VA13 Cells Research Articles

A Knockout of the IFITM3 Gene Increases the Sensitivity of WI-38 VA13 Cells to the Influenza A Virus.

One of the ways to regulate the sensitivity of human cells to the influenza virus is to knock out genes of the innate immune response. Promising targets for the knockout are genes of the interferon-inducible transmembrane protein (IFITM) family, in particular the IFITM3 gene, whose product limits the entry of a virus into the cell by blocking the fusion of the viral and endosomal membranes. In this study, by means of genome-editing system CRISPR/Cas9, monoclonal cell lines with an IFITM3 knockout were obtained based on WI-38 VA13 cells (human origin). It was found that such cell lines are more sensitive to infection by influenza A viruses of various subtypes. Nevertheless, this feature is not accompanied by an increased titer of newly formed viral particles in a culture medium.

4+2]-Cycloaddition to 5-Methylidene-Hydantoins and 5-Methylidene-2-Thiohydantoins in the Synthesis of Spiro-2-Chalcogenimidazolones.

Novel hydantion and thiohydantoin-based spiro-compounds were prepared via theDiels-Alder reactions between 5-methylidene-hydantoins or 5-methylidene-2-thiohydantoins and 1,3-dienes (cyclopentadiene, cyclohexadiene, 2,3-dimethylbutadiene, isoprene). It was shown that the cycloaddition reactions proceed regioselectively and stereoselectively with the formation of exo-isomers in the reactions with cyclic dienes andthe less sterically hindered products in the reactions with isoprene. Reactions of methylideneimidazolones with cyclopentadiene proceed viaco-heating the reactants; reactions with cyclohexadiene, 2,3-dimethylbutadiene, and isoprene require catalysis by Lewis acids. It was demonstrated that ZnI2 is an effective catalyst in the Diels-Alder reactions of methylidenethiohydantoins with non-activated dienes. The possibility of alkylation and acylation of the obtained spiro-hydantoinsat the N(1)nitrogen atoms with PhCH2Cl or Boc2O and the alkylation of the spiro-thiohydantoinsat the S atoms with MeI or PhCH2Cl in high yields have been demonstrated. The preparativetransformation of spiro-thiohydantoins into corresponding spiro-hydantoinsin mild conditions by treating with 35% aqueous H2O2 or nitrile oxide has been carried out. The obtained compounds show moderate cytotoxicity in the MTT test on MCF7, A549, HEK293T, and VA13 cell lines. Some of the tested compounds demonstrated some antibacterial effect against Escherichia coli (E. coli) BW25113 DTC-pDualrep2 but were almost inactive against E. coli BW25113 LPTD-pDualrep2.

Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•- Levels and Glutathione Depletion.

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable, cell-permeable redox-cycling nitroxide water-soluble superoxide dismutase (SOD) mimetic agent. However, little is known about its cytotoxic effects on lung-related cells. Thus, the present study investigated the effects of Tempol on cell growth and death as well as changes in reactive oxygen species (ROS) and glutathione (GSH) levels in Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblast cells. Results showed that Tempol (0.5~4 mM) dose-dependently inhibited the growth of lung cancer and normal cells with an IC50 of approximately 1~2 mM at 48 h. Tempol induced apoptosis in lung cells with loss of mitochondrial membrane potential (MMP; ∆Ψm) and activation of caspase-3. There was no significant difference in susceptibility to Tempol between lung cancer and normal cells. Z-VAD, a pan-caspase inhibitor, significantly decreased the number of annexin V-positive cells in Tempol-treated Calu-6, A549, and WI-38 VA-13 cells. A 2 mM concentration of Tempol increased ROS levels, including O2•- in A549 and WI-38 VA-13 cells after 48 h, and specifically increased O2•- levels in Calu-6 cells. In addition, Tempol increased the number of GSH-depleted cells in Calu-6, A549, and WI-38 VA-13 cells at 48 h. Z-VAD partially downregulated O2•- levels and GSH depletion in Tempol-treated these cells. In conclusion, treatment with Tempol inhibited the growth of both lung cancer and normal cells via apoptosis and/or necrosis, which was correlated with increased O2•- levels and GSH depletion.

