21 This conversion is catalyzed by the acetyl-coA synthetases,22 recently redesignated acyl-coenzyme A synthetase short-chain family members 1 and 2 (ACSS1 [UniProt Q9NUB1] and ACSS2 [Q9NR19]).23 The role of acetyl-coA synthesis in control of inflammation has not previously been investigated and could open

selleckchem a novel field of study into the relationship between cellular energy supply and inflammatory disease. In this study we test the hypothesis that ethanol enhances macrophage cytokine production by uncoupling gene transcription from its normal regulatory mechanisms through increased histone acetylation, and that the conversion of the ethanol metabolite acetate to acetyl-coA is crucial to this process. AAH, acute alcoholic hepatitis; ACSS, acyl-coenzyme A synthetase short-chain; ALD, alcoholic liver disease; ER, endoplasmic reticulum; HAT, histone acetyltransferase; HDAC, histone deacetylase; IL, interleukin; LPS, lipopolysaccharide; NAD, nicotinamide adenine dinucleotide;

ROS, reactive oxygen species; SIRT, sirtuin; SREBP, sterol response element binding protein; TLR, Toll-like receptor; TNF, tumor necrosis factor. The human monoblastic cell line MonoMac6 (DSMZ, Braunschweig, Pifithrin-�� order Germany, ACC124), an established human cell line that displays features of mature macrophages and has been used to model Kupffer cell responses in ethanol,10 was grown in RPMI-1640 Urease medium supplemented with 2 mM l-glutamine (Lonza, Basel, Switzerland), 1 mM sodium pyruvate, 1× nonessential amino acids (both Gibco, Paisley, UK), 10% fetal calf serum (Lonza), and 9 μg/mL human insulin (Sigma, St. Louis, MO). Ethanol exposure was achieved in fresh media with 86 mM (0.5%, 400 mg/dL) ethanol (VWR, Poole, UK). This is five times the legal blood alcohol limit for driving in the UK and equivalent to heavy drinking in humans.24 The ethanol concentration

was maintained by using ethanol vapor in the incubator to prevent evaporation of ethanol from culture media10 and monitored by potassium dichromate reduction assay (BioAssay Systems, Hayward, CA). For acetate culture experiments, media were supplemented with 1 mM sodium acetate (Sigma) and replaced every 48 hours to minimize fluctuations in acetate concentration. One mM is an achievable acetate concentration in an individual metabolizing ethanol at the concentration used.25 After 7 days incubation cells were resuspended in fresh medium at 2 × 106/mL and stimulated with E. coli O111:B4 LPS (InvivoGen) at a final concentration of 10 ng/mL.