Novel IL-4/HB-EGF-dependent crosstalk between eosinophils and macrophages controls liver regeneration after ischaemia and reperfusion injury.

OBJECTIVE: Previous studies indicate that eosinophils are recruited into the allograft following orthotopic liver transplantation and protect from ischaemia reperfusion (IR) injury. In the current studies, we aim to explore whether their protective function could outlast during liver repair. DESIGN: Eosinophil-deficient mice and adoptive transfer of bone marrow-derived eosinophils (bmEos) were employed to investigate the effects of eosinophils on tissue repair and regeneration after hepatic IR injury. Aside from exogenous cytokine or neutralising antibody treatments, mechanistic studies made use of a panel of mouse models of eosinophil-specific IL-4/IL-13-deletion, cell-specific IL-4rα-deletion in liver macrophages and hepatocytes and macrophage-specific deletion of heparin-binding epidermal growth factor-like growth factor (hb-egf). RESULT: We observed that eosinophils persisted over a week following hepatic IR injury. Their peak accumulation coincided with that of hepatocyte proliferation. Functional studies showed that eosinophil deficiency was associated with a dramatic delay in liver repair, which was normalised by the adoptive transfer of bmEos. Mechanistic studies demonstrated that eosinophil-derived IL-4, but not IL-13, was critically involved in the reparative function of these cells. The data further revealed a selective role of macrophage-dependent IL-4 signalling in liver regeneration. Eosinophil-derived IL-4 stimulated macrophages to produce HB-EGF. Moreover, macrophage-specific hb-egf deletion impaired hepatocyte regeneration after IR injury. CONCLUSION: Together, these studies uncovered an indispensable role of eosinophils in liver repair after acute injury and identified a novel crosstalk between eosinophils and macrophages through the IL-4/HB-EGF axis.

Inhibition of acid rock drainage with iron-silicate or phosphate film: in rainy and submerged environments.

Iron phosphate-based coating and iron silicate-based coating were used to inhibit the oxidation of sulfide minerals in rainy and submerged environments. The inhibiting effectiveness of coating agents on the oxidation of iron sulfide minerals was investigated using pyrite and rock samples resulting from acid drainage. The film formed with both surface-coating agents was identified by pyrite surface analysis. It was also confirmed that the formation of coatings varies depending on the crystallographic orientation. The inhibitory effects under rainy and submerged conditions were investigated using column experiments. Submerged conditions accelerated deterioration compared to that under rainy conditions. Iron phosphate coating had a significantly better oxidation-inhibitory effect (84.86-98.70%) than iron silicate coating (56.80-92.36%), and at a concentration of 300 mM, H+ elution was inhibited by more than 90% throughout the experiment. Furthermore, methods for effective film formation were investigated in terms of producing Fe3+; (1) application of coating agents mixed with oxidant (H2O2), (2) application of coating agent after the use of the oxidant. In a rainy environment, applying iron phosphate-based coating using the sequential method showed oxidation inhibition effects for cycles 1-9, whereas applying the mixed material showed effects for cycles 9-13. The use of a surface-coating agent after applying an oxidant did not inhibit oxidation. The surface coating agent and the oxidizing agent should be applied as a mixture to form a film.

Metabolic dysregulation in the Atp7b-/- Wilson's disease mouse model.