TERRA R-LOOPS ARE INCREASED IN ACUTE MYELOID LEUKEMIA

Telomeric repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from subtelomeric regions towards telomere ends. TERRA binds to subtelomeric and telomeric regions, forming R-loops, structures composed of DNA-RNA hybrids and displaced single-stranded DNA. TERRA R-loops protect telomeres from DNA damage and promote telomere elongation in short telomeres by enabling telomerase or by activating alternative telomere lengthening. However, high or low TERRA levels are associated with DNA damage and telomere dysfunction. RNaseH1 and RNaseH2 cleave R-loops at telomeres, and regulate R-loops levels. The role of TERRA R-loops in telomere maintenance in human cells is not well understood, since conflicting results derive from experiments using diverse organisms and commercial cell lines, with few studies applying primary human cells. Here we describe for the first time the TERRA physiology in hematopoietic cells of patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), telomeropathies (TEL), and healthy subjects. We initially screened 17 patients with AML, 5 with ALL, 9 with TEL, and 20 healthy subjects. Additionally, 88 AML patients were studied. HeLa, H1, THP1, NB4, and VA13 cell lines were used for functional experiments, and lentivirus vectors containing GFP or RNaseH1-GFP motifs were employed. ATRX, TRF2, TERT, RNaseH1, RNaseH2, TERRA 10q, 15q, 20q, XqYq, XpYp, and 17p expression levels were measured by RT-qPCR. Telomere length was determined by qPCR. R-loops were assessed by immunofluorescence using the S9.6 antibody. Cell line viability was defined by MTT assay. NGS panel was designed to evaluate variants in splicing factors and epigenetic regulators. In the screening population, TERRA expression (10q, 15q, 20q, and XqYq) was significantly increased in AML samples (p < 0.05) in comparison to healthy controls and VA-13 cells. Telomeres were very short in AML samples and TEL patients compared to controls. R-loop formation was significantly increased in AML samples compared to controls (p < 0.01). To examine the TERRA physiology in AML, we further eveluated 88 AML samples. Hierarchical cluster algorithm based on TERRA expression defined two AML groups, one with high and the other with low TERRA expression. Both groups dysplayed short telomeres, but the high TERRA AML group exhibited low TERT and RNaseH2 expression levels (p < 0.05). There was no difference between groups on somatic variants or RNaseH1, TRF2, ATRX, and TRF2 expression levels. AML cell lines were transfected RNaseH1-GFP-containing or empty vector lentiviruses and treated with cytarabine. AML cells with exogenous RNAseH1 expression showed more sensitivity to cytarabine in MTT assays (p < 0.05) than cells transfected with the empty vector. These results show for the first time that TERRA is dysregulated in AML. High TERRA levels are unique to a subset of AML cases with low TERT expression. High TERRA correlated with the accumulation of R-loops, which may be therapeutic targets. TERRA upregulation in AML due to short telomeres is unlikely, given the low TERRA levels in TEL patients. TERRA upregulation did not appear to be directly related to neoplastic differentiation, given low TERRA in ALL. TERRA expression and R-loops are dysregulated in AML. TERRA R-loops inhibition increases chemotherapy-induced apoptosis in AML cell lines and may serve as a therapeutic target.

Anticancer activity of G4-targeting phenoxazine derivatives in vitro

G4-stabilizing ligands are now being considered as anticancer, antiviral and antibacterial agents. Phenoxazine is a promising scaffold for the development of G4 ligands. Here, we profiled two known phenoxazine-based nucleoside analogs and five new nucleoside and non-nucleoside derivatives against G4 targets from telomere repeats and the KIT promoter region. Leading new derivatives exhibited remarkably high G4-stabilizing effects (comparable or superior to the effects of the commonly used selective G4 ligands PDS and NMM) and selectivity toward G4s over duplex (superior to BRACO-19). All phenoxazine-based ligands inhibited cellular metabolic activity. The phenoxazine derivatives were particularly toxic for lung adenocarcinoma cells A549' and human liver cancer cells HepG2 (CC50 of the nucleoside analogues in the nanomolar range), but also affected breast cancer cells MCF7, as well as immortalized fibroblasts VA13 and embryonic kidney cells HEK293t (CC50 in the micromolar range). Importantly, the CC50 values varied mostly in accordance with G4-binding affinities and G4-stabilizing effects, and the phenoxazine derivatives localized in the cell nuclei, which corroborates G4-mediated mechanisms of action.

