Metabolism of a Selective Serotonin and Norepinephrine Reuptake Inhibitor Duloxetine in Liver Microsomes and Mice.

Duloxetine (DLX) is a dual serotonin and norepinephrine reuptake inhibitor, widely used for the treatment of major depressive disorder. Although DLX has shown good efficacy and safety, serious adverse effects (e.g., liver injury) have been reported. The mechanisms associated with DLX-induced toxicity remain elusive. Drug metabolism plays critical roles in drug safety and efficacy. However, the metabolic profile of DLX in mice is not available, although mice serve as commonly used animal models for mechanistic studies of drug-induced adverse effects. Our study revealed 39 DLX metabolites in human/mouse liver microsomes and mice. Of note, 13 metabolites are novel, including five N-acetyl cysteine adducts and one reduced glutathione (GSH) adduct associated with DLX. Additionally, the species differences of certain metabolites were observed between human and mouse liver microsomes. CYP1A2 and CYP2D6 are primary enzymes responsible for the formation of DLX metabolites in liver microsomes, including DLX-GSH adducts. In summary, a total of 39 DLX metabolites were identified, and species differences were noticed in vitro. The roles of CYP450s in DLX metabolite formation were also verified using human recombinant cytochrome P450 (P450) enzymes and corresponding chemical inhibitors. Further studies are warranted to address the exact role of DLX metabolism in its adverse effects in vitro (e.g., human primary hepatocytes) and in vivo (e.g., Cyp1a2-null mice). SIGNIFICANCE STATEMENT: This current study systematically investigated Duloxetine (DLX) metabolism and bioactivation in liver microsomes and mice. This study provided a global view of DLX metabolism and bioactivation in liver microsomes and mice, which are very valuable to further elucidate the mechanistic study of DLX-related adverse effects and drug-drug interaction from metabolic aspects.

Improving agreement of ASA physical status class between pre-anesthesia screening and day of surgery by adding institutional-specific and ASA-approved examples: a quality improvement project.

BACKGROUND: A successful anesthesia pre-assessment clinic needs to identify patients who need further testing, evaluation, and optimization prior to the day of surgery to avoid delays and cancelations. Although the ASA Physical Status Classification system (ASA PS) has been used widely for over 50 years, it has poor interrater agreement when only using the definitions. In 2014, ASA-approved examples for each ASA physical status class (ASA PS). In this quality improvement study, we developed and evaluated the effectiveness of institutional-specific examples on interrater reliability between anesthesia pre-anesthesia clinic (APAC) and the day of surgery evaluation (DOS). METHODS: A multi-step, multi-year quality improvement project was performed. Step 1, pre-intervention, was a retrospective review to determine the percentage agreement of ASA PS assignment between APAC and DOS for adult and pediatric patients. Step 2 was a retrospective review of the step 1 cases where the ASA PS assignment differed to determine which medical conditions were valued differently and then develop institutional-specific examples for medical conditions not addressed by ASA-approved examples. Step 3 was to educate clinicians about the newly implemented examples and how they should be used as a guide. Step 4, post-intervention, was a retrospective review to determine if the examples improved agreement between APAC and DOS ASA PS assignments. Weighted Kappa coefficient was used to measure of interrater agreement excluding chance agreement. RESULTS: Having only ASA PS definitions available, APAC and DOS agreement was only 74% for adults (n = 737) and 63% for pediatric patients (n = 216). For adults, 20 medical co-morbidity categories and, for pediatric patients, 9 medical co-morbidity categories accounted for > 90% the differences in ASA PS. After development and implementation of institutional-specific examples with ASA-approved examples, the percentage agreement increased for adult patients (n = 795) to 91% and for pediatric patients (n = 239) to 84%. Weighted Kappa coefficients increased significantly for all patients (from 0.62 to 0.85, p < .0001), adult patients (from 0.62 to 0.86, p < .0001), and pediatric patients (from 0.48 to 0.78, p < .0001). CONCLUSIONS: ASA-approved examples do not address all medical conditions that account for differences in the assignment of ASA PS between pre-anesthesia screening and day of anesthesia evaluation at our institution. The process of developing institutional-specific examples addressed the medical conditions that caused differences in assignment at one institution. The implementation of ASA PS examples improved consistency of assignment, and therefore communication of medical conditions of patients presenting for anesthesia care.

