Synergism of mechanisms underlying early-stage changes in retina function in male hyperglycemic db/db mice in the absence and presence of chemically-induced dyslipidemia.

The study was designed to quantify retina function in a spontaneous mutation mouse model of diabetes, in which sustained dyslipidemia was induced chemically. The goal of the study was to identify if dyslipidemia in the presence of hyperglycemia resulted in either a synergistic, or a merely additive, exacerbation of retinal and visual dysfunctions in diabetes. Two cohorts of mice, male C57BL/6 and C57BL/KsJ-db/db mice were divided into two groups each. One group of each strain received the triblock copolymer, poloxamer 407 (P-407), administered by intraperitoneal injection ("WT P-407" and "db/db P-407" groups) with saline as a control in the remaining two groups ("WT" and "db/db" groups). Blood glucose, total cholesterol (TC) and total triglyceride (TG) levels were quantified using enzyme-based colorimetric assays. Retina function was measured using electroretinography (ERG) and visual acuity was determined by behaviorally assessing parameters of the optomotor reflex. TC and TG levels were normal in both saline controls (WT) and db/db mice but were significantly elevated in the WT P-407 group (p < 0.01 for TC; p < 0.001 for TG), while levels of the same lipids were further elevated in the db/db P-407 group when compared to the WT P-407 group levels (p < 0.001 for both TC and TG). Behavioral assessment of the optomotor reflex indicated reduced visual acuity for the db/db P-407 group when compared to either the WT P-407 or the db/db groups (p < 0.001, p < 0.0001). ERG measurements of scotopic retina function showed a significant decline in the scotopic b-wave amplitude of the WT P-407 animals (p < 0.01) and a further reduction for the db/db P-407 group when compared to controls (p < 0.0001). Very significant, strong correlations between scotopic b-wave amplitude and implicit time to TC (r = - 0.8376, p = < 0.0001 and r = 0.7069, p = 0.0022, respectively) and TG levels (r = - 0.8554, p = < 0.0001 and r = 0.7150, p = 0.0019, respectively) were found. Dyslipidemia in the presence of hyperglycemia synergistically exacerbated the severity of retinal dysfunction in diabetes. P-407 administration significantly elevated plasma TC and TG levels in male wild-type (WT) and diabetic mice (db/db), but the resulting hyperlipidemia was more significantly pronounced in the diabetic mice. While elevated plasma lipid and blood glucose levels were individually correlated with a decline in retinal function, the combination of both exacerbated retinal dysfunction. This model of combined hyperglycemia and dyslipidemia can be used to dissect individual contributions of features of the metabolic syndrome to the pathogenesis of retinal dysfunction in diabetes.

Glaucomatous aqueous humor vesicles are smaller and differ in composition compared to controls.

Cells communicate with each other using vesicles of varying sizes, including a specific repertoire known as exosomes. We isolated aqueous humor (AH)-derived vesicles using two different methods: ultracentrifugation and an exosome isolation kit. We confirmed a unique vesicle size distribution in the AH derived from control and primary open-angle glaucoma (POAG) patients using various techniques, including Nanotracker, dynamic light scattering, atomic force imaging, and electron microscopy. Bonafide vesicle and/or exosome markers were present by dot blot in both control and POAG AH-derived vesicles. Marker levels differed between POAG and control samples, while non-vesicle negative markers were absent in both. Quantitative labeled (iTRAQ) proteomics showed a reduced presence of a specific protein, STT3B, in POAG compared to controls, which was further confirmed using dot blot, Western blot, and ELISA assays. Along the lines of previous findings with AH profiles, we found vast differences in the total phospholipid composition of AH vesicles in POAG compared to controls. Electron microscopy further showed that the addition of mixed phospholipids alters the average size of vesicles in POAG. We found that the cumulative particle size of type I collagen decreased in the presence of Cathepsin D, which normal AH vesicles were able to protect against, but POAG AH vesicles did not. AH alone had no effect on collagen particles. We observed a protective effect on collagen particles with an increase in artificial vesicle sizes, consistent with the protective effects observed with larger control AH vesicles but not with the smaller-sized POAG AH vesicles. Our experiments suggest that AH vesicles in the control group provide greater protection for collagen beams compared to POAG, and their increased vesicle sizes are likely contributing factors to this protection.

Molecular Mechanisms Underlying the Therapeutic Role of Vitamin E in Age-Related Macular Degeneration.