Syntheses and structure-activity relationship study of santamarine, douglanine, dihydrosantamarine and four series of 1-/3-oxygenated eudesman-12,6α-olides

Herein, we report the syntheses and structure-activity relationships of Santamarine, Douglanine, dihydrosantamarine, 1-oxygenated and 3-oxygenated eudesman-12,6α-lactones. The anti-inflammatory and anti-cancer activities were measured by the inhibition on induction of intercellular adhesion molecule (ICAM-1), killing function of cytotoxic T lymphocytes (CTLs), and cytotoxic activities to cell lines (VA-13, HepG-2, and WI-38), respectively. α-Methylene-γ-lactones 13 with a 1-oxo-2-en moiety showed significant inhibition toward ICAM-1(IC50 12.8 μM), the killing function of CTLs (IC50 11.0 μM) and the VA-13 cell line (IC50 2.84 μM), 21 with α-bromo-ketone moiety showed significant inhibition to VA-13(IC50 1.85 μM). Both 13 ((cell viability)/(inhibitory activity of ICAM-1) = 6.0-fold, WI-38/VA-13 = 1.28-fold) and 21 (WI-38/VA-13 = 1.28-fold) were within safety satisfaction. To conclude, the α,β-unsaturated-ketone and α-bromo-ketone moieties were essential for α-methylene-eudesman-12,6α-olides' anti-inflammatory and anti-cancer activities.

Dispirooxindole-β-Lactams: Synthesis via Staudinger Ketene-Imine Cycloaddition and Biological Evaluation.

In this work, we present the first synthesis of dispirooxindole-β-lactams employing optimized methodology of one-pot Staudinger ketene-imine cycloaddition with N-aryl-2-oxo-pyrrolidine-3-carboxylic acids as the ketene source. Spiroconjugation of indoline-2-one with β-lactams ring is considered to be able to provide stabilization and wide scope of functionalization to resulting scaffolds. The dispipooxindoles obtained demonstrated medium cytotoxicity in the MTT test on A549, MCF7, HEK293, and VA13 cell lines, and one of the compounds demonstrated antibacterial activity against E. coli strain LPTD.

5‑Aminosalicylic acid attenuates paraquat‑induced lung fibroblast activation and pulmonary fibrosis of rats.

Pulmonary fibrosis is one of the most important pathological processes associated with paraquat (PQ) poisoning. 5‑Aminosalicylic acid (5‑ASA) has been shown to be a promising agent against fibrotic diseases. In the present study, the alleviating role of 5‑ASA was evaluated in a rat model of pulmonary fibrosis induced by PQ intragastric poisoning (80mg/kg). Wistar rats were divided into control, PQ, 5‑ASA (30mg/kg daily, 14days) and PQ + 5‑ASA groups. Histological examination revealed congestion, edema and inflammatory cell infiltration in the bronchial and alveolar walls at3days after PQ exposure. Alveolar septum thickening with alveolar lumen narrowing was observed at14days, while fibroblast proliferation, increase in collagen fiber number and fibrous thickening of the alveolar walls were observed at28day. All the aforementioned pulmonary injury changes in the PQ group were attenuated in the PQ + 5‑ASA group. Hydroxyproline (HYP) content increased in the lung tissues of the rats at14days after PQ treatment and reached a peak at28days. Compared with the PQ group, HYP contents of lung tissue decreased at14and28days after PQ + 5‑ASA treatment. Masson's trichrome staining revealed that the increase in the amount of collagen fibers in the lung tissues of rats in the PQ group was inhibited by 5‑ASA treatment, further confirming the alleviating effect of 5‑ASA on fibrosis. In addition, the results showed that 5‑ASA attenuated the upregulation of transforming growth factor‑β1 and phosphorylated‑SMAD3, and the reduction of peroxisome proliferator activated receptor γ induced by PQ in lung tissue of rats and human lung fibroblast WI‑38 VA13 cells. In conclusion, the results suggested that 5‑ASA had an alleviating effect on PQ‑induced pulmonary fibrosis, partly by suppressing the activation of the TGF‑β1 signaling pathway.

Amphotericin b effect on the sensitivity to influenza infection of WI-38 VA-13 cells with IFITM3 gene knockout

