Publications by authors named "Wen-Xing Ding"

168 Publications

Ripk3 signaling regulates HSCs during stress and represses radiation-induced leukemia in mice.

Stem Cell Reports 2022 Apr 30. Epub 2022 Apr 30.

Department of Cancer Biology, Oncology Research Institute, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL 60153, USA; Department of Radiation Oncology, Loyola University Medical Center, Maywood, IL 60153, USA; Department of Pathology, Loyola University Medical Center, Maywood, IL 60153, USA. Electronic address:

Receptor-interacting protein kinase 3 (Ripk3) is one of the critical mediators of inflammatory cytokine-stimulated signaling. Here we show that Ripk3 signaling selectively regulates both the number and the function of hematopoietic stem cells (HSCs) during stress conditions. Ripk3 signaling is not required for normal homeostatic hematopoiesis. However, in response to serial transplantation, inactivation of Ripk3 signaling prevents stress-induced HSC exhaustion and functional HSC attenuation, while in response to fractionated low doses of ionizing radiation (IR), inactivation of Ripk3 signaling accelerates leukemia/lymphoma development. In both situations, Ripk3 signaling is primarily stimulated by tumor necrosis factor-α. Activated Ripk3 signaling promotes the elimination of HSCs during serial transplantation and pre-leukemia stem cells (pre-LSCs) during fractionated IR by inducing Mlkl-dependent necroptosis. Activated Ripk3 signaling also attenuates HSC functioning and represses a pre-LSC-to-LSC transformation by promoting Mlkl-independent senescence. Furthermore, we demonstrate that Ripk3 signaling induces senescence in HSCs and pre-LSCs by attenuating ISR-mediated mitochondrial quality control.
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http://dx.doi.org/10.1016/j.stemcr.2022.04.009DOI Listing
April 2022

Scramblases as Regulators of Autophagy and Lipid Homeostasis: Implications for NAFLD.

Autophagy Rep 2022 7;1(1):143-160. Epub 2022 Apr 7.

Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160, USA.

Equilibration of phospholipids between the two monolayers of the lipid bilayer of cellular membranes is mediated by scramblases acting as phospholipid shuttling proteins that are critical for cellular function, particularly during inter-organelle contact. Recent work has identified several protein scramblases, including TMEM41B, VMP1 and ATG9 that are critical in autophagy. More recently, ATG9, TMEM41B, and VMP1 have also been discovered to be important regulators of cellular lipid homeostasis. In vivo mouse models involving ablation of TMEM41B in liver have shown that knockout of these proteins can lead to rapid development of non-alcoholic steatohepatitis (NASH) and systemic dyslipidemia, though this has not been explored yet with ATG9. The resulting phenotype is likely due to the combined effects of a severe lipid secretion defect caused by stalled neutral lipids export from the endoplasmic reticulum (ER) membrane bilayer coupled with increased lipogenesis. Here we briefly discuss recent exciting findings on the topic of scramblases in autophagy, their relevance to human non-alcoholic fatty liver disease (NAFLD)/NASH, as well as future directions in this research.
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http://dx.doi.org/10.1080/27694127.2022.2055724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9066413PMC
April 2022

Lack of VMP1 Impairs Hepatic Lipoprotein Secretion and Promotes Nonalcoholic Steatohepatitis.

J Hepatol 2022 Apr 19. Epub 2022 Apr 19.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA. Electronic address:

Background & Aims: Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that regulates the formation of autophagosomes and lipid droplets. Recent evidence suggests that VMP1 plays a critical role in lipoprotein secretion in zebra fish and cultured cells. However, the pathophysiological roles and mechanisms by which VMP1 regulates lipoprotein secretion and lipid accumulation in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are unknown.

Methods: Liver-specific and hepatocyte-specific Vmp1 knockout mice as well as Vmp1 knockin mice were generated by crossing Vmp1 or Vmp1 mice with albumin Cre mice or by injecting AAV8-TBG-cre, respectively. These mice were characterized for lipid and energy metabolism, metabolomics and transcriptome analyses. Hepatic VMP1 overexpressed mice fed on a NASH diet were also characterized.

Results: Hepatocyte-specific deletion of Vmp1 severely impaired VLDL secretion resulting in massive hepatic steatosis, hepatocyte death, inflammation and fibrosis, which are hallmarks of NASH. Mechanistically, loss of Vmp1 led to decreased hepatic levels of phosphatidylcholine and phosphatidylethanolamine as well as the changes of phospholipid composition. Deletion of Vmp1 in mouse liver also led to neutral lipids accumulated in endoplasmic reticulum (ER) bilayer and impaired mitochondrial beta-oxidation. Overexpression of VMP1 ameliorated steatosis in diet-induced NASH by improving VLDL secretion. Importantly, we also show decreased liver VMP1 is associated with NAFLD/NASH in humans.

Conclusions: Our results provide novel insights on the role of VMP1 in regulating hepatic phospholipid synthesis and lipoprotein secretion in the pathogenesis of NAFLD/NASH.

Lay Summary: Lack of hepatic VMP1 in mice decreases levels of phospholipids and causes impaired lipoprotein secretion and fatty acid β oxidation resulting in the development of NASH. Decreased VMP1 is associated with human NAFLD/NASH. Overexpression of VMP1 ameliorates diet-induced impaired VLDL secretion and NAFLD in mice.
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http://dx.doi.org/10.1016/j.jhep.2022.04.010DOI Listing
April 2022

GFP-LC3 High-Content Assay for Screening Autophagy Modulators.