The eye is particularly susceptible to oxidative stress and disruption of the delicate balance between oxygen-derived free radicals and antioxidants leading to many degenerative diseases. Attention has been called to all isoforms of vitamin E, with α-tocopherol being the most common form. Though similar in structure, each is diverse in antioxidant activity. Preclinical reports highlight vitamin E's influence on cell physiology and survival through several signaling pathways by activating kinases and transcription factors relevant for uptake, transport, metabolism, and cellular action to promote neuroprotective effects. In the clinical setting, population-based studies on vitamin E supplementation have been inconsistent at times and follow-up studies are needed. Nonetheless, vitamin E's health benefits outweigh the controversies. The goal of this review is to recognize the importance of vitamin E's role in guarding against gradual central vision loss observed in age-related macular degeneration (AMD). The therapeutic role and molecular mechanisms of vitamin E's function in the retina, clinical implications, and possible toxicity are collectively described in the present review.

Effect of Ubiquinol on Glaucomatous Neurodegeneration and Oxidative Stress: Studies for Retinal Ganglion Cell Survival and/or Visual Function.

Oxidative stress is one of major causal factors in glaucomatous neurodegeneration. Ubiquinol promotes retinal ganglion cell (RGC) survival against glaucomatous insults such as oxidative stress. Here we investigated the effect of ubiquinol on RGC survival and/or visual function in mouse models of glaucoma and oxidative stress. DBA/2J and age-matched DBA/2J-Gpnmb+ (D2-Gpnmb+), which do not develop intraocular pressure elevation, or C57BL/6J mice were fed with ubiquinol (1%) or control diet daily for 5 or 2 months. We assessed RGC survival by Brn3a immunohistochemistry and measured expression levels of active and total BAX, peroxisome proliferator-activated receptor-gamma coactivator 1α, transcription factor A (TFAM) and oxidative phosphorylation (OXPHOS) complex protein. Following induction of oxidative stress by paraquat injection, we also assessed visual function. In glaucomatous retina, ubiquinol supplementation significantly promoted RGC survival, blocked BAX activation and increased TFAM and OXPHOS complex II protein expression. Also, ubiquinol supplementation ameliorated oxidative stress-induced visual dysfunction. These findings indicate that ubiquinol promotes RGC survival by increasing TFAM expression and OXPHOS complex II activity in glaucomatous neurodegeneration, and that ubiquinol enhances RGC survival and preserves visual function against oxidative stress. We propose that ubiquinol has a therapeutic potential for treating oxidative stress-associated glaucomatous neurodegeneration.

Corrigendum to "Inhibition of cAMP/PKA Pathway Protects Optic Nerve Head Astrocytes against Oxidative Stress by Akt/Bax Phosphorylation-Mediated Mfn1/2 Oligomerization".

[This corrects the article DOI: 10.1155/2019/8060962.].

AIBP protects retinal ganglion cells against neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration.

Glaucoma is a leading cause of blindness worldwide in individuals 60 years of age and older. Despite its high prevalence, the factors contributing to glaucoma progression are currently not well characterized. Glia-driven neuroinflammation and mitochondrial dysfunction play critical roles in glaucomatous neurodegeneration. Here, we demonstrated that elevated intraocular pressure (IOP) significantly decreased apolipoprotein A-I binding protein (AIBP; gene name Apoa1bp) in retinal ganglion cells (RGCs), but resulted in upregulation of TLR4 and IL-1ß expression in Müller glia endfeet. Apoa1bp-/- mice had impaired visual function and Müller glia characterized by upregulated TLR4 activity, impaired mitochondrial network and function, increased oxidative stress and induced inflammatory responses. We also found that AIBP deficiency compromised mitochondrial network and function in RGCs and exacerbated RGC vulnerability to elevated IOP. Administration of recombinant AIBP prevented RGC death and inhibited inflammatory responses and cytokine production in Müller glia in vivo. These findings indicate that AIBP protects RGCs against glia-driven neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration and suggest that recombinant AIBP may be a potential therapeutic agent for glaucoma.

Loss of AKAP1 triggers Drp1 dephosphorylation-mediated mitochondrial fission and loss in retinal ganglion cells.

