Murine Hepatocyte Cell Line Research Articles

Dual-Emissive Detection of ATP and Hypochlorite Ions for Monitoring Inflammation-Driven Liver Injury In Vitro and In Vivo.

Reactive oxygen species play a pivotal role in liver disease, contributing to severe liver damage and chronic inflammation. In liver injury driven by inflammation, adenosine-5'-triphosphate (ATP) and hypochlorite ion (ClO-) emerge as novel biomarkers, reflecting mitochondrial dysfunction and amplified oxidative stress, respectively. However, the dynamic fluctuations of ATP and ClO- in hepatocytes and mouse livers remain unclear, and multidetection techniques for these biomarkers are yet to be developed. This study presents RATP-NClO, a dual-channel fluorescent bioprobe capable of synchronously detecting ATP and ClO- ions. RATP-NClO exhibits excellent selectivity and sensitivity for ATP and ClO- ions, demonstrating a dual-channel fluorescence response in a murine hepatocyte cell line. Upon intravenous administration, RATP-NClO reveals synchronized ATP depletion and ClO- amplification in the livers of mice with experimental metabolic dysfunction-associated steatohepatitis (MASH). Through a comprehensive analysis of the principal mechanism of the developed bioprobe and the verification of its reliable detection ability in both in vitro and in vivo settings, we propose it as a unique tool for monitoring changes in intracellular ATP and ClO- level. These findings underscore its potential for practical image-based monitoring and functional phenotyping of MASH pathogenesis.

Regeneration of Non-Alcoholic Fatty Liver Cells Using Chimeric FGF21/HGFR: A Novel Therapeutic Approach.

Non-alcoholic fatty liver disease (NAFLD) has emerged as a significant liver ailment attributed to factors like obesity and diabetes. While ongoing research explores treatments for NAFLD, further investigation is imperative to address this escalating health concern. NAFLD manifests as hepatic steatosis, precipitating insulin resistance and metabolic syndrome. This study aims to validate the regenerative potential of chimeric fibroblast growth factor 21 (FGF21) and Hepatocyte Growth Factor Receptor (HGFR) in NAFLD-afflicted liver cells. AML12, a murine hepatocyte cell line, was utilized to gauge the regenerative effects of chimeric FGF21/HGFR expression. Polysaccharide accumulation was affirmed through Periodic acid-Schiff (PAS) staining, while LDL uptake was microscopically observed with labeled LDL. The expression of FGF21/HGFR and NAFLD markers was analyzed by mRNA analysis with RT-PCR, which showed a decreased expression in acetyl-CoA carboxylase 1 (ACC1) and sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) with increased expression of hepatocellular growth factor (HGF), hepatocellular nuclear factor 4 alpha (HNF4A), and albumin (ALB). These findings affirm the hepato-regenerative properties of chimeric FGF21/HGFR within AML12 cells, opening novel avenues for therapeutic exploration in NAFLD.

Robinetin Alleviates Metabolic Failure in Liver through Suppression of p300-CD38 Axis.

Metabolic abnormalities in the liver are closely associated with diverse metabolic diseases such as non-alcoholic fatty liver disease, type 2 diabetes, and obesity. The aim of this study was to evaluate the ameliorating effect of robinetin (RBN) on the significant pathogenic features of metabolic failure in the liver and to identify the underlying molecular mechanism. RBN significantly decreased triglyceride (TG) accumulation by downregulating lipogenesis-related transcription factors in AML-12 murine hepatocyte cell line. In addition, mice fed with Western diet (WD) containing 0.025% or 0.05% RBN showed reduced liver mass and lipid droplet size, as well as improved plasma insulin levels and homeostatic model assessment of insulin resistance (HOMA-IR) values. CD38 was identified as a target of RBN using the BioAssay database, and its expression was increased in OPA-treated AML-12 cells and liver tissues of WD-fed mice. Furthermore, RBN elicited these effects through its anti-histone acetyltransferase (HAT) activity. Computational simulation revealed that RBN can dock into the HAT domain pocket of p300, a histone acetyltransferase, which leads to the abrogation of its catalytic activity. Additionally, knock-down of p300 using siRNA reduced CD38 expression. The chromatin immunoprecipitation (ChIP) assay showed that p300 occupancy on the promoter region of CD38 was significantly decreased, and H3K9 acetylation levels were diminished in lipid-accumulated AML-12 cells treated with RBN. RBN improves the pathogenic features of metabolic failure by suppressing the p300-CD38 axis through its anti-HAT activity, which suggests that RBN can be used as a new phytoceutical candidate for preventing or improving this condition.