Inactivating mutations in the copper transporter Atp7b result in Wilson's disease. The Atp7b-/- mouse develops hallmarks of Wilson's disease. The activity of several nuclear receptors decreased in Atp7b-/- mice, and nuclear receptors are critical for maintaining metabolic homeostasis. Therefore, we anticipated that Atp7b-/- mice would exhibit altered progression of diet-induced obesity, fatty liver, and insulin resistance. Following 10 wk on a chow or Western-type diet (40% kcal fat), parameters of glucose and lipid homeostasis were measured. Hepatic metabolites were measured by liquid chromatography-mass spectrometry and correlated with transcriptomic data. Atp7b-/- mice fed a chow diet presented with blunted body-weight gain over time, had lower fat mass, and were more glucose tolerant than wild type (WT) littermate controls. On the Western diet, Atp7b-/- mice exhibited reduced body weight, adiposity, and hepatic steatosis compared with WT controls. Atp7b-/- mice fed either diet were more insulin sensitive than WT controls; however, fasted Atp7b-/- mice exhibited hypoglycemia after administration of insulin due to an impaired glucose counterregulatory response, as evidenced by reduced hepatic glucose production. Coupling gene expression with metabolomic analyses, we observed striking changes in hepatic metabolic profiles in Atp7b-/- mice, including increases in glycolytic intermediates and components of the tricarboxylic acid cycle. In addition, the active phosphorylated form of AMP kinase was significantly increased in Atp7b-/- mice relative to WT controls. Alterations in hepatic metabolic profiles and nuclear receptor signaling were associated with improved glucose tolerance and insulin sensitivity as well as with impaired fasting glucose production in Atp7b-/- mice.

The bile acid induced hepatokine orosomucoid suppresses adipocyte differentiation.

Bile acids have recently emerged as key metabolic hormones with beneficial impacts in multiple metabolic diseases. We previously discovered that hepatic bile acid overload distally modulates glucose and fatty acid metabolism in adipose tissues to exert anti-obesity effects. However, the detailed mechanisms that explain the salutary effects of serum bile acid elevation remain unclear. Here, proteomic profiling identified a new hepatokine, Orosomucoid (ORM) that governs liver-adipose tissue crosstalk. Hepatic ORMs were highly induced by both genetic and dietary bile acid overload. To address the direct metabolic effects of ORM, purified ORM proteins were administered during adipogenic differentiation of 3T3-L1 cells and mouse stromal vascular fibroblasts. ORM suppressed adipocyte differentiation and strongly inhibited gene expression of adipogenic transcription factors such as C/EBPß, KLF5, C/EBPα, and PPARγ. Taken together, our data clearly suggest that bile acid-induced ORM secretion from the liver blocks adipocyte differentiation, potentially linked to anti-obesity effect of bile acids.

Vitamin D Receptor Activation in Liver Macrophages Protects Against Hepatic Endoplasmic Reticulum Stress in Mice.

BACKGROUND AND AIMS: Hepatic endoplasmic reticulum (ER) stress, whether triggered by intrinsic or extrinsic factors, can be resolved by the unfolded protein response (UPR). Sustained UPR activation leads to cell death and inflammatory response and contributes to liver disease progression. Hepatic tissue macrophages are key players in orchestrating liver inflammation, and ER stress can enhance macrophage activation. However, it is not well defined how the interplay between ER stress and inflammation is regulated during hepatic stress response. APPROACH AND RESULTS: Here we demonstrate that vitamin D receptor (VDR) activation mitigates hepatic ER stress response, whereas VDR knockout mice undergo persistent UPR activation and apoptosis in response to chemical ER stress inducer. Moreover, VDR deficiency promotes hepatic macrophage infiltration and increases gene expression and systematic levels of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor α. VDR expression is induced in hepatic macrophages by ER stress, and VDR plays a dual regulatory role in macrophages by protecting against ER stress and promoting anti-inflammatory polarization. Co-culture with VDR-activated bone marrow-derived macrophages suppresses UPR target genes in primary hepatocytes treated with ER stress inducers. Thus, the immunomodulatory functions of VDR in macrophages are critical in hepatic ER stress resolution in mice. CONCLUSIONS: VDR signaling in macrophages regulates a shift between proinflammatory and anti-inflammatory activation during ER stress-induced inflammation to promote hepatic ER stress resolution.

Methyl-Sensing Nuclear Receptor Liver Receptor Homolog-1 Regulates Mitochondrial Function in Mouse Hepatocytes.