Methods Mol Biol 2022 ;2474:83-89

National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.

Autophagy plays an important role in maintaining cellular homeostasis. Defects in autophagy have been linked to various human diseases, such as cancer, neurodegenerative diseases, and cardiovascular diseases. Therefore, it is useful to develop an assay that can measure the functions of autophagy and also be used to identify autophagy modulators by screening a large number of compounds. This chapter describes a cell-based high content green fluorescent protein (GFP)-LC3 assay using mouse embryonic fibroblasts (MEF) stably expressing GFP-LC3.
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http://dx.doi.org/10.1007/978-1-0716-2213-1_9DOI Listing
March 2022

The role of MLKL in Hepatic Ischemia-Reperfusion Injury of Alcoholic Steatotic Livers.

Int J Biol Sci 2022 1;18(3):1096-1106. Epub 2022 Jan 1.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.

Alcohol-related liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the primary causes of chronic liver disease in western countries. Liver transplantation is currently one of the most efficient approaches to save patients with liver failure, which is often associated with hepatic ischemia-reperfusion (IR) injury. IR injury is exacerbated by hepatic steatosis, yet the mechanism remains elusive. Necroptosis is a form of regulated cell death mediated by receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL) protein, which has been implicated in the pathogenesis of ALD and NAFLD. Though necroptosis plays an important role in IR injury of high fat diet - induced steatotic livers, the role of necroptosis in IR injury of ethanol - induced steototic livers has not been investigated. In the present study, we used chronic plus binge alcohol (Gao-binge) feeding followed by IR surgery to investigate IR liver injury with ethanol-associated steatosis. We found that the levels of key necroptotic proteins MLKL and RIP3 increased in alcohol-fed mouse livers. Moreover, we observed increased liver injury after IR in control diet-fed mice, which was further exacerbated by alcohol feeding based on serum alanine aminotransferase (ALT) levels and TUNEL staining of necrotic cells. Hepatic neutrophil infiltration also increased in alcohol-fed mice after IR surgery. However, deletion of did not protect against IR liver injury in alcohol-fed mice compared with matched wild-type mice. In conclusion, alcoholic steatosis promotes IR injury, which seems to be independent of MLKL-mediated necroptosis.
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http://dx.doi.org/10.7150/ijbs.67533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8771841PMC
April 2022

Acute exercise rapidly activates hepatic mitophagic flux.

J Appl Physiol (1985) 2022 Mar 10;132(3):862-873. Epub 2022 Feb 10.

Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, Missouri.

Exercise is critical for improving metabolic health and putatively maintains or enhances mitochondrial quality control in metabolic tissues. Although previous work has shown that exercise elicits hepatic mitochondrial biogenesis, it is unknown if acute exercise activates hepatic mitophagy, the selective degradation of damaged or low-functioning mitochondria. We tested if an acute bout of treadmill running increased hepatic mitophagic flux both right after and 2-h postexercise in 15- to 24-wk-old C57BL/6J female mice. Acute exercise did not significantly increase markers of autophagic flux, however, mitophagic flux was activated 2-h post-treadmill running as measured by accumulation of both LC3-II and p62 in isolated mitochondria in the presence of leupeptin, an inhibitor of autophagosome degradation. Furthermore, mitochondrial-associated ubiquitin, which recruits the autophagy receptor protein p62, was also significantly increased at 2 h. Further examination via Western blot and proteomics analysis revealed that acute exercise elicits a time-dependent, dynamic activation of mitophagy pathways. Moreover, the results suggest that exercise-induced hepatic mitophagy is likely mediated by both polyubiquitination and receptor-mediated signaling pathways. Overall, we provide evidence that acute exercise activates hepatic mitophagic flux while also revealing specific receptor-mediated proteins by which exercise maintains mitochondrial quality control in the liver. This study provides evidence that acute exercise activates hepatic mitophagic flux and mitochondrial polyubiquitination while additionally revealing specific receptor-mediated proteins by which exercise maintains mitochondrial quality control in the liver.
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http://dx.doi.org/10.1152/japplphysiol.00704.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8934677PMC
March 2022

Dual roles of p62/SQSTM1 in the injury and recovery phases of acetaminophen-induced liver injury in mice.

Acta Pharm Sin B 2021 Dec 16;11(12):3791-3805. Epub 2021 Nov 16.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA.

Acetaminophen (APAP) overdose can induce liver injury and is the most frequent cause of acute liver failure in the United States. We investigated the role of p62/SQSTM1 (referred to as p62) in APAP-induced liver injury (AILI) in mice. We found that the hepatic protein levels of p62 dramatically increased at 24 h after APAP treatment, which was inversely correlated with the hepatic levels of APAP-adducts. APAP also activated mTOR at 24 h, which is associated with increased cell proliferation. In contrast, p62 knockout (KO) mice showed increased hepatic levels of APAP-adducts detected by a specific antibody using Western blot analysis but decreased mTOR activation and cell proliferation with aggravated liver injury at 24 h after APAP treatment. Surprisingly, p62 KO mice recovered from AILI whereas the wild-type mice still sustained liver injury at 48 h. We found increased number of infiltrated macrophages in p62 KO mice that were accompanied with decreased hepatic von Willebrand factor (VWF) and platelet aggregation, which are associated with increased cell proliferation and improved liver injury at 48 h after APAP treatment. Our data indicate that p62 inhibits the late injury phase of AILI by increasing autophagic selective removal of APAP-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation.
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http://dx.doi.org/10.1016/j.apsb.2021.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8727897PMC
December 2021

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls.