GSK3β/NF-κB -dependent transcriptional regulation of homeostatic hepatocyte Tnf production.

Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-α (TNFα) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 β (GSK3β) and nuclear factor κB (NF-κB) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3β and NF-κB inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-κB activation. Pharmacological inhibition of GSK3β resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3β inhibition decreased nuclear levels of the NF-κB subunits p65 and p50. We determined that NF-κB transcription factor subunit p65 binds to consensus sequence elements present in the murine TNFα promoter and inhibition of GSK3β decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3β and NF-κB inhibition. These results demonstrate that GSK3β and NF-κB are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNFα mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver.NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-α (TNFα)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.

The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

Transforming growth factor-β1 (TGF-β1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis and hepatocellular carcinoma. However, the reciprocal regulation of TGF-β1 signaling and cholesterol metabolism in MASLD is yet unknown. Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA sequencing of murine hepatocyte cell line (alpha mouse liver 12/AML12) and mouse primary hepatocytes treated with TGF-β1. Functional assays were performed on AML12 cells (untreated, TGF-β1 treated, or subjected to cholesterol enrichment [CE] or cholesterol depletion [CD]), and on mice injected with adenovirus-associated virus 8-control/TGF-β1. TGF-β1 inhibited messenger RNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, mouse primary hepatocytes, and adenovirus-associated virus-TGF-β1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue also were reduced upon TGF-β1 treatment. Smad2/3 phosphorylation after 2 hours of TGF-β1 treatment persisted after CE or CD and was mildly increased after CD, whereas TGF-β1-mediated AKT phosphorylation (30min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by72hours of incubation with TGF-β1, including epithelial-mesenchymal transition, actin polymerization, and apoptosis. CD mimicked the outcome of long-term TGF-β1 administration, an effect that was blocked by an inhibitor of the type I TGF-β receptor. In addition, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. TGF-β1 inhibits cholesterol metabolism whereas cholesterol attenuates TGF-β1 downstream effects in hepatocytes.

Gemigliptin exerts protective effects against doxorubicin-induced hepatotoxicity by inhibiting apoptosis via the regulation of fibroblast growth factor 21 expression

AimDoxorubicin is a highly effective anticancer agent that causes hepatotoxicity and cardiotoxicity in patients. Fibroblast growth factor 21, a well-known regulator of glucose and lipid metabolism, exerts cardioprotective effects. Gemigliptin and dipeptidyl peptidase-4 inhibitors are widely used in the treatment of patients with type 2 diabetes. The protective effects of gemigliptin on hepatotoxicity via the increase in fibroblast growth factor 21 expression has not yet been elucidated. This study was designed to investigate the protective effects of gemigliptin against doxorubicin-induced hepatotoxicity via the upregulation of fibroblast growth factor 21 expression in the cultured murine hepatocyte cell line, AML12. MethodsMurine hepatocyte AML12 cells were treated with doxorubicin, fibroblast growth factor 21 and gemigliptin in 0.5% fetal bovine serum medium for 24 h at the indicated doses. Cells were transfected with the fibroblast growth factor 21 small interfering RNA for 24 h, followed by protein isolation. ResultsFibroblast growth factor 21 expression levels were increased during doxorubicin-induced hepatotoxicity in the murine hepatocyte AML12 cells. Fibroblast growth factor 21 treatment prevented doxorubicin-induced hepatotoxicity by attenuating apoptosis. Gemigliptin prevented doxorubicin-induced hepatotoxicity by upregulating fibroblast growth factor 21 expression. However, the protective effects of gemigliptin were blocked by fibroblast growth factor 21 inhibition in doxorubicin-treated AML12 cells. ConclusionThese results indicate that gemigliptin exhibits protective effects against doxorubicin-induced hepatotoxicity by upregulating the fibroblast growth factor 21 expression levels in the cultured murine hepatocyte AML12 cells.

