et al.Differential effects of reticulophagy and mitophagy on nonalcoholic fatty liver disease.Cell Death Dis. 2018; 9: 90Crossref PubMed Scopus (34) Google Scholar). Recently, it has been proposed that NAFLD is a mitochondrial disease because besides being the cell's power plants, mitochondria are also responsible for cholesterol transport, fatty acid oxidation, and generation of reactive oxygen species. Mitochondrial dysfunction renders liver cells susceptible to damage and extracellular risk factors by disrupting intracellular metabolism and signal transduction (6Grattagliano I. Montezinho L.P. Oliveira P.J. Fruhbeck G. Gomez-Ambrosi J. Montecucco F. et al.Targeting mitochondria to oppose the progression of nonalcoholic fatty liver disease.Biochem. 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A. et mitochondrial to a membrane 2015; PubMed Scopus Google Scholar). with and showed FC in human the of ATAD3A in the of cholesterol homeostasis I. et cause dysfunction with mitochondrial and cholesterol PubMed Scopus Google Scholar). In ATAD3A was as an inner mitochondrial membrane the of ATAD3 was proposed to into the outer mitochondrial membrane and such as the J. ATAD3 proteins: brokers of a mitochondria-endoplasmic reticulum connection in mammalian cells.Biol. Rev. Camb Philos. Soc. 2018; 93: 827-844Crossref PubMed Scopus (54) Google Scholar, 10Issop L. Fan J. Lee S. Rone M.B. Basu K. Mui J. et al.Mitochondria-associated membrane formation in hormone-stimulated Leydig cell steroidogenesis: role of ATAD3.Endocrinology. 2015; 156: 334-345Crossref PubMed Scopus (102) Google interactions with was that ATAD3A with protein a protein in the the role of ATAD3A in the of stress Y. A. J. Mitochondrial ATAD3A with to the PubMed Scopus Google Scholar). ATAD3A has been shown to promote cell through the of the in cells D. Liu L. M. B. Z. et ATAD3A and of cells from the Biol. 2018; PubMed Scopus Google Scholar). ATAD3A to mitochondrial structure, cholesterol mitochondrial interactions with and cell the role of ATAD3A under excess fat and relationship with a mitochondrial have been into the function of ATAD3A in the progression of we characterized ATAD3A expression in liver from rats with NASH with controls and liver from patients with SS, ATAD3A was found to upregulated with the progression of the of Moreover, loss of ATAD3A increased FC and TG accumulation under FC overload in Huh7 cells and autophagosome in Huh7 cells and fresh human hepatocytes isolated from humanized liver mice was largely the loss of ATAD3A under FC overload in Huh7 ATAD3A KO Huh7 cells under FC overload also showed mitochondrial results that ATAD3A plays an essential role in the progression of NAFLD. assess the expression levels of ATAD3A in the progression of we rats a for to induce a NASH Y. L. L. A. et protein steatosis and acid in nonalcoholic fatty liver PubMed Scopus Google Scholar). rats were used as livers were isolated and to to assess the levels of and showed that ATAD3A expression was significantly upregulated in livers of rats the to the this is to we liver from and patients with SS, cirrhosis with human liver were first the NAFLD by a of were as of liver stages by of and cirrhosis for for further results showed that in patients with SS, and the expression of ATAD3A in the liver was increased to this that ATAD3A a role in the of NAFLD of the disease the role of ATAD3A in the progression of CRISPR-Cas9 was to an ATAD3A KO Huh7 cell loss of ATAD3A was by that most of ATAD3A with the mitochondrial in Huh7 showed a of and a of of ATAD3A with ATAD3A was in the ATAD3A KO Huh7 cells, the loss of ATAD3A a deletion of and a in in ATAD3A at the in a mitochondrial and mitochondrial in the ATAD3A KO Huh7 cells is with the mitochondrial in cells in to ATAD3A M.B. L. G. S. Fan J. et of a mitochondrial protein cholesterol and metabolism to steroid Endocrinol. PubMed Scopus Google Scholar). results that we an ATAD3A KO Huh7 cell FC cholesterol to the progression of NAFLD (3Arguello G. Balboa E. Arrese M. Zanlungo S. Recent insights on the role of cholesterol in non-alcoholic fatty liver disease.Biochim. Biophys. Acta. 2015; 1852: 1765-1778Crossref PubMed Scopus (211) Google we induced FC overload in Huh7 cells by treatment with cholesterol and 58035, an inhibitor of acetyl-CoA a responsible for the of FC to cholesterol (3Arguello G. Balboa E. Arrese M. Zanlungo S. Recent insights on the role of cholesterol in non-alcoholic fatty liver disease.Biochim. Biophys. Acta. 2015; 1852: 1765-1778Crossref PubMed Scopus (211) Google Scholar). treatment of cholesterol plus 58035, FC levels were significantly increased in and KO cells, that the treatment induced FC overload in Huh7 cells FC levels in KO cells were significantly under FC overload Huh7 cells were also with a used to FC, and under shown in the were and in KO cells under FC overload, as as were and to the of shown in the with cell was significantly in KO cells under FC overload and the ATAD3A KO FC accumulation under FC overload, we cholesterol metabolism was the loss of results show in levels of the for cholesterol