Acta Pharm Sin B 2021 Dec 30;11(12):3740-3755. Epub 2021 Sep 30.

Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.
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http://dx.doi.org/10.1016/j.apsb.2021.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8727921PMC
December 2021

SOX9 acts downstream of YAP to decide liver cell fate and tumor types.

J Hepatol 2022 03 17;76(3):503-505. Epub 2021 Dec 17.

Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2021.12.008DOI Listing
March 2022

Bile Acid-Mediated Activation of Brown Fat Protects From Alcohol-Induced Steatosis and Liver Injury in Mice.

Cell Mol Gastroenterol Hepatol 2022 9;13(3):809-826. Epub 2021 Dec 9.

Department of Diabetes Complications and Metabolism, Duarte, California; Graduate School of Biological Science, Beckman Research Institute, City of Hope National Medical Center, Duarte, California. Electronic address:

Background & Aims: Alcohol-associated liver disease (AALD) is one of the most common causes of liver injury and failure. Limited knowledge of the mechanisms underlying AALD impedes the development of efficacious therapies. Bile acid (BA) signaling was shown to participate in the progression of AALD. However, the mechanisms remain poorly understood.

Methods: C57BL/6J wild-type (WT), Takeda G-protein-coupled bile acid receptor 5 (TGR5) knockout (KO) and brown adipose tissue (BAT)-specific TGR5 knockdown mice were subjected to ethanol feeding-induced AALD. Liver samples from alcoholic hepatitis patients were used to examine the BA circulation signaling. Human Embryonic Kidney Cells 293 were used for the TGR5 reporter assay. 23(S)-methyl-lithocholic acid was used as a molecular tool to confirm the regulatory functions of BAT in the AALD mouse model.

Results: Ethanol feeding increased the expression of the thermogenesis genes downstream of TGR5 in BAT of WT, but not TGR5 KO, mice. TGR5 deficiency significantly blocked BAT activity and energy expenditure in mice after ethanol feeding. Alcohol increased serum BA levels in mice and human beings through altering BA transportation, and the altered BAs activated TGR5 signaling to regulate metabolism. Compared with ethanol-fed WT mice, ethanol-fed TGR5 KO mice showed less free fatty acid (FFA) β-oxidation in BAT, leading to higher levels of FFA in the circulation, increased liver uptake of FFAs, and exacerbated AALD. BAT-specific TGR5 knockdown mice showed similar results with TGR5 KO mice in AALD. Agonist treatment significantly activated TGR5 signaling in BAT, increased thermogenesis, reduced serum FFA level, and ameliorated hepatic steatosis and injury in AALD mice, while these effects were lost in TGR5 KO mice.

Conclusions: BA signaling plays a protective role in AALD by enhancing BAT thermogenesis. Targeting TGR5 in BAT may be a promising approach for the treatment of AALD.
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http://dx.doi.org/10.1016/j.jcmgh.2021.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8802063PMC
April 2022

An unexpected tumor suppressor role of SQSTM1/p62 in liver tumorigenesis.

Autophagy 2022 02 6;18(2):459-461. Epub 2021 Dec 6.

Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS USA.

SQSTM1/p62 (sequestosome 1) is a macroautophagy/autophagy receptor protein that is degraded by selective autophagy. Intracellular accumulation of SQSTM1 activates multiple cell survival signaling pathways including NFΚB/NF-κB (nuclear factor kappa B), MTOR (mechanistic target of rapamycin kinase) and NFE2L2/Nrf2 (nuclear factor, erythroid derived 2, like 2). Both SQSTM1 and NFE2L2 have been considered as oncogenic, and increased accumulation of SQSTM1 and NFE2L2 activation have been frequently observed in various cancers including hepatocellular carcinoma. In a recent study, we found that deletion of improved hepatic metabolic reprogramming and cell repopulation resulting in the attenuation of liver injury in mice with liver-specific deletion of and that have defective hepatic autophagy and persistent MTOR complex 1 (MTORC1) activation. To our surprise, hepatocytic deletion of promotes liver tumorigenesis in liver-specific and double-knockout mice. Overall, these findings reveal a complex interplay among autophagy, SQSTM1 and MTORC1 and their differential roles either as oncogenic or tumor suppressor in liver tumorigenesis depending on the disease stage and context.
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http://dx.doi.org/10.1080/15548627.2021.2008693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942560PMC
February 2022

Loss of Hepatic Transcription Factor EB Attenuates Alcohol-Associated Liver Carcinogenesis.

Am J Pathol 2022 01 27;192(1):87-103. Epub 2021 Oct 27.

Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas. Electronic address:

Alcohol is a well-known risk factor for hepatocellular carcinoma. Autophagy plays a dual role in liver cancer, as it suppresses tumor initiation and promotes tumor progression. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy, which is impaired in alcohol-related liver disease. However, the role of TFEB in alcohol-associated liver carcinogenesis is unknown. Liver-specific Tfeb knockout (KO) mice and their matched wild-type (WT) littermates were injected with the carcinogen diethylnitrosamine (DEN), followed by chronic ethanol feeding. The numbers of both total and larger tumors increased significantly in DEN-treated mice fed ethanol diet than in mice fed control diet. Although the number of tumors was not different between WT and L-Tfeb KO mice fed either control or ethanol diet, the number of larger tumors was less in L-Tfeb KO mice than in WT mice. No differences were observed in liver injury, steatosis, inflammation, ductular reaction, fibrosis, and tumor cell proliferation in DEN-treated mice fed ethanol. However, the levels of glypican 3, a marker of malignant hepatocellular carcinoma, markedly decreased in DEN-treated L-Tfeb KO mice fed ethanol in comparison to the WT mice. These findings indicate that chronic ethanol feeding promotes DEN-initiated liver tumor development, which is attenuated by genetic deletion of hepatic TFEB.
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http://dx.doi.org/10.1016/j.ajpath.2021.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8747011PMC
January 2022

Hepatocytic p62 suppresses ductular reaction and tumorigenesis in mouse livers with mTORC1 activation and defective autophagy.

J Hepatol 2022 03 25;76(3):639-651. Epub 2021 Oct 25.

Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160, USA. Electronic address:

Background & Aims: Either activation of mTORC1 due to loss of Tsc1 (tuberous sclerosis complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms by which autophagy contributes to the hepatic metabolic changes and tumorigenesis mediated by mTORC1 activation.

Methods: Atg5 Flox/Flox (Atg5) and Tsc1 mice were crossed with albumin-Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1 (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1-Nrf2 triple KO mice. These mice were housed for various periods up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis but increased mortality and was accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L-Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity were also observed in a subset of human hepatocellular carcinomas.

Conclusions: These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy.

Lay Summary: Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy and increased mTOR activation. Using multiple genetically engineered mouse models of defective hepatic autophagy and persistent mTOR activation, we dissected the complex mechanisms behind this observation. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1, which regulated liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.
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http://dx.doi.org/10.1016/j.jhep.2021.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8858859PMC
March 2022

Loss of acinar cell VMP1 triggers spontaneous pancreatitis in mice.

Autophagy 2021 Oct 28:1-11. Epub 2021 Oct 28.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA.

The pathogenesis of pancreatitis has been linked to disruption of organelle homeostasis including macroautophagy/autophagy dysfunction and endoplasmic reticulum (ER) stress. However, the direct impact of aberrant organelle function on pancreatitis initiation and progression is largely unknown. Recently an ER membrane protein, VMP1 (vacuole membrane protein 1), has been reported to play a crucial role in autophagosome formation. Notably, we found that VMP1 is downregulated in both human chronic pancreatitis (CP) and experimental mouse acute pancreatitis (AP). Pancreatic acinar cell-specific deletion promotes inflammation, acinar-to-ductal metaplasia, and fibrosis in mice, sharing histological similarities with human CP. Mechanistically, loss of pancreatic VMP1 leads to defective autophagic degradation and ER stress as well as activation of the NFE2L2/Nrf2 pathway. Genetic ablation of NFE2L2 attenuated pancreatitis in VMP1-deficient mice. Our data highlight the importance of VMP1 in modulating an integrated organelle stress response and its functional role in maintaining pancreas homeostasis in the context of CP. AMY: amylase; ADM: acinar-to-ductal metaplasia; AP: acute pancreatitis; CASP3: caspase 3; CP: chronic pancreatitis; DDIT3/CHOP: DNA damage inducible transcript 3; DKO, double knockout; ER: endoplasmic reticulum; GCLC: glutamate-cysteine ligase catalytic subunit; GCLM: glutamate-cysteine ligase modifier subunit; HSPA5/BIP: heat shock protein family A (Hsp70) member 5; KO: knockout; KRT19/CK19: keratin 19; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MPO: myeloperoxidase; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; ND: normal donor; NQO1: NAD(P)H quinone dehydrogenase 1; PCNA: proliferating cell nuclear antigen; RIPA: radio-immunoprecipitation; SQSTM1/p62: sequestosome 1; SOX9: SRY-box transcription factor 9; TAP: trypsinogen activation peptide; TFEB: transcription factor EB; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; UB: ubiquitin; VMP1: vacuole membrane protein 1; XBP1: X-box binding protein 1; YAP1, Yes1 associated transcriptional regulator; ZG: zymogen granule.
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http://dx.doi.org/10.1080/15548627.2021.1990672DOI Listing
October 2021

Perspectives on Mitochondria-ER and Mitochondria-Lipid Droplet Contact in Hepatocytes and Hepatic Lipid Metabolism.

Cells 2021 09 1;10(9). Epub 2021 Sep 1.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA.

Emerging evidence suggests that mitochondrion-endoplasmic reticulum (ER) and mitochondrion-lipid droplet (LD) contact sites are critical in regulating lipid metabolism in cells. It is well established that intracellular organelles communicate with each other continuously through membrane contact sites to maintain organelle function and cellular homeostasis. The accumulation of LDs in hepatocytes is an early indicator of non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ALD), which may indicate a breakdown in proper inter-organelle communication. In this review, we discuss previous findings in mitochondrion-ER and mitochondrion-LD contact, focusing on their roles in lipid metabolism in hepatocytes. We also present evidence of a unique mitochondrion-LD contact structure in hepatocytes under various physiological and pathological conditions and propose a working hypothesis to speculate about the role of these structures in regulating the functions of mitochondria and LDs and their implications in NAFLD and ALD.
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http://dx.doi.org/10.3390/cells10092273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8472694PMC
September 2021

Liver-specific deletion of mechanistic target of rapamycin does not protect against acetaminophen-induced liver injury in mice.