PREX1 depletion ameliorates high-fat diet-induced non-alcoholic fatty liver disease in mice and mitigates palmitic acid-induced hepatocellular injury via suppressing the NF-κB signaling pathway

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver diseases worldwide. Oxidative stress has been considered a key factor in the pathogenesis of NAFLD. Phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 (PREX1), a guanine nucleotide exchange factor for Rac, has been associated with inflammation and oxidative stress. This study aimed to investigate the biological function of PREX1 in the progression of NAFLD. Male C57BL/6 mice were fed a high-fat diet for 12 weeks to induce NAFLD in vivo. Adeno-associated virus type 8-mediated liver-specific PREX1 depletion was employed to investigate the role of PREX1 in the progression of high-fat diet-induced NAFLD. Murine hepatocyte cell line AML-12 was stimulated with palmitic acid for 24 h to induce steatosis in vitro. PREX1 depletion was carried out by transfection with PREX1 small interfering RNA. Results showed that PREX1 depletion exerted protective effects against lipid accumulation, oxidative stress and inflammation and inhibited activation of the nuclear factor-κB (NF-κB) signaling pathway in vivo and in vitro. Subsequently, NF-κB inhibitor BAY11–7082 was applied to investigate the role of the NF-κB signaling pathway in the protective effect of PREX1 inhibition against NAFLD. We confirmed that PREX1 inhibition mitigated palmitic acid-induced hepatocellular inflammation mainly via the NF-κB signaling pathway and lipid accumulation and oxidative stress at least partly via the NF-κB signaling pathway. This study highlights the biological function of PREX1 in the pathogenesis of NAFLD.

Murine AML12 hepatocytes allow Salmonella Typhimurium T3SS1-independent invasion and intracellular fate

Numerous studies have demonstrated the key role of the Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) apparatus as well as its associated effectors in the invasion and intracellular fate of Salmonella in the host cell. Several T3SS1 effectors work together to control cytoskeleton networks and induce massive membrane ruffles, allowing pathogen internalization. Salmonella resides in a vacuole whose maturation requires that the activity of T3SS1 subverts early stages of cell signaling. Recently, we identified five cell lines in which Salmonella Typhimurium enters without using its three known invasion factors: T3SS1, Rck and PagN. The present study investigated the intracellular fate of Salmonella Typhimurium in one of these models, the murine hepatocyte cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway leading to the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho-GTPases. Maturation of the SCV continued through an acidified phase that led to Salmonella multiplication as well as the formation of a tubular network resembling Salmonella induced filaments (SIF). The fact that in the murine AML12 hepatocyte, the T3SS1 mutant induced an intracellular fate resembling to the wild-type strain highlights the fact that Salmonella Typhimurium invasion and intracellular survival can be completely independent of T3SS1.

Activation of p53 After Irradiation Impairs the Regenerative Capacity of the Mouse Liver.

Radiation therapy is one of the treatment methods for hepatocellular carcinoma. However, radiation tolerance of the liver is low, and the detailed effect of radiation on liver regeneration has not been clarified. C57BL/6J mice or hepatocyte‐specific p53 knockout (KO) mice (albumin [Alb]‐Cre Trp53flox/flox ) were irradiated with a single fraction of 10 Gy localized to the upper abdomen. We performed 70% partial hepatectomy (PHx) 24 hours after irradiation. Liver regeneration was assessed by proliferation cell nuclear antigen (PCNA)‐ and Ki‐67‐positive hepatocyte ratios and liver‐to‐body weight ratio after PHx. To establish a fibrosis model, CCl4 was orally administered for 8 weeks. The murine hepatocyte cell line BNL CL.2 (CL2) was irradiated with 10 Gy. Irradiation activated p53, induced downstream p21 in the liver, and delayed liver regeneration after PHx. While PHx increased hepatocyte growth factor (HGF) levels and activated Met with or without irradiation in the regenerative liver, it activated Akt and extracellular kinase 1 and 2 (Erk 1/2) less in irradiated mice than in nonirradiated mice. In CL2 cells cultured with HGF, irradiation suppressed cell growth by decreasing phosphorylated Akt and Erk 1/2 levels, which was abolished by small interfering RNA‐mediated p53 knockdown but not by p21 knockdown. Hepatocyte‐specific knockout of p53 in mice abolished the irradiation‐induced suppression of both liver regeneration and Akt and Erk 1/2 activation after PHx. In the fibrotic mouse model, the survival rate after PHx of irradiated p53 KO mice was higher than that of wild‐type mice. Conclusion: p53 but not p21 is involved in the impaired regenerative ability of the irradiated liver.

Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

Defined as the dysfunction and/or cell death caused by toxic lipids accumulation in hepatocytes, hepatic lipotoxicity plays a pathological role in nonalcoholic fatty liver disease. The cellular and molecular mechanisms underlying lipotoxicity remain to be elucidated. In this study, using AML12 cells, a nontransformed murine hepatocyte cell line, exposed to palmitate (a 16-C saturated fatty acid) as an experimental model, we investigated the role and mechanisms of nicotinamide N-methyltransferase (NNMT), a methyltransferase catalyzing nicotinamide methylation and degradation, in hepatic lipotoxicity. We initially identified activating transcription factor 4 (ATF4) as a major transcription factor for hepatic NNMT expression. Here, we demonstrated that palmitate upregulates NNMT expression via activating ATF4 in a mechanistic target of rapamycin complex 1 (mTORC1)-dependent mechanism in that mTORC1 inhibition by both Torin1 and rapamycin attenuated ATF4 activation and NNMT upregulation. We further demonstrated that the mTORC1-dependent ATF4 activation is an integral signaling event of unfolded protein response (UPR) as both ATF4 activation and NNMT upregulation by tunicamycin, a well-documented endoplasmic reticulum (ER) stress inducer, are blunted when hepatocytes were pretreated with Torin1. Importantly, our data uncovered that NNMT upregulation contributes to palmitate-induced hepatotoxicity as NNMT inhibition, via either pharmacological (NNMT inhibitors) or genetic approach (siRNA transfection), provided protection against palmitate lipotoxicity. Our further mechanistic exploration identified protein kinase A (PKA) activation to contribute, at least, partially to the protective effect of NNMT inhibition against lipotoxicity. Collectively, our data demonstrated that NNMT upregulation by the mTORC1-ATF4 pathway activation contributes to the development of lipotoxicity in hepatocytes.

MTORC1-IRE1α pathway activation contributes to palmitate-elicited triglyceride secretion and cell death in hepatocytes.

Lipotoxicity induced by saturated fatty acids (SFA) plays a pivotal role in the pathogenesis of a variety of obesity-related metabolic disorders; however, the exact mechanism(s) underlying lipotoxicity development remains elusive. The liver plays a central role in regulating intrahepatic and circulatory lipid homeostasis. In the current study, we identified that mammalian target of rapamycin complex 1 (mTORC1) activation plays an important role in regulating the detrimental effects of SFA palmitate in hepatocytes, in specific cell death, and TG overproduction. Furthermore, our data confirmed that palmitate-induced mTORC1 activation is attributable to its stimulatory effect on IRE1α, one of three canonical pathways activated during ER stress. Importantly, IRE1α inhibition prevented palmitate-triggered cell death and TG overproduction, suggesting mTORC1-IRE1α pathway is mechanistically implicated in palmitate lipotoxicity. The data obtained in the current investigation support future study to explore the therapeutic potential of targeting the mTORC1-IRE1α pathway as a novel clinical strategy for the treatment of metabolic disorders involving lipotoxicity.

CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation

Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.