BACKGROUND AND AIMS: Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates metabolic homeostasis in the liver. Previous studies identified phosphatidylcholines as potential endogenous agonist ligands for LRH-1. In the liver, distinct subsets of phosphatidylcholine species are generated by two different pathways: choline addition to phosphatidic acid through the Kennedy pathway and trimethylation of phosphatidylethanolamine through phosphatidylethanolamine N-methyl transferase (PEMT). APPROACH AND RESULTS: Here, we report that a PEMT-LRH-1 pathway specifically couples methyl metabolism and mitochondrial activities in hepatocytes. We show that the loss of Lrh-1 reduces mitochondrial number, basal respiration, beta-oxidation, and adenosine triphosphate production in hepatocytes and decreases expression of mitochondrial biogenesis and beta-oxidation genes. In contrast, activation of LRH-1 by its phosphatidylcholine agonists exerts opposite effects. While disruption of the Kennedy pathway does not affect the LRH-1-mediated regulation of mitochondrial activities, genetic or pharmaceutical inhibition of the PEMT pathway recapitulates the effects of Lrh-1 knockdown on mitochondria. Furthermore, we show that S-adenosyl methionine, a cofactor required for PEMT, is sufficient to induce Lrh-1 transactivation and consequently mitochondrial biogenesis. CONCLUSIONS: A PEMT-LRH-1 axis regulates mitochondrial biogenesis and beta-oxidation in hepatocytes.

Protective role of cardiac-specific overexpression of caveolin-3 in cirrhotic cardiomyopathy.

Cirrhotic cardiomyopathy is a clinical syndrome in patients with liver cirrhosis characterized by blunted cardiac contractile responses to stress and/or heart rate-corrected QT (QTc) interval prolongation. Caveolin-3 (Cav-3) plays a critical role in cardiac protection and is an emerging therapeutic target for heart disease. We investigated the protective role of cardiac-specific overexpression (OE) of Cav-3 in cirrhotic cardiomyopathy. Biliary fibrosis was induced in male Cav-3 OE mice and transgene negative (TGneg) littermates by feeding a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; 0.1%) for 3 wk. Liver pathology and blood chemistries were assessed, and stress echocardiography, telemetry, and isolated heart perfusion studies to assess adrenergic responsiveness were performed. Cav-3 OE mice showed a similar degree of hyperdynamic contractility, pulmonary hypertension, and QTc interval prolongation as TGneg mice after 3 wk of DDC diet. Blunted systolic responses were shown in both DDC-fed Cav-3 OE and TGneg hearts after in vivo isoproterenol challenge. However, QTc interval prolongation after in vivo isoproterenol challenge was significantly less in DDC-fed Cav-3 OE hearts compared with DDC-fed TGneg hearts. In ex vivo perfused hearts, where circulatory factors are absent, isoproterenol challenge showed hearts from DDC-fed Cav-3 OE mice had better cardiac contractility and relaxation compared with DDC-fed TGneg hearts. Although Cav-3 OE in the heart did not prevent cardiac alterations in DDC-induced biliary fibrosis, cardiac expression of Cav-3 reduced QTc interval prolongation after adrenergic stimulation in cirrhosis.NEW & NOTEWORTHY Prevalence of cirrhotic cardiomyopathy is up to 50% in cirrhotic patients, and liver transplantation is the only treatment. However, cirrhotic cardiomyopathy is associated with perioperative morbidity and mortality after liver transplantation; therefore, management of cirrhotic cardiomyopathy is crucial for successful liver transplantation. This study shows cardiac myocyte specific overexpression of caveolin-3 (Cav-3) provides better cardiac contractile responses and less corrected QT prolongation during adrenergic stress in a cirrhotic cardiomyopathy model, suggesting beneficial effects of Cav-3 expression in cirrhotic cardiomyopathy.

Nutrient-sensing nuclear receptors coordinate autophagy.