Efficacy of motivational-interviewing and guided opioid tapering support for patients undergoing orthopedic surgery (MI-Opioid Taper): A prospective, assessor-blind, randomized controlled pilot trial.

BACKGROUND: Postoperative opioid use can lead to chronic use and misuse. Few studies have examined effective approaches to taper postoperative opioid use while maintaining adequate analgesia. METHODS: This randomized, assessor-blinded, pilot trial of postoperative motivational interviewing and guided opioid tapering support (MI-Opioid Taper) added to usual care (UC) enrolled patients undergoing total hip or knee arthroplasty at a single U.S. academic medical center. MI-Opioid Taper involved weekly (to seven weeks) and monthly (to one year) phone calls until patient-reported opioid cessation. Opioid tapering involved 25% weekly dose reductions. The primary feasibility outcome was study completion in the group to which participants were randomized. The primary efficacy outcome, time to baseline opioid use, was the first of five consecutive days of return to baseline preoperative dose. Intention-to-treat analysis with Cox proportional hazards regression was adjusted for operation. ClinicalTrials.gov registration: NCT02070003. FINDINGS: From November 26, 2014, to April 27, 2018, 209 patients were screened, and 104 patients were assigned to receive MI-Opioid Taper (49 patients) or UC only (55 patients). Study completion after randomization was similar between groups (96.4%, 53 patients receiving UC, 91.8%, 45 patients receiving MI-Opioid Taper). Patients receiving MI-Opioid Taper had a 62% increase in the rate of return to baseline opioid use after surgery (HR 1.62; 95%CI 1.06-2.46; p = 0•03). No trial-related adverse events occurred. INTERPRETATION: In patients undergoing total joint arthroplasty, MI-Opioid Taper is feasible and future research is needed to establish the efficacy of MI-Opioid Taper to promote postoperative opioid cessation. FUNDING: National Institute on Drug Abuse.

Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions.

Measurement of the electron transfer cascade (ETC) enzyme activities and their kinetic profiles is important in assessing mitochondrial function in the nervous system in health and disease or following exposure to toxic agents. The optimization of enzymatic assays for brain tissues and neurons is critical to the development of high-throughput assay formats. This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perform these assays in under 3 hr. © 2019 by John Wiley & Sons, Inc.

Double deletion of PINK1 and Parkin impairs hepatic mitophagy and exacerbates acetaminophen-induced liver injury in mice.

Mitochondria damage plays a critical role in acetaminophen (APAP)-induced necrosis and liver injury. Cells can adapt and protect themselves by removing damaged mitochondria via mitophagy. PINK1-Parkin pathway is one of the major pathways that regulate mitophagy but its role in APAP-induced liver injury is still elusive. We investigated the role of PINK1-Parkin pathway in hepatocyte mitophagy in APAP-induced liver injury in mice. Wild-type (WT), PINK1 knockout (KO), Parkin KO, and PINK1 and Parkin double KO (DKO) mice were treated with APAP for different time points. Liver injury was determined by measuring serum alanine aminotransferase (ALT) activity, H&E staining as well as TUNEL staining of liver tissues. Tandem fluorescent-tagged inner mitochondrial membrane protein Cox8 (Cox8-GFP-mCherry) can be used to monitor mitophagy based on different pH stability of GFP and mCherry fluorescent proteins. We overexpressed Cox8-GFP-mCherry in mouse livers via tail vein injection of an adenovirus Cox8-GFP-mCherry. Mitophagy was assessed by confocal microscopy for Cox8-GFP-mCherry puncta, electron microscopy (EM) analysis for mitophagosomes and western blot analysis for mitochondrial proteins. Parkin KO and PINK1 KO mice improved the survival after treatment with APAP although the serum levels of ALT were not significantly different among PINK1 KO, Parkin KO and WT mice. We only found mild defects of mitophagy in PINK1 KO or Parkin KO mice after APAP, and improved survival in PINK1 KO and Parkin KO mice could be due to other functions of PINK1 and Parkin independent of mitophagy. In contrast, APAP-induced mitophagy was significantly impaired in PINK1-Parkin DKO mice. PINK1-Parkin DKO mice had further elevated serum levels of ALT and increased mortality after APAP administration. In conclusion, our results demonstrated that PINK1-Parkin signaling pathway plays a critical role in APAP-induced mitophagy and liver injury.