Prohibitin 1 Deficiency Causes Impairment of Lipid Metabolism and the Disruption Is Deteriorated by Excessive Palmitic Acids in Hepatocytes (P08-106-19)

ObjectivesNon-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid in hepatocytes. In spite of the growing number of NAFLD patients with the increased prevalence of obesity, the pathogenesis of NAFLD remains to be fully elucidated. Prohibitin1 (Phb1) is an evolutionarily conserved protein, which is expressed predominantly on the inner mitochondrial membrane. Several studies have provided evidences that the Phb1 plays a crucial role not only in proliferation of hepatocytes, but also in lipid metabolism in adipocytes. Since the liver is the major site for lipid metabolism, this study was aimed to understand the lipid metabolism regulated by Phb1 in hepatocytes. MethodsNormal murine hepatocyte cell line, AML12 was used in this study and Phb1 was disrupted by small interfering RNA (siRNA). These siRNA transfected cells were further treated with saturated fatty acids (SFAs), palmitic acid (PA) for 24 hours. Then the cell viability and the mRNA expression level of key enzymes involved in lipid metabolism were determined. ResultsApproximately 20% cell viability decreased when Phb1 was silenced and the cell viability diminished dramatically after PA treatment in both control and Phb1 deficiency groups. Without excessive fatty acids (FAs) inside the cells, Phb1 deficiency led to significant mRNA expression of stearoyl-CoA desaturase-1 (Scd1) to generate triglycerides (TG) and activated the endoplasmic reticulum (ER) stress response by upregulating the mRNA expression level of C/EBP homologous protein (Chop). The elevated expression of peroxisome proliferator activated receptor gamma (PPARγ), which was reported to be upregulated in the livers of NAFLD patients, was also found in this study. When intracellular FAs were surplus, the catabolism of FAs was inhibited by downregulation of carnitine palmitoyltransferase 1a (Ctp1a), while the synthesis of TG was further promoted by significantly increased mRNA expression level of Scd1. The elevated mRNA expression level of Chop and Pparγ induced by Phb1 disruption were further elevated. ConclusionsThis study, Phb1 deficiency led to altered lipid metabolism, resulting in the of lipid accumulation inside the cells and ER stress, and these cytotoxic effects were further deteriorated upon the excessive PA treatment. Funding SourcesNational Research Foundation of Korea, 2-1604-001.

Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication.

Hepatitis B virus (HBV) is a major cause of chronic liver diseases, including hepatitis, cirrhosis, and hepatocellular carcinoma. HBV research has been hampered by the lack of robust cell culture and small animal models of HBV infection. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor has been a landmark advance in HBV research in recent years. Ectopic expression of NTCP in nonpermissive HepG2, Huh7, and AML12 cell lines confers HBV susceptibility. However, HBV replication in these human and murine hepatocyte cell lines appeared suboptimal. In the present study, we constructed stable NTCP-expressing HepG2 and AML12 cell lines and found that HBV permissiveness is correlated with NTCP expression. More significantly, we developed robust HBV cell culture models by treating the HBV-infected cells with dimethyl sulfoxide (DMSO) and hydrocortisone, which significantly promoted HBV replication and production. Mechanistic studies suggested that hydrocortisone significantly enhanced the transcription and expression of PGC1α and HNF4α, which are known to promote HBV transcription and replication. These new human and murine hepatocyte culture systems of HBV infection and replication will accelerate the determination of molecular aspects underlying HBV infection, replication, and morphogenesis in human and murine hepatocytes. We anticipate that our HBV cell culture models will also facilitate the discovery and development of antiviral drugs towards the ultimate eradication of chronic hepatitis B virus infection.IMPORTANCE HBV research has been greatly hampered by the lack of robust cell culture and small animal models of HBV infection and propagation. The discovery of NTCP as an HBV receptor has greatly impacted the field of HBV research. Although HBV infection of NTCP-expressing human and murine hepatocyte cell lines has been demonstrated, its replication in cell culture appeared inefficient. To further improve cell culture systems of HBV infection and replication, we constructed NTCP-expressing HepG2 and AML12 cell lines that are highly permissive to HBV infection. More significantly, we found that DMSO and hydrocortisone markedly enhanced HBV transcription and replication in human and murine hepatocytes when added to the cell culture medium. These new cell culture models of HBV infection and replication will facilitate HBV research and antiviral drug discovery towards the ultimate elimination of chronic hepatitis B virus infection.

DNase II activated by the mitochondrial apoptotic pathway regulates RIP1-dependent non-apoptotic hepatocyte death via the TLR9/IFN-β signaling pathway.