Liver Res 2021 Jun 19;5(2):79-87. Epub 2021 Mar 19.

Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS, USA.

Background: Acetaminophen (APAP) overdose can cause liver injury and liver failure, which is one of the most common causes of drug-induced liver injury in the United States. Pharmacological activation of autophagy by inhibiting mechanistic target of rapamycin (mTOR) protects against APAP-induced liver injury likely via autophagic removal of APAP-adducts and damaged mitochondria. In the present study, we aimed to investigate the role of genetic ablation of mTOR pathways in mouse liver in APAP-induced liver injury and liver repair/regeneration.

Methods: Albumin-Cre (Alb-Cre) mice, mTOR and Raptor mice (C57BL/6J background) were crossbred to produce liver-specific mTOR knockout (L-mTOR KO, Alb Cre+/-, mTOR) and liver-specific Raptor KO (L-Raptor, Alb Cre+/-, Raptor ) mice. Alb-Cre littermates were used as wild-type (WT) mice. These mice were treated with APAP for various time points for up to 48 h. Liver injury, cell proliferation, autophagy and mTOR activation were determined.

Results: We found that genetic deletion of neither Raptor, an important adaptor protein in mTOR complex 1, nor mTOR, in the mouse liver significantly protected against APAP-induced liver injury despite increased hepatic autophagic flux. Genetic deletion of Raptor or mTOR in mouse livers did not affect APAP metabolism and APAP-induced c-Jun N-terminal kinase (JNK) activation, but slightly improved mouse survival likely due to increased hepatocyte proliferation.

Conclusions: Our results indicate that genetic ablation of mTOR in mouse livers does not protect against APAP-induced liver injury but may slightly improve liver regeneration and mouse survival after APAP overdose.
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http://dx.doi.org/10.1016/j.livres.2021.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425470PMC
June 2021

Trehalose activates hepatic transcription factor EB (TFEB) but fails to ameliorate alcohol-impaired TFEB and liver injury in mice.

Alcohol Clin Exp Res 2021 10 5;45(10):1950-1964. Epub 2021 Sep 5.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, USA.

Background: Recent evidence demonstrates that alcohol activates the mechanistic target of rapamycin (mTOR) and impairs hepatic transcription factor EB (TFEB) reducing autophagy and contributing to alcohol-induced liver injury. Trehalose, a disaccharide, activates TFEB and protects against diet-induced nonalcoholic fatty liver disease in mice. The aim of the present study was to investigate whether trehalose would reverse the impairment of TFEB induced by alcohol and protect against alcohol-induced liver injury.

Methods: Male C57BL/6J mice were subjected to chronic-plus-binge (Gao-binge) alcohol feeding with and without trehalose supplementation. Some mice were also administrered Alda-1, an aldehyde dehydrogenase 2 agonist.

Results: We found that Alda-1 did not affect Gao-binge alcohol-induced mTOR activation and impaired TFEB in mouse livers. Trehalose increased TFEB nuclear translocation, elevated levels of LC3-II and lysosomal proteins in mouse livers and cultured AML12 cells, confirming the activation of TFEB by trehalose. However, trehalose did not improve the impairment in TFEB induced by Gao-binge alcohol. Both Alda-1 and trehalose failed to protect against Gao-binge alcohol-induced steatosis and liver injury, based on the serum levels of alanine aminotransferase (ALT), histological analysis, and levels of hepatic triglyceride. Interestingly, trehalose increased expression of pro-inflammatory genes in mouse macrophage RAW264.7 cells and slightly increased the infiltration of hepatic neutrophils and inflammatory cytokine gene expression in Gao-binge alcohol-fed mice livers.

Conclusions: Trehalose fails to improve the impaired TFEB induced by Gao-binge alcohol and does not protect against alcohol-induced liver injury.
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http://dx.doi.org/10.1111/acer.14695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8602756PMC
October 2021

A fluorescence imaging based-assay to monitor mitophagy in cultured hepatocytes and mouse liver.

Liver Res 2021 Mar 28;5(1):16-20. Epub 2021 Jan 28.

Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS, USA.

Background And Aim: Mitophagy is a lysosomal degradation pathway that selectively removes damaged, aged and dysfunctional mitochondria. Recent advances in understanding mitophagy highlight its importance in various physiological and pathological conditions including liver diseases. However, reliable quantitative assays to monitor mitophagy in cultured cells and in tissues are still scarce.

Methods: We describe a detailed protocol for monitoring mitophagy in primary cultured hepatocytes and mouse livers using cytochrome C oxidase subunit 8 (Cox8)-enhanced green fluorescent protein (EGFP)-mCherry, a dual color fluorescence based-imaging method.

Results: Mitochondria are visualized in yellow fluorescence due to the merged EGFP and mCherry signal. In contrast, autolysosome enclosed mitochondria are shown as red puncta due to quenching of EGFP green fluorescence in acidic compartments. Quantifying the number of red-only puncta in each cell can obtain a quantitative measure for mitophagy.

Conclusions: Cox8-EGFP-mCherry assay can specifically target to mitochondria and be used to monitor mitophagy and .
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http://dx.doi.org/10.1016/j.livres.2020.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8330398PMC
March 2021

Autophagy in liver diseases: A review.