Alternol eliminates excessive ATP production by disturbing Krebs cycle in prostate cancer.

BACKGROUND: Alternol is a natural compound isolated from fermentation products of a mutant fungus. Our previous studies demonstrated that Alternol specifically kills cancer cells but spares benign cells. METHODS: To investigate the mechanism underlying alternol-induced cancer cell-specific killing effect, we took a comprehensive strategy to identify Alternol's protein targets in prostate cancer cells, including PC-3, C4-2, and 22RV1, plus benign BPH1 cell lines. Major experimental techniques included biotin-streptavidin pulldown assay coupled with mass-spectrometry, in vitro enzyme activity assay for Krebs cycle enzymes and gas chromatography-mass spectrometry (GC-MS) for metabolomic analysis. RESULTS: Among 14 verified protein targets, four were Krebs cycle enzymes, fumarate hydratase (FH), malate dehydrogenase-2 (MDH2), dihydrolipoamide acetyltransferase (DLAT) in pyruvate dehydrogenase complex (PDHC) and dihydrolipoamide S-succinyltransferase (DLST) in a-ketoglutarate dehydrogenase complex (KGDHC). Functional assays revealed that PDHC and KGDHC activities at the basal level were significantly higher in prostate cancer cells compared to benign prostate BPH1 cells, while alternol treatment reduced their activities in cancer cells close to the levels in BPH1 cells. Although FH and MDH2 activities were comparable among prostate cancer and benign cell lines at the basal level, Alternol treatment largely increased their activities in cancer cells. Metabolomic analysis revealed that Alternol treatment remarkably reduced the levels of malic acid, fumaric acid, and isocitric acid and mitochondrial respiration in prostate cancer cells. Alternol also drastically reduced mitochondrial respiration and ATP production in PC-3 cells in vitro or in xenograft tissues but not in BPH1 cells or host liver tissues. CONCLUSIONS: Alternol interacts with multiple Krebs cycle enzymes, resulting in reduced mitochondrial respiration and ATP production in prostate cancer cells and xenograft tissues, providing a novel therapeutic strategy for prostate cancer treatment.

Preliminary observations of mitochondrial dysfunction in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a complex multisystem disorder because of errors in genomic imprinting with severe hypotonia, decreased muscle mass, poor suckling, feeding problems and failure to thrive during infancy, growth and other hormone deficiency, childhood-onset hyperphagia, and subsequent obesity. Decreased energy expenditure in PWS is thought to contribute to reduced muscle mass and physical activity but may also relate to cellular metabolism and disturbances in mitochondrial function. We established fibroblast cell lines from six children and adults with PWS and six healthy controls for mitochondrial assays. We used Agilent Seahorse XF extracellular flux technology to determine real-time measurements of several metabolic parameters including cellular substrate utilization, Adenosine Triphosphate (ATP)-linked respiration, and mitochondrial capacity in living cells. Decreased mitochondrial function was observed in the PWS patients compared to the healthy controls with significant differences in basal respiration, maximal respiratory capacity, and ATP-linked respiration. These results suggest disturbed mitochondrial bioenergetics in PWS although the low number of studied subjects will require a larger subject population before a general consensus can be reached to identify if mitochondrial dysfunction is a contributing factor in PWS.