Cell death, including apoptotic and non-apoptotic cell death, is frequently observed in liver disease. Upon activation of the mitochondrial apoptotic pathway, mitochondria release not only apoptogenic cytochrome c but also mitochondrial DNA (mtDNA) into the cytosol. The impact of DNase II, a lysosomal acid DNase that degrades mtDNA, on hepatocyte death remains unclear. Administration of ABT-737, a Bcl-xL inhibitor, upregulated DNase II activity in murine hepatocyte cell line BNL CL.2 cells and induced apoptosis. In cells treated with DNase II siRNA, ABT-737 led to accumulation of mtDNA in the cytosol and increased expression of interferon (IFN)-β and induction of propidium iodide (PI)-positive cells, in addition to apoptosis. Induced PI-positive cells were suppressed by RIP1 inhibitor, Necrostatin-1, but not by pan-caspase inhibitor, ZVAD-FMK, suggesting non-apoptotic cell death. Both the increase in IFN-β and the induction of non-apoptotic cell death were abolished by administering a TLR9 antagonist, ODN2088, or by the removal of mtDNA from cells with ethidium bromide. Hepatocyte-specific Mcl-1 knockout mice developed hepatocyte apoptosis accompanied by upregulated DNase II activity in their livers. Further knockout of DNase II induced IFN-β expression and RIP1-dependent non-apoptotic hepatocyte death, both of which were suppressed by the administration of ODN2088. Mice fed a high-fat diet (HFD), an obesity-associated fatty liver model, showed increased expression of IFN-β with suppression of DNase II activity in their livers and developed not only hepatocyte apoptosis but also non-apoptotic hepatocyte death. Hepatocyte-specific knockout of DNase II exacerbated HFD-induced non-apoptotic hepatocyte death and liver fibrosis. In conclusion, without DNase II, apoptotic stimulation on hepatocytes induces TLR9-dependent IFN-β production and RIP1-dependent non-apoptotic cell death originating from mtDNA. In fatty livers, DNase II activity is suppressed in contrast to simple inactivation of Bcl-xL or Mcl-1, and both apoptotic and non-apoptotic hepatocyte death can develop, leading to the progression of liver fibrosis.

The pseudophosphatase phogrin enables glucose-stimulated insulin signaling in pancreatic β cells

Autocrine insulin signaling is critical for pancreatic β-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic β cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin-IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic β-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment-induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic β cells.

315. Optimization of the Capsid of Recombinant Adeno-Associated Virus 6 (AAV6) Vectors for Liver-, and Muscle-Directed Gene Therapy

Recombinant adeno-associated virus (AAV) vectors are currently in use in a number of Phase I/II clinical trials. However, large vector doses are needed to achieve therapeutic effects. The ubiquitinproteasome pathway plays a critical role in the intracellular trafficking of AAV vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. We have previously reported that substitution of critical amino acid residues dramatically increases the transduction efficiency of vectors based on AAV serotypes 2, 3, 5, and 8 by preventing capsid phosphorylation and subsequent proteasomal degradation.In the present studies, we document that the combination of mutations of specific tyrosine (Y), serine (S), threonine (T) and lysine (K) residues on the capsid of AAV6 vectors also significantly increases the transduction efficiency compared with the wild-type (WT) AAV6 vectors. AAV6 capsid contains 7 tyrosine, 17 serine, 19 threonine and 11 lysine residues, which are surface-exposed. Initially, 17 single-mutants were generated based on the AAV6 crystal structure and overlap of amino acid positions with previously studied serotypes: Y445F, Y705F, Y731F, S455V, S472V, S499V, S551V, S663V, S669V, T251V, T492V, T665V, T722V, K531E, K567E, K666E, K707E. Next, the best performing Y-, S-, T-, and K- mutations were combined onto a single AAV6 capsid to generate 9 double-mutants: Y705+731F, Y731F+T492V, T492V+S663V, T492V+S551V, T665V+S551V, S663V+S551V, T492V+K531E, S663V+K531E, T492V+K666E; 7 triple-mutants: Y445+705+731F, Y705+731F+S663V, Y705+731F+S551V, Y705+731F+T492V, Y705+731F+K531E, Y705+731F+K666E, T492V+S663V+K531E; and two quadruple-mutants: Y705+731F+T492V+S663V, and Y705+731F+T492V+K531E. We identified several AAV6-mutant vectors, such as S663+551V, Y705+731F+T492V and Y705+731F+K531E, which were particularly efficient. These vectors increased the transduction efficiency by approximately 5- to 8-fold in a murine hepatocyte cell line, H2.35, and approximately 3- to 5-fold in a murine myoblast cell line, C2C12, over the WT AAV6 vector in vitro. Similar results were obtained in murine hepatocytes and muscle in vivo after tail-vein or intra-muscular injections of these vectors, followed by whole-body bioluminescence imaging of C57BL/6 mice.These residues are localized towards the base of the protrusions surrounding the icosahedral 3-fold axes of the capsid (T492S, S551V and K531E), in the depression surrounding the 2-fold axes (Y705F and Y731Y), or the HI loop (Y663S). This study thus identifies these capsid surface regions as being critical for the trafficking and transduction properties of the AAV6 vectors.In summary, our studies have led to the generation of novel capsid-optimized AAV6 vectors for their potential use in liver-directed, and muscle-directed gene therapy in humans.