Mol Aspects Med 2021 Dec 11;82:100973. Epub 2021 Jun 11.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS, 66160, USA. Electronic address:

The liver is a highly dynamic metabolic organ that plays critical roles in plasma protein synthesis, gluconeogenesis and glycogen storage, cholesterol metabolism and bile acid synthesis as well as drug/xenobiotic metabolism and detoxification. Research from the past decades indicate that autophagy, the cellular catabolic process mediated by lysosomes, plays an important role in maintaining cellular and metabolic homeostasis in the liver. Hepatic autophagy fluctuates with hormonal cues and the availability of nutrients that respond to fed and fasting states as well as circadian activities. Dysfunction of autophagy in liver parenchymal and non-parenchymal cells can lead to various liver diseases including non-alcoholic fatty liver diseases, alcohol associated liver disease, drug-induced liver injury, cholestasis, viral hepatitis and hepatocellular carcinoma. Therefore, targeting autophagy may be a potential strategy for treating these various liver diseases. In this review, we will discuss the current progress on the understanding of autophagy in liver physiology. We will also discuss several forms of selective autophagy in the liver and the molecular signaling pathways in regulating autophagy of different cell types and their implications in various liver diseases.
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http://dx.doi.org/10.1016/j.mam.2021.100973DOI Listing
December 2021

Combined ASBT Inhibitor and FGF15 Treatment Improves Therapeutic Efficacy in Experimental Nonalcoholic Steatohepatitis.

Cell Mol Gastroenterol Hepatol 2021 7;12(3):1001-1019. Epub 2021 May 7.

Harold Hamm Diabetes Center, Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. Electronic address:

Background & Aims: Pharmacologic agents targeting bile acid signaling show promise for treating nonalcoholic steatohepatitis (NASH). However, clinical findings suggest that new treatment strategies with enhanced therapeutic efficacy and minimized undesired effects are needed. This preclinical study investigates whether combining an apical sodium-bile acid transporter (ASBT) inhibitor GSK233072 (GSK672) and fibroblast growth factor-15 (FGF15) signaling activation improves anti-NASH efficacy.

Methods: Mice with high fat, cholesterol, and fructose (HFCFr) diet-induced NASH and stage 2 fibrosis are used as a NASH model. GSK672 or AAV8-TBG-FGF15 interventions are administered alone or in combination to HFCFr diet-fed mice.

Results: The combined treatment significantly enhances therapeutic efficacy against steatosis, inflammation, ballooning, and fibrosis than either single treatment. Mechanistically, the synergistic actions of GSK672 and FGF15 on inhibiting gut bile acid reuptake and hepatic bile acid synthesis achieve greater magnitude of bile acid pool reduction that not only decreases bile acid burden in NASH livers but also limits intestinal lipid absorption, which, together with FGF15 signaling activation, produces weight loss, reduction of adipose inflammation, and attenuated hepatocellular organelle stress. Furthermore, the combined treatment attenuates increased fecal bile acid excretion and repressed bile acid synthesis, which underlie diarrhea and hypercholesterolemia associated with ASBT inhibition and FGF19 analogue, respectively, in clinical settings.

Conclusions: Concomitant ASBT inhibition and FGF15 signaling activation produce metabolic changes that partially mimic the bariatric surgery condition whereby lipid malabsorption and increased FGF15/19 signaling synergistically mediate weight loss and metabolic improvement. Further clinical studies may be warranted to investigate whether combining ASBT inhibitor and FGF19 analogue enhances anti-NASH efficacy and reduced treatment-associated adverse events in humans.
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http://dx.doi.org/10.1016/j.jcmgh.2021.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346663PMC
March 2022

Gut-restricted apical sodium-dependent bile acid transporter inhibitor attenuates alcohol-induced liver steatosis and injury in mice.

Alcohol Clin Exp Res 2021 06 11;45(6):1188-1199. Epub 2021 May 11.

Harold Hamm Diabetes Center, Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

Background: Recent studies have shown that human and experimental alcohol-related liver disease (ALD) is robustly associated with dysregulation of bile acid homeostasis, which may in turn modulate disease severity. Pharmacological agents targeting bile acid metabolism and signaling may be potential therapeutics for ALD.

Methods: The potential beneficial effects of a gut-restricted apical sodium-dependent bile acid transporter (ASBT) inhibitor were studied in a chronic-plus-binge ALD mouse model.

Results: Blocking intestinal bile acid reabsorption by the gut-restricted ASBT inhibitor GSK2330672 attenuated hepatic steatosis and liver injury in a chronic-plus-binge ALD mouse model. Alcohol feeding is associated with intestinal bile acid accumulation but paradoxically impaired ileal farnesoid × receptor (FXR) function, and repressed hepatic cholesterol 7α-hydrolase (CYP7A1) expression despite decreased hepatic small heterodimer partner (SHP) and ileal fibroblast growth factor 15 (FGF15) expression. ASBT inhibitor treatment decreased intestinal bile acid accumulation and increased hepatic CYP7A1 expression, but further decreased ileal FXR activity. Alcohol feeding induces serum bile acid concentration that strongly correlates with a liver injury marker. However, alcohol-induced serum bile acid elevation is not due to intrahepatic bile acid accumulation but is strongly and positively associated with hepatic multidrug resistance-associated protein 3 (MRP4) and MRP4 induction but poorly associated with sodium-taurocholate cotransporting peptide (NTCP) expression. ASBT inhibitor treatment decreases serum bile acid concentration without affecting hepatocyte basolateral bile acid uptake and efflux transporters.