Impairment of mitochondrial structure and function is strongly linked to glaucoma pathogenesis. Despite the widely appreciated disease relevance of mitochondrial dysfunction and loss, the molecular mechanisms underlying mitochondrial fragmentation and metabolic stress in glaucoma are poorly understood. We demonstrate here that glaucomatous retinal ganglion cells (RGCs) show loss of A-kinase anchoring protein 1 (AKAP1), activation of calcineurin (CaN) and reduction of dynamin-related protein 1 (Drp1) phosphorylation at serine 637 (Ser637). These findings suggest that AKAP1-mediated phosphorylation of Drp1 at Ser637 has a critical role in RGC survival in glaucomatous neurodegeneration. Male mice lacking AKAP1 show increases in CaN and total Drp1 levels, as well as a decrease in Drp1 phosphorylation at Ser637 in the retina. Ultrastructural analysis of mitochondria shows that loss of AKAP1 triggers mitochondrial fragmentation and loss, as well as mitophagosome formation in RGCs. Loss of AKAP1 deregulates oxidative phosphorylation (OXPHOS) complexes (Cxs) by increasing CxII and decreasing CxIII-V, leading to metabolic and oxidative stress. Also, loss of AKAP1 decreases Akt phosphorylation at Serine 473 (Ser473) and threonine 308 (Thr308) and activates the Bim/Bax signaling pathway in the retina. These results suggest that loss of AKAP1 has a critical role in RGC dysfunction by decreasing Drp1 phosphorylation at Ser637, deregulating OXPHOS, decreasing Akt phosphorylation at Ser473 and Thr308, and activating the Bim/Bax pathway in glaucomatous neurodegeneration. Thus, we propose that overexpression of AKAP1 or modulation of Drp1 phosphorylation at Ser637 are potential therapeutic strategies for neuroprotective intervention in glaucoma and other mitochondria-related optic neuropathies.

Endogenous ocular lipids as potential modulators of intraocular pressure.

Elevated intraocular pressure (IOP) is a risk factor in glaucoma, a group of irreversible blinding diseases. Endogenous lipids may be involved in regulation of IOP homeostasis. We present comparative fold analysis of phospholipids and sphingolipids of aqueous humour and trabecular meshwork from human control vs primary open-angle glaucoma and mouse control (normotensive) vs ocular hypertensive state. The fold analysis in control vs disease state was based on ratiometric mass spectrometric data for above classes of lipids. We standardized in vitro assays for rapid characterization of lipids undergoing significant diminishment in disease state. Evaluation of lipids using in vitro assays helped select a finite number of lipids that may potentially expand cellular interstitial space embedded in an artificial matrix or increase fluid flow across a layer of cells. These assays reduced a number of lipids for initial evaluation using a mouse model, DBA/2J with spontaneous IOP elevation. These lipids were then used in other mouse models for confirmation of IOP lowering potential of a few lipids that were found promising in previous assessments. Our results provide selected lipid molecules that can be pursued for further evaluation and studies that may provide insight into their function.

Comparison of liver regeneration after partial hepatectomy and acetaminophen-induced acute liver failure: A global picture based on transcriptome analysis.

Liver regenerates following surgical removal and after drug-induced liver injury (DILI). However, most of the mechanisms of liver regeneration were identified using partial hepatectomy (PHX) model rather than using DILI models. We compared mechanisms of liver regeneration following PHX and after acetaminophen (APAP) overdose, a DILI model, using transcriptomic approach. Kinetics of hepatocyte proliferation and global gene expression profiles were studied in male C57BL/6J mice either subjected to PHX or following APAP overdose. Liver regeneration was much more synchronized after PHX as compared to APAP overdose. Transcriptomics analysis revealed activation of common upstream regulators in both models including growth factors HGF, EGF and VEGF; and cytokines IL6 and TNFα. However, magnitude of activation and temporality was significantly differed between the two models. HGF and VEGF showed similar activation between PHX and APAP but activation of EGF was significantly stronger in the APAP model. Activation of IL6 and TNFα transcriptional programs was delayed but remarkably higher in APAP. These dissimilarities could be attributed to inherent differences in the two models including significant injury and inflammation exclusively in the APAP model. This study highlights need to study mechanisms of liver regeneration after DILI separately from the mechanisms of regeneration PHX.

Inhibition of cAMP/PKA Pathway Protects Optic Nerve Head Astrocytes against Oxidative Stress by Akt/Bax Phosphorylation-Mediated Mfn1/2 Oligomerization.