Regulation of energy metabolism during early mammalian development: TEAD4 controls mitochondrial transcription.

Early mammalian development is crucially dependent on the establishment of oxidative energy metabolism within the trophectoderm (TE) lineage. Unlike the inner cell mass, TE cells enhance ATP production via mitochondrial oxidative phosphorylation (OXPHOS) and this metabolic preference is essential for blastocyst maturation. However, molecular mechanisms that regulate establishment of oxidative energy metabolism in TE cells are incompletely understood. Here, we show that conserved transcription factor TEAD4, which is essential for pre-implantation mammalian development, regulates this process by promoting mitochondrial transcription. In developing mouse TE and TE-derived trophoblast stem cells (TSCs), TEAD4 localizes to mitochondria, binds to mitochondrial DNA (mtDNA) and facilitates its transcription by recruiting mitochondrial RNA polymerase (POLRMT). Loss of TEAD4 impairs recruitment of POLRMT, resulting in reduced expression of mtDNA-encoded electron transport chain components, thereby inhibiting oxidative energy metabolism. Our studies identify a novel TEAD4-dependent molecular mechanism that regulates energy metabolism in the TE lineage to ensure mammalian development.

Cancer-Associated Fibroblasts Drive Glycolysis in a Targetable Signaling Loop Implicated in Head and Neck Squamous Cell Carcinoma Progression.

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR.Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769-82. ©2018 AACR.

Impaired TFEB-Mediated Lysosome Biogenesis and Autophagy Promote Chronic Ethanol-Induced Liver Injury and Steatosis in Mice.

BACKGROUND & AIMS: Defects in lysosome function and autophagy contribute to the pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes by evaluating the functions of transcription factor EB (TFEB), which regulates lysosomal biogenesis. METHODS: We performed studies with GFP-LC3 mice, mice with liver-specific deletion of TFEB, mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB and TFE3 double-knockout mice), and Tfebflox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of regular ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice also were given injections of torin-1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time polymerase chain reaction to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. RESULTS: Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB compared with control mice, and hepatocytes had decreased lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting in increased mTOR activation. Administration of torin-1 increased liver levels of TFEB and decreased steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in the liver developed less severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared with mice carrying a control vector. Mice with knockdown of TFEB and TFEB-TFE3 double-knockout mice developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues. CONCLUSIONS: We found that ethanol feeding plus an acute binge decreased hepatic expression of TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect the liver from ethanol-induced damage.

Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism.

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with ß-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2.

Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell's ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.

Targeting the Enterohepatic Bile Acid Signaling Induces Hepatic Autophagy via a CYP7A1-AKT-mTOR Axis in Mice.

BACKGROUND & AIMS: Hepatic cholesterol accumulation and autophagy defects contribute to hepatocyte injury in fatty liver disease. Bile acid synthesis is a major pathway for cholesterol catabolism in the liver. This study aims to understand the molecular link between cholesterol and bile acid metabolism and hepatic autophagy activity. METHODS: The effects of cholesterol and cholesterol 7α-hydroxylase (CYP7A1) expression on autophagy and lysosome function were studied in cell models. The effects and mechanism of disrupting enterohepatic bile acid circulation on hepatic autophagy were studied in mice. RESULTS: The results first showed differential regulation of hepatic autophagy by free cholesterol and cholesterol ester, whereby a modest increase of cellular free cholesterol, but not cholesterol ester, impaired lysosome function and caused marked autolysosome accumulation. We found that CYP7A1 induction, either by cholestyramine feeding in mice or adenovirus-mediated CYP7A1 expression in hepatocytes, caused strong autophagy induction. Mechanistically, we showed that CYP7A1 expression markedly attenuated growth factor/AKT signaling activation of mechanistic target of rapamycin (mTOR), but not amino acid signaling to mTOR in vitro and in vivo. Metabolomics analysis further found that CYP7A1 induction not only decreased hepatic cholesterol but also altered phospholipid and sphingolipid compositions. Collectively, these results suggest that CYP7A1 induction interferes with growth factor activation of AKT/mTOR signaling possibly by altering membrane lipid composition. Finally, we showed that cholestyramine feeding restored impaired hepatic autophagy and improved metabolic homeostasis in Western diet-fed mice. CONCLUSIONS: This study identified a novel CYP7A1-AKT-mTOR signaling axis that selectively induces hepatic autophagy, which helps improve hepatocellular integrity and metabolic homeostasis.