Conclusion: ASBT inhibitor treatment corrects alcohol-induced bile acid dysregulation and attenuates liver injury in experimental ALD.
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http://dx.doi.org/10.1111/acer.14619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8717856PMC
June 2021

New Glance at the Role of TM6SF2 in Lipid Metabolism and Liver Cancer.

Hepatology 2021 09 19;74(3):1141-1144. Epub 2021 Jun 19.

Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS.

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http://dx.doi.org/10.1002/hep.31851DOI Listing
September 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Heat Treatment Improves Hepatic Mitochondrial Respiratory Efficiency via Mitochondrial Remodeling.

Function (Oxf) 2021 22;2(2):zqab001. Epub 2021 Jan 22.

Molecular and Integrative Physiology, The University of Kansas Medical Center, Kansas City, KS 66160, USA.

Nonacholic fatty liver disease, or hepatic steatosis, is the most common liver disorder affecting the western world and currently has no pharmacologic cure. Thus, many investigations have focused on alternative strategies to treat or prevent hepatic steatosis. Our laboratory has shown that chronic heat treatment (HT) mitigates glucose intolerance, insulin resistance, and hepatic steatosis in rodent models of obesity. Here, we investigate the direct bioenergetic mechanism(s) surrounding the metabolic effects of HT on hepatic mitochondria. Utilizing mitochondrial proteomics and respiratory function assays, we show that one bout of acute HT (42°C for 20 min) in male C57Bl/6J mice ( = 6/group) triggers a hepatic mitochondrial heat shock response resulting in acute reductions in respiratory capacity, degradation of key mitochondrial enzymes, and induction of mitophagy via mitochondrial ubiquitination. We also show that chronic bouts of HT and recurrent activation of the heat shock response enhances mitochondrial quality and respiratory function via compensatory adaptations in mitochondrial organization, gene expression, and transport even during 4 weeks of high-fat feeding ( = 6/group). Finally, utilizing a liver-specific heat shock protein 72 (HSP72) knockout model, we are the first to show that HSP72, a protein putatively driving the HT metabolic response, does not play a significant role in the hepatic mitochondrial adaptation to acute or chronic HT. However, HSP72 is required for the reductions in blood glucose observed with chronic HT. Our data are the first to suggest that chronic HT (1) improves hepatic mitochondrial respiratory efficiency via mitochondrial remodeling and (2) reduces blood glucose in a hepatic HSP72-dependent manner.
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http://dx.doi.org/10.1093/function/zqab001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886620PMC
January 2021

The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway.

Cell Mol Gastroenterol Hepatol 2021 19;11(5):1369-1385. Epub 2021 Jan 19.

Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey; Cancer Metabolism and Growth Program, Rutgers Cancer Institute of New Jersey, New Brunswick, New Jersey. Electronic address:

Background And Aims: TRIM21 is a ubiquitin E3 ligase that is implicated in numerous biological processes including immune response, cell metabolism, redox homeostasis, and cancer development. We recently reported that TRIM21 can negatively regulate the p62-Keap1-Nrf2 antioxidant pathway by ubiquitylating p62 and prevents its oligomerization and protein sequestration function. As redox homeostasis plays a pivotal role in many cancers including liver cancer, we sought to determine the role of TRIM21 in hepatocarcinogenesis.

Methods: We examined the correlation between TRIM21 expression and the disease using publicly available data sets and 49 cases of HCC clinical samples. We used TRIM21 genetic knockout mice to determine how TRIM21 ablation impact HCC induced by the carcinogen DEN plus phenobarbital (PB). We explored the mechanism that loss of TRIM21 protects cells from DEN-induced oxidative damage and cell death.

Results: There is a positive correlation between TRIM21 expression and HCC. Consistently, TRIM21-knockout mice are resistant to DEN-induced hepatocarcinogenesis. This is accompanied by decreased cell death and tissue damage upon DEN treatment, hence reduced hepatic tissue repair response and compensatory proliferation. Cells deficient in TRIM21 display enhanced p62 sequestration of Keap1 and are protected from DEN-induced ROS induction and cell death. Reconstitution of wild-type but not the E3 ligase-dead and the p62 binding-deficient mutant TRIM21 impedes the protection from DEN-induced oxidative damage and cell death in TRIM21-deficient cells.

Conclusions: Increased TRIM21 expression is associated with human HCC. Genetic ablation of TRIM21 leads to protection against oxidative hepatic damage and decreased hepatocarcinogenesis, suggesting TRIM21 as a preventive and therapeutic target.
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http://dx.doi.org/10.1016/j.jcmgh.2021.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024979PMC
March 2022

Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice.

Arch Toxicol 2021 04 17;95(4):1463-1473. Epub 2021 Jan 17.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, MS 1018, Kansas City, KS, 66160, USA.

Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75 mg/kg and 150 mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150 mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75 mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.
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http://dx.doi.org/10.1007/s00204-021-02985-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035234PMC
April 2021

S100A11 Overexpression Promotes Fatty Liver Diseases via Increased Autophagy?

Cell Mol Gastroenterol Hepatol 2021 5;11(3):885-886. Epub 2020 Dec 5.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas. Electronic address:

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http://dx.doi.org/10.1016/j.jcmgh.2020.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900834PMC
July 2021

Autophagy and liver cancer.

Clin Mol Hepatol 2020 10 1;26(4):606-617. Epub 2020 Oct 1.

Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA.