Glaucoma is characterized by a progressive optic nerve degeneration and retinal ganglion cell loss, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role in glaucomatous neurodegeneration. Here, we investigate the impact of activation of cyclic adenosine 3',5'-monophosphate (cAMP)/protein kinase A (PKA) pathway on mitochondrial dynamics of ONH astrocytes exposed to oxidative stress. ONH astrocytes showed a significant loss of astrocytic processes in the glial lamina of glaucomatous DBA/2J mice, accompanied by basement membrane thickening and collagen deposition in blood vessels and axonal degeneration. Serial block-face scanning electron microscopy data analysis demonstrated that numbers of total and branched mitochondria were significantly increased in ONH astrocytes, while mitochondrial length and volume density were significantly decreased. We found that hydrogen peroxide- (H2O2-) induced oxidative stress compromised not only mitochondrial bioenergetics by reducing the basal and maximal respiration but also balance of mitochondrial dynamics by decreasing dynamin-related protein 1 (Drp1) protein expression in rat ONH astrocytes. In contrast, elevated cAMP by dibutyryl-cAMP (dbcAMP) or isobutylmethylxanthine treatment significantly increased Drp1 protein expression in ONH astrocytes. Elevated cAMP exacerbated the impairment of mitochondrial dynamics and reduction of cell viability to oxidative stress in ONH astrocytes by decreasing optic atrophy type 1 (OPA1), and mitofusin (Mfn)1/2 protein expression. Following combined treatment with H2O2 and dbcAMP, PKA inhibition restored mitochondrial dynamics by increasing mitochondrial length and decreasing mitochondrial number, and this promoted cell viability in ONH astrocytes. Also, PKA inhibition significantly promoted Akt/Bax phosphorylation and Mfn1/2 oligomerization in ONH astrocytes. These results suggest that modulation of the cAMP/PKA signaling pathway may have therapeutic potential by activating Akt/Bax phosphorylation and promoting Mfn1/2 oligomerization in glaucomatous ONH astrocytes.

Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress.

Over 11 million people in the United States alone have some form of age-related macular degeneration (AMD). Oxidative stress, cell death, and the degeneration of retinal pigment epithelial (RPE) cells contribute to AMD pathology. Recent evidence suggests that ceramide (Cer), a cellular sphingolipid mediator that acts as a second messenger to induce apoptosis, might play a role in RPE cell death. The lysosomal breakdown of Cer by acid ceramidase [N-acylsphingosine amidohydrolase (ASAH)1] into sphingosine (Sph) is the major source for Sph 1-phosphate production, which has an opposing role to Cer and provides cytoprotection. Here, we investigated the role of Cer in human RPE-derived ARPE19 cells under hydrogen peroxide-induced oxidative stress, and show that Cer and hexosyl-Cer levels increase in the oxidatively stressed ARPE19 cells, which can be prevented by overexpression of lysosomal ASAH1. This study demonstrates that oxidative stress generates sphingolipid death mediators in retinal cells and that induction of ASAH1 could rescue retinal cells from oxidative stress by hydrolyzing excess Cers.

Shotgun Sphingolipid Analysis of Human Aqueous Humor.

This protocol provides a step-by-step guide to shotgun sphingolipid analysis of aqueous humor. We describe the Bligh and Dyer crude lipid extraction method and electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled with MZmine 2.21 data processing for identification and ratiometric quantitation of sphingosine, sphingosine-1-phosphate, sphingomyelin, and ceramide.

Increased YAP Activation Is Associated With Hepatic Cyst Epithelial Cell Proliferation in ARPKD/CHF.

Autosomal recessive polycystic kidney disease/congenital hepatic fibrosis (ARPKD/CHF) is a rare but fatal genetic disease characterized by progressive cyst development in the kidneys and liver. Liver cysts arise from aberrantly proliferative cholangiocytes accompanied by pericystic fibrosis and inflammation. Yes-associated protein (YAP), the downstream effector of the Hippo signaling pathway, is implicated in human hepatic malignancies such as hepatocellular carcinoma, cholangiocarcinoma, and hepatoblastoma, but its role in hepatic cystogenesis in ARPKD/CHF is unknown. We studied the role of the YAP in hepatic cyst development using polycystic kidney (PCK) rats, an orthologous model of ARPKD, and in human ARPKD/CHF patients. The liver cyst wall epithelial cells (CWECs) in PCK rats were highly proliferative and exhibited expression of YAP. There was increased expression of YAP target genes, Ccnd1 (cyclin D1) and Ctgf (connective tissue growth factor), in PCK rat livers. Extensive expression of YAP and its target genes was also detected in human ARPKD/CHF liver samples. Finally, pharmacological inhibition of YAP activity with verteporfin and short hairpin (sh) RNA-mediated knockdown of YAP expression in isolated liver CWECs significantly reduced their proliferation. These data indicate that increased YAP activity, possibly through dysregulation of the Hippo signaling pathway, is associated with hepatic cyst growth in ARPKD/CHF.