Autophagy is an evolutionarily conserved catabolic process that recycles nutrients upon starvation and maintains cellular energy homeostasis. Its acute regulation by nutrient-sensing signalling pathways is well described, but its longer-term transcriptional regulation is not. The nuclear receptors peroxisome proliferator-activated receptor-α (PPARα) and farnesoid X receptor (FXR) are activated in the fasted and fed liver, respectively. Here we show that both PPARα and FXR regulate hepatic autophagy in mice. Pharmacological activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipid degradation, or lipophagy. This response is lost in PPARα knockout (Ppara(-/-), also known as Nr1c1(-/-)) mice, which are partially defective in the induction of autophagy by fasting. Pharmacological activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in the fasting state, and this response is absent in FXR knockout (Fxr(-/-), also known as Nr1h4(-/-)) mice, which show a partial defect in suppression of hepatic autophagy in the fed state. PPARα and FXR compete for binding to shared sites in autophagic gene promoters, with opposite transcriptional outputs. These results reveal complementary, interlocking mechanisms for regulation of autophagy by nutrient status.

Rapid Disruption of Genes Specifically in Livers of Mice Using Multiplex CRISPR/Cas9 Editing.

BACKGROUND & AIMS: Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts. METHODS: In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product. RESULTS: We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse. CONCLUSIONS: This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.

Hepatic FXR/SHP axis modulates systemic glucose and fatty acid homeostasis in aged mice.

The nuclear receptors farnesoid X receptor (FXR; NR1H4) and small heterodimer partner (SHP; NR0B2) play crucial roles in bile acid homeostasis. Global double knockout of FXR and SHP signaling (DKO) causes severe cholestasis and liver injury at early ages. Here, we report an unexpected beneficial impact on glucose and fatty acid metabolism in aged DKO mice, which show suppressed body weight gain and adiposity when maintained on normal chow. This phenotype was not observed in single Fxr or Shp knockouts. Liver-specific Fxr/Shp double knockout mice fully phenocopied the DKO mice, with lower hepatic triglyceride accumulation, improved glucose/insulin tolerance, and accelerated fatty acid use. In both DKO and liver-specific Fxr/Shp double knockout livers, these metabolic phenotypes were associated with altered expression of fatty acid metabolism and autophagy-machinery genes. Loss of the hepatic FXR/SHP axis reprogrammed white and brown adipose tissue gene expression to boost fatty acid usage. CONCLUSION: Combined deletion of the hepatic FXR/SHP axis improves glucose/fatty acid homeostasis in aged mice, reversing the aging phenotype of body weight gain, increased adiposity, and glucose/insulin tolerance, suggesting a central role of this axis in whole-body energy homeostasis. (Hepatology 2017;66:498-509).

Regulation of Liver Energy Balance by the Nuclear Receptors Farnesoid X Receptor and Peroxisome Proliferator Activated Receptor α.

BACKGROUND: The liver undergoes major changes in substrate utilization and metabolic output over the daily feeding and fasting cycle. These changes occur acutely in response to hormones such as insulin and glucagon, with rapid changes in signaling pathways mediated by protein phosphorylation and other post-translational modifications. They are also reflected in chronic alterations in gene expression in response to nutrient-sensitive transcription factors. Among these, the nuclear receptors farnesoid X receptor (FXR) and peroxisome proliferator activated receptor α (PPARα) provide an intriguing, coordinated response to maintain energy balance in the liver. FXR is activated in the fed state by bile acids returning to the liver, while PPARα is activated in the fasted state in response to the free fatty acids produced by adipocyte lipolysis or possibly other signals. Key Messages: Previous studies indicate that FXR and PPARα have opposing effects on each other's primary targets in key metabolic pathways including gluconeogenesis. Our more recent work shows that these 2 nuclear receptors coordinately regulate autophagy: FXR suppresses this pathway of nutrient and energy recovery, while PPARα activates it. Another recent study indicates that FXR activates the complement and coagulation pathway, while earlier studies identify this as a negative target of PPARα. Since secretion is a very energy- and nutrient-intensive process for hepatocytes, it is possible that FXR licenses it in the nutrient-rich fed state, while PPARα represses it to spare resources in the fasted state. Energy balance is a potential connection linking FXR and PPARα regulation of autophagy and secretion, 2 seemingly unrelated aspects of hepatocyte function. CONCLUSIONS: FXR and PPARα act coordinately to promote energy balance and homeostasis in the liver by regulating autophagy and potentially protein secretion. It is quite likely that their impact extends to additional pathways relevant to hepatic energy balance, and that these pathways will in turn interface with other well-known nutrient-responsive mechanisms of energy control.