Arsenite Effects on Mitochondrial Bioenergetics in Human and Mouse Primary Hepatocytes Follow a Nonlinear Dose Response.

Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis.

Dual Role of Epidermal Growth Factor Receptor in Liver Injury and Regeneration after Acetaminophen Overdose in Mice.

Epidermal growth factor receptor (EGFR) plays a crucial role in hepatocyte proliferation. Its role in acetaminophen (APAP)-mediated hepatotoxicity and subsequent liver regeneration is completely unknown. Role of EGFR after APAP-overdose in mice was studied using pharmacological inhibition strategy. Rapid, sustained and dose-dependent activation of EGFR was noted after APAP-treatment in mice, which was triggered by glutathione depletion. EGFR-activation was also observed in primary human hepatocytes after APAP-treatment, preceding elevation of toxicity markers. Treatment of mice with an EGFR-inhibitor (EGFRi), Canertinib, 1h post-APAP resulted in robust inhibition of EGFR-activation and a striking reduction in APAP-induced liver injury. Metabolic activation of APAP, formation of APAP-protein adducts, APAP-mediated JNK-activation and its mitochondrial translocation were not altered by EGFRi. Interestingly, EGFR rapidly translocated to mitochondria after APAP-treatment. EGFRi-treatment abolished mitochondrial EGFR activity, prevented APAP-mediated mitochondrial dysfunction/oxidative-stress and release of endonucleases from mitochondria, which are responsible for DNA-damage/necrosis. Treatment with N-acetylcysteine (NAC), 4h post-APAP in mice did not show any protection but treatment of EGFRi in combination with NAC showed decrease in liver injury. Finally, delayed treatment with EGFRi, 12-h post-APAP, did not alter peak injury but caused impairment of liver regeneration resulting in sustained injury and decreased survival after APAP overdose in mice. Impairment of regeneration was due to inhibition of cyclinD1 induction and cell cycle arrest. Our study has revealed a new dual role of EGFR both in initiation of APAP-injury and in stimulation of subsequent compensatory regeneration after APAP-overdose.

Sortilin 1 Modulates Hepatic Cholesterol Lipotoxicity in Mice via Functional Interaction with Liver Carboxylesterase 1.

The liver plays a key role in cholesterol metabolism. Impaired hepatic cholesterol homeostasis causes intracellular free cholesterol accumulation and hepatocyte injury. Sortilin 1 (SORT1) is a lysosomal trafficking receptor that was identified by genome-wide association studies (GWAS) as a novel regulator of cholesterol metabolism in humans. Here we report that SORT1 deficiency protected against cholesterol accumulation-induced liver injury and inflammation in mice. Using an LC-MS/MS-based proteomics approach, we identified liver carboxylesterase 1 (CES1) as a novel SORT1-interacting protein. Mechanistic studies further showed that SORT1 may regulate CES1 lysosomal targeting and degradation and that SORT1 deficiency resulted in higher liver CES1 protein abundance. Previous studies have established an important role of hepatic CES1 in promoting intracellular cholesterol mobilization, cholesterol efflux, and bile acid synthesis. Consistently, high cholesterol atherogenic diet-challenged Sort1 knock-out mice showed less hepatic free cholesterol accumulation, increased bile acid synthesis, decreased biliary cholesterol secretion, and the absence of gallstone formation. SORT1 deficiency did not alter hepatic ceramide and fatty acid metabolism in high cholesterol atherogenic diet-fed mice. Finally, knockdown of liver CES1 in mice markedly increased the susceptibility to high cholesterol diet-induced liver injury and abolished the protective effect against cholesterol lipotoxicity in Sort1 knock-out mice. In summary, this study identified a novel SORT1-CES1 axis that regulates cholesterol-induced liver injury, which provides novel insights that improve our current understanding of the molecular links between SORT1 and cholesterol metabolism. This study further suggests that therapeutic inhibition of SORT1 may be beneficial in improving hepatic cholesterol homeostasis in metabolic and inflammatory liver diseases.