between KO and under FC overload, ATAD3A KO significantly upregulated expression of in controls protein levels were loss of ATAD3A under the between and ATAD3A KO cells was under FC overload ATAD3A KO to expression of liver a in cholesterol protein levels under FC overload and Taken together, ATAD3A KO FC FC accumulation to cholesterol and metabolism under FC overload, suggesting that ATAD3A in cholesterol such as cholesterol we are to ATAD3A KO TG this was to the expression of in Huh7 cells, and fatty acid is the that the of for fatty acid is the for fatty acid J. of in liver S. A. PubMed Scopus Google Scholar). was found that and levels were to the loss of ATAD3A under FC overload, being and the protein was downregulated in ATAD3A KO cells under FC overload, was for and These the free fatty production in the ATAD3A KO cells under FC ATAD3A KO significantly increased TG accumulation under FC overload and in Huh7 cells, suggesting that lipolysis in ATAD3A KO cells TG is in lipid we cells with a used to intracellular in lipid showed an increased of lipid in ATAD3A KO cells to Huh7 cells under and FC overload and a for is a protein with lipid and from of and liver steatosis in mice with in cells lipid accumulation S. M.B. et loss of diet-induced and J. PubMed Scopus Google Scholar, Y. Y. L. Liu Y. Z. oxygen species lipid accumulation in cells by J. 2018; Scopus Google Scholar). expression was significantly upregulated in ATAD3A KO Huh7 cells to under FC overload, the lipolysis in the ATAD3A KO Huh7 cells Moreover, human hepatocytes were isolated from the liver of and to ATAD3A to ATAD3A as the by the treatment for Huh7 FC overload protein ATAD3A by increased expression to a in human hepatocytes Taken together, ATAD3A KO TG accumulation under FC overload as as inhibition of fatty acid as by the downregulation of expression following the loss of ATAD3A KO significantly upregulated and protein expression of the for fatty acid oxidation, and the to protein of of the of under FC overload in Huh7 cells, the of for free fatty and of production plays an essential role in the progression of SS and is by FC overload of in nonalcoholic fatty liver Dis. PubMed Scopus Google Scholar, K. Y. M. J. plays a role in free cholesterol of Lipid 2010; PubMed Scopus Google we the loss of ATAD3A influence under FC shown in FC overload upregulated the expression of protein chain 3-II a for and ATAD3A KO this expression levels of was also significantly increased in ATAD3A KO Huh7 cells to under FC overload and a in p62 protein levels following the loss of ATAD3A and further and p62 protein levels were also in human hepatocytes with ATAD3A by under the treatment as Huh7 ATAD3A upregulated and p62 protein levels in human hepatocytes and were in ATAD3A KO Huh7 cells in under FC overload, as shown in the the upregulation of and accumulation of can to in autophagosome formation of autophagosome degradation. further was by the loss of we cells with a an inhibitor of for cholesterol and shown in levels increased by FC overload in cells 1, and suggesting that FC overload autophagosome the of further increased induced by FC overload in cells 1, and that is under FC overload FC overload and p62 expression in cells under and FC overload and the upregulation of and p62 in ATAD3A KO cells was by suggesting that ATAD3A KO induce autophagosome formation and the the upregulation of in KO cells was in the of under FC overload 1, and suggesting that was and autophagosome was loss of In the of ATAD3A FC accumulation with cholesterol and the by increased and p62 to has been that is a for cells under FC overload and inhibition of cell K. Y. M. J. plays a role in free cholesterol of Lipid 2010; PubMed Scopus Google Scholar). we that loss of ATAD3A cell through of the results showed that cell was significantly in ATAD3A KO Huh7 cells human hepatocytes to under FC overload and Taken together, that ATAD3A KO autophagosome under FC overload and cell survival. ATAD3A is in inner mitochondrial we a of was the loss of ATAD3A under FC shown in the expression of PTEN-induced kinase the for mediated was downregulated the loss of ATAD3A under FC overload and basal suggesting that ATAD3A is essential for the mitophagy and loss of ATAD3A mitophagy. an indicator of mitochondrial was significantly increased the loss of ATAD3A under and FC overload, suggesting accumulation of mitochondria deletion of ATAD3A upregulation of mitochondrial and of outer mitochondrial membrane further the increased mitochondrial in ATAD3A KO cells under FC overload of mitochondrial function a was to the accumulation of mitochondria ATAD3A deletion is to the of results showed that basal respiration and ATP production significantly the loss of ATAD3A under FC overload, a was for suggesting that ATAD3A KO to accumulate mitochondria in cells under FC overload the mitochondrial respiration and ATP production loss of ATAD3A were to the mitochondrial oxidative the expression of oxidative was shown in ATAD3A KO and and were upregulated in ATAD3A KO cells to under and FC the ATP responsible for ATP was downregulated in