Inhibition of pyrite oxidation by surface coating: a long-term field study.

Pyrite and other iron sulfides are readily oxidized by dissolved oxygen in aqueous phase, producing acidity and Fe(2+), which causes significant environmental problems. Applications of surface coating agents (Na2SiO3 and KH2PO4) were conducted at Boeun (Chungbuk, South Korea) outcrop site, and their efficiencies to inhibit the oxidation of sulfide minerals were monitored for a long-term period (449 days). The rock sample showed positive Net Acid Production Potential (NAPP = 20.23) and low Net Acid Generation pH (NAGpH = 2.42) values, suggesting that the rock sample was categorized in the potential acid-forming group. For the monitored time period (449 days), field study results showed that the application of Na2SiO3 effectively inhibited the pyrite oxidation as compared to KH2PO4. Na2SiO3 as a surface coating agent maintained pH 5-6 and reduced oxidation of pyrite surface up to 99.95 and 97.70 % indicated by Fe(2+) and SO4 (2-) release, respectively. The scanning electron microscope and energy-dispersive X-ray spectrometer analysis indicated that the morphology of rock surface was completely changed attributable to formation of iron silicate coating. The experimental results suggested that the treatment with Na2SiO3 was highly effective and it might be applicable on field for inhibition of iron sulfide oxidation.

Short-Term Impact of Low-Intensity Exercise with Blood Flow Restriction on Mild Knee Osteoarthritis in Older Adults: A Pilot Study.

This pilot study aimed to investigate the immediate impact of low-intensity exercises with blood flow restriction (BFR) on older adults with knee osteoarthritis (KOA). Fifteen patients with KOA who were over 50 years old, participated and underwent low-intensity resistance knee exercises at 30% of their one-repetition maximum with BFR three times/week for two weeks. Pre- and post-exercise assessments included pain levels, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores, isokinetic knee strength, lower extremity muscle volume (via leg circumference and muscle thickness), functional performance tests (timed up-and-go [TUG] and sit-to-stand [STS]), skeletal muscle index (SMI) using bioelectrical impedance analysis, and handgrip strength (HGS). Post-exercise, there was a significant reduction in pain. WOMAC scores showed significant improvements across all three domains: pain, stiffness, and physical function. In the TUG and STS tests, completion times were significantly reduced. Thigh and calf circumferences, as well as thigh muscle thickness significantly increased after exercise. Post-exercise SMI and HGS also significantly increased. However, isokinetic knee strength did not show significant changes. In conclusion, low-intensity BFR exercises provide immediate benefits in symptoms and physical performance for patients with KOA, potentially inducing local and systemic muscle mass increase, even after a short-term intervention.

Probiotic Limosilactobacillus reuteri DSM 17938 Changes Foxp3 Deficiency-Induced Dyslipidemia and Chronic Hepatitis in Mice.

The probiotic Limosilactobacillus reuteri DSM 17938 produces anti-inflammatory effects in scurfy (SF) mice, a model characterized by immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (called IPEX syndrome in humans), caused by regulatory T cell (Treg) deficiency and is due to a Foxp3 gene mutation. Considering the pivotal role of lipids in autoimmune inflammatory processes, we investigated alterations in the relative abundance of lipid profiles in SF mice (± treatment with DSM 17938) compared to normal WT mice. We also examined the correlation between plasma lipids and gut microbiota and circulating inflammatory markers. We noted a significant upregulation of plasma lipids associated with autoimmune disease in SF mice, many of which were downregulated by DSM 17938. The upregulated lipids in SF mice demonstrated a significant correlation with gut bacteria known to be implicated in the pathogenesis of various autoimmune diseases. Chronic hepatitis in SF livers responded to DSM 17938 treatment with a reduction in hepatic inflammation. Altered gene expression associated with lipid metabolism and the positive correlation between lipids and inflammatory cytokines together suggest that autoimmunity leads to dyslipidemia with impaired fatty acid oxidation in SF mice. Probiotics are presumed to contribute to the reduction of lipids by reducing inflammatory pathways.