Sample Preparation and Analysis for Imaging Mass Spectrometry.

Imaging mass spectrometry (IMS) is a novel quantitative technique used to investigative diverse biomolecules in tissue sections. Specifically, IMS uses analytical separation of mass spectrometry to determine the spatial distribution of certain lipids and/or proteins located directly on biological sections from a single tissue sample. Typically, IMS is combined with histological analysis to reveal additional distribution details of characterized biomolecules including cell type and/or subcellular localization. In this chapter, we describe the use of Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-Of-Flight/Time-Of-Flight (TOF/TOF) to analyze various cholesterol and phosphatidylcholine species in atherosclerotic plaque of murine heart aortic valves. In particular, we detail animals used, tissue collection, preparation, matrix application, spectra acquisition for generating a color-coded image based on IMS spectral characteristics.

Segmental outflow of aqueous humor in mouse and human.

The main and only modifiable risk factor in glaucoma, the group of usually late onset progressive and irreversible blinding optic neuropathies, is elevated intraocular pressure (IOP). The increase in IOP is due to impeded aqueous humor (AH) outflow through the conventional pathway. The aberrant increased resistance at the trabecular meshwork (TM), the filter-like region in the anterior eye chamber is the major contributory factor in causing the impeded outflow. In normal as well as in glaucoma eyes the regions of the TM are divided into areas of high and low flow. The collector channels and distal outflow regions are now increasingly being recognized as potential players in contributing to impede AH outflow. Structural and molecular make-up contributing to the segmental blockage to outflow is likely to provide greater insight. Establishing segmental blockage to outflow in model systems of glaucoma such as the mouse in parallel to human eyes will expand our repertoire of tools for investigation. Further study into this area of interest has the potential to ultimately lead to the development of new therapeutics focused on lowering IOP by targeting the various components of segmental blockage of outflow in the TM and in the distal outflow region.

Bile acids promote diethylnitrosamine-induced hepatocellular carcinoma via increased inflammatory signaling.

Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC, but the mechanisms are not known. We investigated the mechanisms of HCC tumor promotion by bile acids the diethylnitrosamine (DEN)-initiation-cholic acid (CA)-induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in threefold increase in number and size of DEN-induced liver tumors. All tumors observed in DEN-treated mice were well-differentiated HCCs. The HCCs observed in DEN-treated CA-fed mice exhibited extensive CD3-, CD20-, and CD45-positive inflammatory cell aggregates. Microarray-based global gene expression studies combined with Ingenuity Pathway Analysis revealed significant activation of NF-κB and Nanog in the DEN-treated 0.2% CA-fed livers. Further studies showed significantly higher TNF-α and IL-1ß mRNA, a marked increase in total and phosphorylated-p65 and phosphorylated IκBα (degradation form) in livers of DEN-treated 0.2% CA-fed mice. Treatment of primary mouse hepatocytes with various bile acids showed significant induction of stemness genes including Nanog, KLF4, Sox2, and Oct4. Quantification of total and 20 specific bile acids in liver, and serum revealed a tumor-associated bile acid signature. Finally, quantification of total serum bile acids in normal, cirrhotic, and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.

Liver-Specific Deletion of Integrin-Linked Kinase in Mice Attenuates Hepatotoxicity and Improves Liver Regeneration After Acetaminophen Overdose.