Low-Density Lipoprotein-Mediated Delivery of Docosahexaenoic Acid Selectively Kills Murine Liver Cancer Cells

The natural omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), has recently been credited for possessing anticancer properties. Herein, we investigate the cytotoxic actions of DHA-loaded low-density lipoprotein (LDL) nanoparticles in normal and liver cancer cells. LDL-DHA nanoparticles were prepared and subjected to extensive biophysical characterization. The therapeutic utility of LDL-DHA nanoparticles was evaluated in normal and malignant murine hepatocyte cell lines, TIB-73 and TIB-75, respectively. The engineered LDL-DHA nanoparticles possessed enhanced physical and oxidative stabilities over native LDL and free DHA. Dose-response studies showed that therapeutic doses of LDL-DHA nanoparticles that completely killed TIB-75 were innocuous to TIB-73. The selective induction of lipid peroxidation and reactive oxygen species in the cancer cells was shown to play a central role in LDL-DHA nanoparticle-mediated cytotoxicity. In summary, these findings indicate that LDL-DHA nanoparticles show great promise as a selective anticancer agent against hepatocellular carcinoma.

Optimization of the capsid of recombinant adeno-associated virus 2 (AAV2) vectors: the final threshold?

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2–3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.

The regulatory role of microRNA-1187 in TNF-α-mediated hepatocyte apoptosis in acute liver failure

In the current study, we aimed at elucidating the regulatory mechanisms through which microR-1187 (miR-1187) participates in hepatocyte apoptosis in acute liver failure (ALF). An ALF model was induced with D-galactosamine (D-GalN) plus lipopolysaccharide (LPS) in BALB/c mice. The hepatic miRNA expression profile was detected by microarray analysis and verified by quantitative real-time PCR (qRT-PCR). The possible underlying mechanism was investigated in vitro using an embryonic murine hepatocyte cell line (BNLCL2) and miR-1187 mimic. Caspase-8 protein was detected by Western blotting and cell apoptosis was assayed by flow cytometry. Hepatic miR-1187 was down-regulated in ALF mice based on microarray data (P<0.001) and verified by qRT-PCR (P<0.01). Target scan revealed that caspase-8 was the putative target of miR-1187. In an in vitro study, miR-1187 showed the highest up-regulation in BNLCL2 cells transfected with the miR-1187 mimic at a 50 nM concentration for 12 h compared with cells transfected with the non-specific mimic (P<0.001). miR-1187 was down-regulated (P<0.01) but caspase-8 mRNA (P<0.01) as well as protein (P<0.05) were up-regulated in the BNLCL2 cells treated with D-GalN/TNF. Furthermore, overexpressed miR-1187 reduced caspase-8 expression at both the mRNA and protein levels significantly (P<0.01 and P<0.05 respectively), and significantly attenuated the apoptotic rate of BNLCL2 cells (P<0.05). We show that miR-1187 regulates hepatocyte apoptosis by targeting caspase-8. miR-1187 may serve as a potential therapeutic target for the treatment of ALF.