The Hepatokine Orosomucoid 2 Mediates Beneficial Metabolic Effects of Bile Acids.

Bile acids (BAs) are pleiotropic regulators of metabolism. Elevated levels of hepatic and circulating BAs improve energy metabolism in peripheral organs, but the precise mechanisms underlying the metabolic benefits and harm still need to be fully understood. In the current study, we identified orosomucoid 2 (ORM2) as a liver-secreted hormone (i.e., hepatokine) induced by BAs and investigated its role in BA-induced metabolic improvements in mouse models of diet-induced obesity. Contrary to our expectation, under a high-fat diet (HFD), our Orm2 knockout (Orm2-KO) exhibited a lean phenotype compared with C57BL/6J control, partly due to the increased energy expenditure. However, when challenged with a HFD supplemented with cholic acid, Orm2-KO eliminated the antiobesity effect of BAs, indicating that ORM2 governs BA-induced metabolic improvements. Moreover, hepatic ORM2 overexpression partially replicated BA effects by enhancing insulin sensitivity. Mechanistically, ORM2 suppressed interferon-γ/STAT1 activities in inguinal white adipose tissue depots, forming the basis for anti-inflammatory effects of BAs and improving glucose homeostasis. In conclusion, our study provides new insights into the molecular mechanisms of BA-induced liver-adipose cross talk through ORM2 induction.

Radiosensitization of brain metastasis by targeting c-MET.

Radiotherapy is the most widely used therapeutic modality in brain metastasis; however, it only provides palliation due to inevitable tumor recurrence. Resistance of tumor cells to ionizing radiation is a major cause of treatment failure. A critical unmet need in oncology is to develop rationale driven approaches that can enhance the efficacy of radiotherapy against metastatic tumor. Utilizing in vivo orthotopic primary tumor and brain metastasis models that recapitulate clinical situation of the patients with metastatic breast cancer, we investigated a molecular mechanism through which metastatic tumor cells acquire resistance to radiation. Recent studies have demonstrated that the hepatocyte growth factor (HGF)-c-Met pathway is essential for the pathologic development and progression of many human cancers such as proliferation, invasion and resistance to anticancer therapies. In this study, c-Met signaling activity as well as total c-Met expression was significantly upregulated in both breast cancer cell lines irradiated in vitro and ex vivo radio-resistant cells derived from breast cancer brain metastatic xenografts. To interrogate the role of c-Met signaling in radioresistance of brain metastasis, we evaluated the effects on tumor cell viability, clonogenicity, sensitivity to radiation, and in vitro/in vivo tumor growth after targeting c-Met by small-hairpin RNA (shRNA) or small-molecule kinase inhibitor (PF-2341066). Although c-Met silencing or radiation alone demonstrated a modest decrease in clonogenic growth of parental breast cancers and brain metastatic derivatives, combination of two modalities showed synergistic antitumor effects resulting in significant prolongation of overall survival in tumor-bearing mice. Taken together, optimizing c-Met targeting in combination with radiation is critical to enhance the effectiveness of radiotherapy in the treatments of brain metastasis.

Updates on the Immune Cell Basis of Hepatic Ischemia-Reperfusion Injury.

Liver ischemia-reperfusion injury (IRI) is the main cause of organ dysfunction and failure after liver surgeries including organ transplantation. The mechanism of liver IRI is complex and numerous signals are involved but cellular metabolic disturbances, oxidative stress, and inflammation are considered the major contributors to liver IRI. In addition, the activation of inflammatory signals exacerbates liver IRI by recruiting macrophages, dendritic cells, and neutrophils, and activating NK cells, NKT cells, and cytotoxic T cells. Technological advances enable us to understand the role of specific immune cells during liver IRI. Accordingly, therapeutic strategies to prevent or treat liver IRI have been proposed but no definitive and effective therapies exist yet. This review summarizes the current update on the immune cell functions and discusses therapeutic potentials in liver IRI. A better understanding of this complex and highly dynamic process may allow for the development of innovative therapeutic approaches and optimize patient outcomes.

Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice.

OBJECTIVE: Mitophagy removes damaged mitochondria to maintain cellular homeostasis. Aryl hydrocarbon receptor (AhR) expression in the liver plays a crucial role in supporting normal liver functions, but its impact on mitochondrial function is unclear. Here, we identified a new role of AhR in the regulation of mitophagy to control hepatic energy homeostasis. METHODS: In this study, we utilized primary hepatocytes from AhR knockout (KO) mice and AhR knockdown AML12 hepatocytes. An endogenous AhR ligand, kynurenine (Kyn), was used to activate AhR in AML12 hepatocytes. Mitochondrial function and mitophagy process were comprehensively assessed by MitoSOX and mt-Keima fluorescence imaging, Seahorse XF-based oxygen consumption rate measurement, and Mitoplate S-1 mitochondrial substrate utilization analysis. RESULTS: Transcriptomic analysis indicated that mitochondria-related gene sets were dysregulated in AhR KO liver. In both primary mouse hepatocytes and AML12 hepatocyte cell lines, AhR inhibition strongly suppressed mitochondrial respiration rate and substrate utilization. AhR inhibition also blunted the fasting response of several essential autophagy genes and the mitophagy process. We further identified BCL2 interacting protein 3 (BNIP3), a mitophagy receptor that senses nutrient stress, as an AhR target gene. AhR is directly recruited to the Bnip3 genomic locus, and Bnip3 transcription was enhanced by AhR endogenous ligand treatment in wild-type liver and abolished entirely in AhR KO liver. Mechanistically, overexpression of Bnip3 in AhR knockdown cells mitigated the production of mitochondrial reactive oxygen species (ROS) and restored functional mitophagy. CONCLUSIONS: AhR regulation of the mitophagy receptor BNIP3 coordinates hepatic mitochondrial function. Loss of AhR induces mitochondrial ROS production and impairs mitochondrial respiration. These findings provide new insight into how endogenous AhR governs hepatic mitochondrial homeostasis.

Vitamin B2 enables regulation of fasting glucose availability.

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.

Wnt activation is implicated in glioblastoma radioresistance.

Glioblastoma (GBM) patients have dismal median survival even with the most rigorous treatments currently available. Radiotherapy is the most effective non-surgical therapy for GBM patients; however, patients succumb due to tumor recurrence within a year. To develop a curative therapeutic approach, we need to better understand the underlying molecular mechanism of radiation resistance in GBM. Towards this goal, we developed an in vivo orthotopic GBM model system that mimics the radiation response of human GBM, using both established-GBM cell line and patient-derived freshly dissociated GBM specimen. In-vivo ionizing radiation (IR) treatment prolonged the survival of mice with intracranical tumor derived from U373MG, but failed to prevent tumor recurrence. U373MG and GBM578 cells isolated after in-vivo IR (U373-IR and 578-IR) were more clonogenic and enriched with stem cell-like characteristics, compared with mock-treated control tumor cells. Transcriptomic analyses and quantitative real-time reverse-transcription PCR analyses using these matched GBM cells before and after radiation treatment revealed that Wnt pathways were preferentially activated in post-IR GBM cells. U373-IR cells and 578-IR were enriched with cells positive for both active ß-catenin (ABC) and Sox2 population, and this subpopulation was further increased after additional in-vitro radiation treatment, suggesting that radiation resistance of GBM is mediated due, in part, to the activation of stem cell-associated pathways including Wnt. Finally, pharmacological and siRNA inhibition of Wnt pathway significantly decreased the survival and clonogenicity of GBM cells and reduced their ABC(+)/Sox2(+) population. Together, these data suggest that Wnt activation is a molecular mechanism to confer GBM radioresistance and an important therapeutic target.