BACKGROUND: The application of CRISPR/Cas9 is one of the most rapidly developing areas in biotechnology. This method was used to obtain clones of а human origin cell line with knockout of one or more genes of the IFITM family, representing host restriction factors for influenza infection. Amphotericin B has previously been shown to promote influenza infection by blocking IFITM3 function.&#x0D; AIM: The aim of this study was to evaluate the effect of amphotericin B on the sensitivity of IFITM knockout cells to influenza A virus infection.&#x0D; MATERIALS AND METHODS: WI-38 VA-13 cells and mutant clones with IFITM3 knockout (F3 clone) or IFITM1, IFITM3 knockout (clone E12) were infected with influenza virus A/PR/8/34 (H1N1) in the presence or absence of amphotericin B. Forty-four hours after infection, the culture medium was taken to determine the infectious activity of the virus by titration in the MDCK cell culture, as well as the hemagglutinating activity of the virus. The infected cells were stained with fluorescently labeled antibodies against the viral NP protein, and the number of NP-positive cells was determined by flow cytometry.&#x0D; RESULTS: The addition of amphotericin B increased the hemagglutinating and infectious activity of the virus in WI-38 VA-13cells, while the difference was insignificant for clones with IFITM gene knockout. A similar dependency was obtained for the percent of infected cells.&#x0D; CONCLUSIONS: Mutant cells with a knockout of one or several genes of the IFITM family were equally susceptible to influenza infection regardless of the addition of amphotericin B, which confirms the crucial importance of a defect in the IFITM3 protein in increasing the permissiveness of cells to influenza A virus.

Tempol differently affects cellular redox changes and antioxidant enzymes in various lung-related cells

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a potential redox agent in cells. The present study investigated changes in cellular reactive oxygen species (ROS) and glutathione (GSH) levels and in antioxidant enzymes, in Tempol-treated Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblasts. Results demonstrated that Tempol (0.5–4 mM) either increased or decreased general ROS levels in lung cancer and normal cells at 48 h and specifically increased O2•− levels in these cells. In addition, Tempol differentially altered the expression and activity of antioxidant enzymes such as superoxide dismutase, catalase, and thioredoxin reductase1 (TrxR1) in A549, Calu-6, and WI-38 VA-13 cells. In particular, Tempol treatment increased TrxR1 protein levels in these cells. Tempol at 1 mM inhibited the growth of lung cancer and normal cells by about 50% at 48 h but also significantly induced cell death, as evidenced by annexin V-positive cells. Furthermore, down-regulation of TrxR1 by siRNA had some effect on ROS levels as well as cell growth inhibition and death in Tempol-treated or -untreated lung cells. In addition, some doses of Tempol significantly increased the numbers of GSH-depleted cells in both cancer cells and normal cells at 48 h. In conclusion, Tempol differentially increased or decreased levels of ROS and various antioxidant enzymes in lung cancer and normal cells, and induced growth inhibition and death in all lung cells along with an increase in O2•− levels and GSH depletion.

Synthesis of 4,4′-substituted 2,2′-[ethane-1,2-diylbis(selanediyl)]bis(1H-imidazol-5(4H)-ones)

Synthesis of new bis(seleno-imidazolone) derivatives bearing the alkyl and aryl moieties at the N(1) atom of the five-membered ring was developed. Cytotoxicity of the synthesized compounds against A549, VA-13, MCF-7, and HEK293T cell lines was evaluated.

TERT and TERC mutations detected in cryptic dyskeratosis congenita suppress telomerase activity.

A cryptic form of dyskeratosis congenita (cDKC) has a gradual onset without the characteristic physical findings of DKC. cDKC is distinguished from other forms of bone marrow failure (BMF) through analysis of telomere shortening and gene mutations. Mutations in the telomerase reverse transcriptase (TERT) and telomere RNA component (TERC) genes have been detected in most Japanese cDKC patients. Therefore, we investigated the impact of each TERT and TERC mutation on telomerase activity. TERT and TERC mutants observed in DKC or cDKC patients were transfected into Saos-2 or VA13+TERT (TERT-expressing VA13 cells) cells to measure telomerase activity. Telomerase activity in cells expressing a mutant detected in cDKC patients was significantly lower (P<.0001) than in cells expressing the wild-type genes. In addition, some TERT mutations seen in cDKC (p.P632R, p.T726M) caused weaker (P=.0013) suppression of telomerase activity than others (p.G106W and p.G682D). In contrast, telomerase activity in cells expressing a TERT or TERC mutant detected in DKC patients did not significantly differ from cells expressing the wild-type genes. These findings suggest that TERT and TERC mutations detected in cDKC patients could potentially contribute to the pathogenesis of cDKC by blocking telomerase activity. However, TERT and TERC mutations detected in DKC patients did not affect telomerase activities, which means studying the telomerase activity of mutants are not always useful for the diagnosis of DKC.

Ullmann-type C-Se Cross-Coupling in the Hydantoin Family: Synthesis, Mechanistic Studies, and Tests of Biological Activity.