Autophagy is a highly conserved catabolic process that degrades cytosolic proteins and organelles via formation of autophagosomes that fuse with lysosomes to form autolysosomes, whereby autophagic cargos are degraded. Numerous studies have demonstrated that autophagy plays a critical role in the regulation of liver physiology and homeostasis, and impaired autophagy leads to the pathogenesis of various liver diseases such as viral hepatitis, alcohol associated liver diseases (AALD), non-alcoholic fatty liver diseases (NAFLD), and liver cancer. Recent evidence indicates that autophagy may play a dual role in liver cancer: inhibiting early tumor initiation while promoting progression and malignancy of already formed liver tumors. In this review, we summarized the progress of current understanding of how hepatic viral infection, alcohol consumption and diet-induced fatty liver diseases impair hepatic autophagy. We also discussed how impaired autophagy promotes liver tumorigenesis, and paradoxically how autophagy is required to promote the malignancy and progression of liver cancer. Understanding the molecular mechanisms underlying how autophagy differentially affects liver cancer development and progression may help to design better therapeutic strategies for prevention and treatment of liver cancer.
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http://dx.doi.org/10.3350/cmh.2020.0169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641568PMC
October 2020

An FGF15/19-TFEB regulatory loop controls hepatic cholesterol and bile acid homeostasis.

Nat Commun 2020 07 17;11(1):3612. Epub 2020 Jul 17.

Harold Hamm Diabetes Center, Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.

Bile acid synthesis plays a key role in regulating whole body cholesterol homeostasis. Transcriptional factor EB (TFEB) is a nutrient and stress-sensing transcriptional factor that promotes lysosomal biogenesis. Here we report a role of TFEB in regulating hepatic bile acid synthesis. We show that TFEB induces cholesterol 7α-hydroxylase (CYP7A1) in human hepatocytes and mouse livers and prevents hepatic cholesterol accumulation and hypercholesterolemia in Western diet-fed mice. Furthermore, we find that cholesterol-induced lysosomal stress feed-forward activates TFEB via promoting TFEB nuclear translocation, while bile acid-induced fibroblast growth factor 19 (FGF19), acting via mTOR/ERK signaling and TFEB phosphorylation, feedback inhibits TFEB nuclear translocation in hepatocytes. Consistently, blocking intestinal bile acid uptake by an apical sodium-bile acid transporter (ASBT) inhibitor decreases ileal FGF15, enhances hepatic TFEB nuclear localization and improves cholesterol homeostasis in Western diet-fed mice. This study has identified a TFEB-mediated gut-liver signaling axis that regulates hepatic cholesterol and bile acid homeostasis.
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http://dx.doi.org/10.1038/s41467-020-17363-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368063PMC
July 2020

Kupffer cells promote T-cell hepatitis by producing CXCL10 and limiting liver sinusoidal endothelial cell permeability.

Theranostics 2020 1;10(16):7163-7177. Epub 2020 Jun 1.

Division of Comparative Pathology, Tulane National Primate Research Center, Covington, LA 70433, USA.

Kupffer cells (KCs) play a crucial role in liver immune homeostasis through interacting with other immune cells and liver sinusoidal endothelial cells (LSECs). However, how KCs exactly interact with these cells for maintaining the homeostasis still require the further investigation. CXCL10 is a chemokine that has been implicated in chemoattraction of monocytes, T cells, NK cells, and dendritic cells, and promotion of T cell adhesion to endothelial cells. Although CXCL10 is also known to participate in the pathogenesis of hepatic inflammation, the degree to which it is functionally involved in the crosstalk between immune cells and regulation of immune response is still unclear. To dynamically investigate the function of KCs, we used our recently developed rapid cell ablation model, intermedilysin (ILY)/human CD59 (hCD59)-mediated cell ablation tool, to selectively ablate KC pool under normal condition or concanavalin A (Con A)- induced hepatitis. At certain time points after KCs ablation, we performed flow cytometry to monitor the amount of hepatic infiltrating immune cells. mRNA array was used to detect the change of hepatic cytokines and chemokines levels. Cytokines and chemokines in the serum were further measured by LEGENDplex mouse proinflammatory chemokine panel and inflammation panel. Evans blue staining and transmission electron microscopy were used to investigate the interaction between KCs and LSECs in steady condition. CXCL10 neutralizing antibody and CXCL10 deficient mouse were used to study the role of CXCL10 in immune cell migration and pathogenesis of Con A-induced hepatitis. At steady state, elimination of KCs results in a reduction of hepatic infiltrating monocytes, T, B, and NK cells and a list of cytokines and chemokines at transcriptional level. In the meantime, the depletion of KCs resulted in increased sinusoidal vascular permeability. In the pathological condition, the KCs elimination rescues Con A-induced acute hepatitis through suppressing proinflammatory immune responses by down-regulation of hepatitis-associated cytokines/chemokines in serum such as CXCL10, and recruitment of infiltrating immune cells (monocytes, T, B, and NK cells). We further documented that deficiency or blockade of CXCL10 attenuated the development of Con A-induced hepatitis associated with reduction of the infiltrating monocytes, especially inflammatory Ly6C monocytes. This study supports the notion that KCs actively interact with immune cells and LSECs for maintaining immune response and liver homeostasis. Our data indicate that the interplay between KCs and infiltrated monocytes via CXCL10 contribute to Con A-induced hepatitis.
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http://dx.doi.org/10.7150/thno.44960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330839PMC
May 2021
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