ATAD3A KO cells to under and FC These that ATAD3A is for the and function of oxidative and loss of ATAD3A ATP production through of the oxidative damaged mitochondrial and mitochondrial in the ATAD3A KO cells under FC overload can also by ATAD3A has been to in cholesterol transport, the function of ATAD3A in NAFLD we show the upregulation of ATAD3A in the progression of NAFLD in livers from and it has been shown that cholesterol homeostasis and FC overload contribute to NAFLD it is to the function of ATAD3A under FC overload (3Arguello G. Balboa E. Arrese M. Zanlungo S. Recent insights on the role of cholesterol in non-alcoholic fatty liver disease.Biochim. Biophys. Acta. 2015; 1852: 1765-1778Crossref PubMed Scopus (211) Google Scholar). We induced FC overload in Huh7 cells by treatment with cholesterol and 58035, an inhibitor of acetyl-CoA the role of ATAD3A under FC overload, we a Huh7 ATAD3A KO cell CRISPR-Cas9 results showed that ATAD3A KO induced FC and TG accumulation under FC overload Moreover, an increased of lipid was in ATAD3A KO cells to Huh7 cells under and FC overload In the of lipid is used to and the disease fats of liver that following the loss of is an in TG levels in liver cells treatment that loss of ATAD3A promote NAFLD In NAFLD steatosis of fat with and steatosis formation of that the can in the liver M. Lipid and liver disease: from to Rev. Gastroenterol. Hepatol. PubMed Scopus Google Scholar). of ATAD3A upregulated and p62 under FC overload in Huh7 cells and human hepatocytes and downregulated suggesting ATAD3A KO under FC overload autophagosome and mitophagy. loss of ATAD3A impaired mitochondrial basal respiration and ATP production under FC overload in Huh7 that is upregulated in cirrhosis M. Oliveira et role for mammalian of in steatohepatitis 2010; Google Scholar) and is by ATAD3A in cells D. Liu L. M. B. Z. et ATAD3A and of cells from the Biol. 2018; PubMed Scopus Google we to upregulation of ATAD3A the progression of NAFLD. results showed that in the NASH and NAFLD human liver ATAD3A levels were In Huh7 cells, we found that the loss of mitochondria and were is with S. S. F. S. N. et controls mitochondrial in and cholesterol Cell 2018; Google Scholar). of mitochondria and the loss of ATAD3A in mitochondrial of mitochondria the impaired mitochondrial basal respiration and ATP production mitochondrial Taken together, that ATAD3A is for the mitochondrial and the loss of ATAD3A induced significantly FC accumulation under FC overload, in cholesterol in cholesterol metabolism showed of ATAD3A significantly upregulated expression under expression under FC overload, was a in expression in ATAD3A KO cells under FC protein levels were downregulated upregulated to contribute to the FC loss of ATAD3A under FC expression was significantly by the loss of ATAD3A under FC overload a was suggesting that ATAD3A cholesterol from cells to the intracellular cholesterol is that ATAD3A levels et protein ATPase AAA 3A with in 2013; 15: PubMed Scopus Google Scholar, Y. E. et mitochondrial inner membrane protein ATAD3A mitochondrial dynamics in PubMed Scopus Google Scholar). In this study, we showed that autophagosome was loss of a the mitophagy was impaired the loss of ATAD3A under and FC overload with the mitochondrial and impaired mitochondrial respiration the accumulation of damaged is impaired with the progression of and in mitophagy has been shown to liver steatosis Y. F. J. Liu J. et mitophagy in the effects of 2018; Scopus Google the of mitophagy for the of Our that ATAD3A is for the mitophagy and loss of ATAD3A the it a for NAFLD. upregulation of the loss of ATAD3A that lipolysis to the TG from lipolysis at the of and is as a for NAFLD. has been shown that loss of in hepatocytes mice from steatosis and largely from of TG and J. et promotes and fatty liver disease in mice through hepatocyte and 2019; PubMed Scopus Google Scholar, M. M. M. S. K. et protein lipid accumulation and lipid formation in Cell Biol. PubMed Google Scholar). of ATAD3A to further relationship with NAFLD. has also been shown to lipid it is TG have shown that of In it has also been proposed that also contribute to the TG by of to for TG in hepatocytes D. lipid homeostasis for the of in J. Cell 2019; PubMed Scopus Google Scholar, A. and of in a Dis. 2020; PubMed Scopus Google Scholar). In this study, FC overload induced and TG stores in hepatocytes. However, ATAD3A KO and TG in the cells, suggesting that also contribute to TG accumulation ATAD3A In the loss of ATAD3A FC and autophagosome and of damaged mitochondria with impaired mitochondrial and induced significantly liver cell with FC overload, the essential role of ATAD3A in NAFLD through the of lipid homeostasis and mitochondrial of is shown in in lipid accumulation the dynamics the progression of NAFLD in between liver cell such as cells, cells, and in are to the in a role for ATAD3A in NAFLD progression and to the that ATAD3A a for NAFLD NAFLD was in with the by was from in the in this by the of of the human liver used was and is in Y. L. L. 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