An attractive strategy for C-Se bond formation by Ullmann-type copper(I)-promoted cross-coupling is developed. A wide range of aryliodides reacts with various disubstituted 2-selenohydantoins under mild conditions and provides Se-arylated imidazolines in moderate to high yields. Computational mechanistic studies show the oxidative addition/intramolecular reductive elimination likely to be the lowest-energy pathway. Cytotoxic activity of all 43 reaction products has been tested in vitro against MCF7 and A549 cancer cell lines with VA13 and MCF10a control cells.

Nagilactone D ameliorates experimental pulmonary fibrosis in vitro and in vivo via modulating TGF-β/Smad signaling pathway

Pulmonary fibrosis is a prototypic chronic progressive lung disease with high morbidity and mortality worldwide. Novel effective therapeutic agents are urgently needed owing to the limited treatment options in clinic. Herein, nagilactone D (NLD), a natural dinorditerpenoid obtained from Podocarpus nagi, was found to suppress transforming growth factor-β1 (TGF-β1)-mediated fibrotic process in vitro and bleomycin (BLM)-induced pulmonary fibrosis in vivo. NLD attenuated TGF-β1-induced expression of fibrotic markers including type I and III collagen, fibronectin, α-SMA, and CTGF in human pulmonary fibroblasts (WI-38 VA-13 and HLF-1 cells). Mechanism study indicated that NLD suppressed TGF-β1-induced up-regulation of TβR I, and Smad2 phosphorylation, nuclear translocation, and transcriptional activation. Moreover, NLD ameliorated BLM-induced histopathological abnormalities in the lungs of experimental fibrotic mice, suppressed synthesis of relative fibrotic markers and fibroblast-to-myofibroblast transition, as well as BLM-induced up-regulation of TβR I expression and Smad signaling in mouse lungs. These data collectively support NLD to be a potential therapeutic agent for pulmonary fibrosis.

Nitrosylsulfuric acid in the synthesis of 5-chloroisoxazoles from 1,1-dichlorocyclopropanes

Nitrosylsulfuric acid is shown to be a usable reagent for the synthesis of 5-chloroisoxazoles from readily available 1,1-di-chlorocyclopropanes via nitrosation–heterocyclization reaction. A cytotoxicity of some of the prepared 5-chloroisoxazoles towards MCF7, A549, HEK293T and VA13 cell lines was evaluated.

Synthesis of dispirooxindoles containing N-unsubstituted heterocyclic moieties and study of their anticancer activity

A convenient method is proposed for the synthesis of N-unsubstituted spiroxindoles with different heterocyclic moieties (2-thiohydantoin, hydantoin, and thiazolidine) by the regio-selective 1,3-dipolar cycloaddition of azomethine ylides, generated from isatins and sarcosine, to arylidene derivatives of corresponding heterocycles. The cytotoxicity of compounds was tested by the MTT method against MCF7, A549, HEK, and VA13 cell lines and compared with the anticancer drug Nutlin-3a. The best bioactivity was observed for hydantoin-based dispiroindolinones, the most cytotoxic compound demonstrated selectivity against A549 lung cancer cells with an IC50 value of 6.6±1.6 μmol L−1.

Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts

Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.

Giantin Is Required for Post-Alcohol Recovery of Golgi in Liver Cells

In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Perinuclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieber–DeCarli diet)). For recovery, EtOH was removed and replenished with control medium (48 h for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.

Synthesis and cytotoxicity of novel dispiro derivatives of 5-arylidenoxazolones, potential inhibitors of p53—MDM2 protein-protein interaction

Regioselective synthesis of new dispiro indolinones combining both an indolinone and an oxazolone fragment in their structure comprised the 1,3-dipolar cycloaddition of azomethine ylides, generated in situ from isatin and sarcosine, at 2-aryl-5-arylmethylidene-substituted 1,3-oxazol-5(4H)-ones. When ortho and para halogen atoms were present in the aromatic substituents of the starting oxazolones, complex mixtures containing large amounts of oxazoline ring opening products and their dispiro derivatives were formed. The cytotoxicity of compounds was tested by MTT on LNCaP, PC3, HCT116, MCF7, A549, HEK, and VA13 cell lines. The compound possessing the best cytotoxicity revealed the IC50 = 1.08±0.96 μM towards the p53- expressing LNCaP cells and lower activity (IC50 = 3.21±1.45 μM) towards the non-expressing p53 protein PC3 cells, however, it has proved inactive towards the HCT cells, both expressing (HCТ+/+) and non-expressing (HCT–/–) p53.

β-Adrenergic induction of lipolysis in hepatocytes is inhibited by ethanol exposure

In liver steatosis (i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the β-adrenergic (β-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to β-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the β-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). β-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this β-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in β-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that β-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.