Editor's Highlight: Metformin Protects Against Acetaminophen Hepatotoxicity by Attenuation of Mitochondrial Oxidant Stress and Dysfunction.

Overdose of acetaminophen (APAP) causes severe liver injury and even acute liver failure in both mice and human. A recent study by Kim et al. (2015, Metformin ameliorates acetaminophen hepatotoxicity via Gadd45ß-dependent regulation of JNK signaling in mice. J. Hepatol. 63, 75-82) showed that metformin, a first-line drug to treat type 2 diabetes mellitus, protected against APAP hepatotoxicity in mice. However, its exact protective mechanism has not been well clarified. To investigate this, C57BL/6J mice were treated with 400 mg/kg APAP and 350 mg/kg metformin was given 0.5 h pre- or 2 h post-APAP. Our data showed that pretreatment with metformin protected against APAP hepatotoxicity, as indicated by the over 80% reduction in plasma alanine aminotransferase (ALT) activities and significant decrease in centrilobular necrosis. Metabolic activation of APAP, as indicated by glutathione depletion and APAP-protein adducts formation, was also slightly inhibited. However, 2 h post-treatment with metformin still reduced liver injury by 50%, without inhibition of adduct formation. Interestingly, neither pre- nor post-treatment of metformin inhibited c-jun N-terminal kinase (JNK) activation or its mitochondrial translocation. In contrast, APAP-induced mitochondrial oxidant stress and dysfunction were greatly attenuated in these mice. In addition, mice with 2 h post-treatment with metformin also showed significant inhibition of complex I activity, which may contribute to the decreased mitochondrial oxidant stress. Furthermore, the protection was reproduced in JNK activation-absent HepaRG cells treated with 20 mM APAP followed by 0.5 or 1 mM metformin 6 h later, confirming JNK-independent protection mechanisms. Thus, metformin protects against APAP hepatotoxicity by attenuating the mitochondrial oxidant stress and subsequent mitochondrial dysfunction, and may be a potential therapeutic option for APAP overdose patients.

Metabolomics reveals the formation of aldehydes and iminium in gefitinib metabolism.

Gefitinib (GEF), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is widely used for the treatment of cancers, particularly non-small cell lung cancer. However, its clinical use is limited by multiple adverse effects associated with GEF, such as liver and lung injuries, severe nausea, and diarrhea. Although, the exact mechanism of GEF adverse effects are still unknown, xenobiotic-induced bioactivation is thought to play a significant role in GEF induced toxicity. Using a metabolomic approach, we investigated the metabolic pathways of GEF in human and mouse liver microsomes. Thirty four GEF metabolites and adducts were identified and half of them are novel. The potential reactive metabolites, two aldehydes and one iminium, were identified for the first time. The previously reported GSH adducts and primary amines were observed as well. The aldehyde and iminium pathways were further confirmed by using methoxylamine and potassium cyanide as trapping reagents. Using recombinant CYP450 isoforms, CYP3A4 inhibitor, and S9 from Cyp3a-null mice, we confirmed CYP3A is the major enzyme contributing to the formation of aldehydes, GSH adducts, and primary amines in liver. Multiple enzymes contribute to the formation of iminium. This study provided us more knowledge of GEF bioactivation and enzymes involved in metabolic pathways, which can be utilized for understanding the mechanism of adverse effects associated with GEF and predicting possible drug-drug interactions. Further studies are suggested to determine the roles of these bioactivation pathways in GEF toxicity.

Functional coupling of ATP-binding cassette transporter Abcb6 to cytochrome P450 expression and activity in liver.

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.