Acetaminophen (APAP) overdose is the major cause of acute liver failure in the US. Prompt liver regeneration is critical for recovery after APAP hepatotoxicity, but mechanisms remain elusive. Extracellular matrix (ECM)-mediated signaling via integrin-linked kinase (ILK) regulates liver regeneration after surgical resection. However, the role of ECM signaling via ILK in APAP toxicity and compensatory regeneration is unknown, which was investigated in this study using liver-specific ILK knockout (KO) mice. ILK KO and wild-type (WT) mice were treated with 300 mg/kg APAP, and injury and regeneration were studied at 6 and 24 h after APAP treatment. ILK KO mice developed lower liver injury after APAP overdose, which was associated with decreased JNK activation (a key mediator of APAP toxicity). Further, higher glutathione levels after APAP treatment and lower APAP protein adducts levels, along with lower levels of CYP2E1, suggest decreased metabolic activation of APAP in ILK KO mice. Interestingly, despite lower injury, ILK KO mice had rapid and higher liver regeneration after APAP overdose accompanied with increased ß-catenin signaling. In conclusion, liver-specific deletion of ILK improved regeneration, attenuated toxicity after APAP overdose, and decreased metabolic activation of APAP. Our study also indicates that ILK-mediated ECM signaling plays a role in the regulation of CYP2E1 and may affect toxicity of several centrilobular hepatotoxicants including APAP.

Aqueous humor phospholipids of DBA/2J and DBA/2J-Gpnmb(+)/SjJ mice.

PURPOSE: To compare phospholipid profiles [phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI)] of normotensive and hypertensive aqueous humor (AH) from DBA/2J and compare them with phospholipid profiles of DBA/2J-Gpnmb(+)/SjJ mice. METHODS: AH was obtained from young-normotensive DBA/2J, old -hypertensive DBA/2J mice, young and old DBA/2J-Gpnmb(+)/SjJ mice (aging control). Lipids were extracted using modified Bligh and Dyer method and subjected to mass spectrometric identification using appropriate class-specific lipid standards and ratiometric quantification. Corresponding aqueous phase (of extraction) protein concentrations were measured using Bradford method. RESULTS: The total amount of phospholipids showed a decrease in the hypertensive state compared to normotensive state. The total PE and total PS contributed over 50% of the total amount. Total PS showed a remarkable decrease in hypertensive compared to normotensive state. In contrast, total PE in the hypertensive stage presented an increase in amount. Unique lipid species were found encompassing all four phospholipid classes in normotensive as well as in the hypertensive state. Several phospholipid species were found common to both states but with remarkable differences in amount in individual states. The ratio of lysophospholipids to total phospholipids is significantly reduced in the hypertensive state. Commensurate with reduced level of lysophospholipids, we found an increased level of lysophospholipase D (Autotaxin) in the hypertensive state. The difference of total phospholipids between young and old was 35.4% in DBA/2J group but 10% in DBA/2J-Gpnmb(+)/SjJ mice. CONCLUSION: The significant change of phospholipids including lysophospholipids was found commensurate with the elevated intraocular pressure (IOP).

Pro-regenerative signaling after acetaminophen-induced acute liver injury in mice identified using a novel incremental dose model.

Acetaminophen (APAP) overdose results in acute liver failure and has limited treatment options. Previous studies show that stimulating liver regeneration is critical for survival after APAP overdose, but the mechanisms remain unclear. In this study, we identified major signaling pathways involved in liver regeneration after APAP-induced acute liver injury using a novel incremental dose model. Liver injury and regeneration were studied in C57BL/6 mice treated with either 300 mg/kg (APAP300) or 600 mg/kg (APAP600) APAP. Mice treated with APAP300 developed extensive liver injury and robust liver regeneration. In contrast, APAP600-treated mice exhibited significant liver injury but substantial inhibition of liver regeneration, resulting in sustained injury and decreased survival. The inhibition of liver regeneration in the APAP600 group was associated with cell cycle arrest and decreased cyclin D1 expression. Several known regenerative pathways, including the IL-6/STAT-3 and epidermal growth factor receptor/c-Met/mitogen-activated protein kinase pathways, were activated, even at APAP600, where regeneration was inhibited. However, canonical Wnt/ß-catenin and NF-κB pathways were activated only in APAP300-treated mice, where liver regeneration was stimulated. Furthermore, overexpression of a stable form of ß-catenin, where serine 45 is mutated to aspartic acid, in mice resulted in improved liver regeneration after APAP overdose. Taken together, our incremental dose model has identified a differential role of several signaling pathways in liver regeneration after APAP overdose and highlighted canonical Wnt signaling as a potential target for regenerative therapies for APAP-induced acute liver failure.