Volume 122, Issue 1, Pages 106-118 (January 2002) Ethanol metabolism and transcription factor activation in pancreatic acinar cells in rats  Anna S. Gukovskaya, Michelle Mouria, Ilya Gukovsky, Christopher N. Reyes, Vladimir N. Kasho, Larry D. Faller, Stephen J. Pandol  Gastroenterology  Volume 122, Issue 1, Pages 106-118 (January 2002) DOI: 10.1053/gast.2002.30302 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Activities of FAEE synthase and ADH in rat pancreatic and liver tissue, and isolated pancreatic acini. (A) FAEE synthase activities were measured in cell or tissue lysates incubated for 1 hour with 0.4 mmol/L [14C] oleate and ethanol at indicated concentrations. FAEE accumulation was measured as described in the Materials and Methods section. Values are means ± SE from 3 separate experiments. *P < 0.02 compared with liver. (B) ADH activities were measured in cell or tissue lysates incubated for 1 hour with 4 mmol/L NAD and ethanol at indicated concentrations. ADH activity was measured by conversion of NAD into NADH as described in the Materials and Methods section. Values are means ± SE (n = 3). *P < 0.0001 compared with liver. (C) ADH activities were measured in acinar cell or liver lysates incubated for 1 hour with 4 mmol/L NAD and 200 mmol/L ethanol, in the absence or presence of MP. ADH activity was measured by conversion of NAD into NADH as described in the Materials and Methods section. Values are means ± SE (n = 3). *P < 0.001 compared with liver ADH activity in the absence of MP. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of ethanol concentration on FAEE synthase and ADH activities in pancreatic acinar cells. (A) FAEE synthase activities were measured in acinar cell lysates incubated for 1 hour with 0.4 mmol/L [14C] oleate and different concentrations of ethanol. Values are means ± SE from 3 separate experiments. K0.5 = 550 ± 75 mmol/L, Vmax = 8.5 ± 0.8 nmol/mg protein/hr. (B) ADH activities were measured in acinar cell lysates incubated for 1 hour with 4 mmol/L NAD and different concentrations of ethanol, in the presence of 1 mmol/L cyanamide. Values are means ± SE from 3 separate experiments. Km = 270 ± 70 mmol/L, Vmax = 46 ± 7 nmol/mg protein/hr. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Activation of NF-κB and AP-1 by FAEEs in pancreatic acinar cells. Isolated pancreatic acini were incubated for indicated times without (control) or with 3 mmol/L FAEEs (a mixture of 1 mmol/L each of ethyl oleate, ethyl linoleate, and ethyl palmitate). FAEEs were either dissolved in dimethyl sulfoxide and directly added into the cell suspension (d) or first reconstituted in LDL (a-c) as described in the Materials and Methods section. (A) Nuclear extracts from these cells were subjected to electrophoretic mobility shift assay for NF-κB or AP-1 by using consensus oligonucleotide probes. Positions of NF-κB or AP-1 complexes (single arrowhead) and the free probe (double arrowhead) are indicated. (B) The extent of NF-κB and AP-1 activation in FAEE-treated cells (open bars) was determined by densitometry of NF-κB and AP-1 bands in the PhosphorImager, and is given as means ± SE (n = 3-5) relative to control (closed bars). *P < 0.05 compared with control. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Specificity and composition of NF-κB and AP-1 complexes in FAEE-treated pancreatic acinar cells. Isolated pancreatic acini were incubated for 3 hours with 3 mmol/L FAEEs (a mixture of 1 mmol/L each of ethyl oleate, ethyl linoleate, and ethyl palmitate). Nuclear extracts from these cells were subjected to EMSA for NF-κB or AP-1. Left panels: cold competition experiments confirming the specificity of binding by adding 100× molar excess of unlabeled wild-type (wt) or mutated (mut) NF-κB or AP-1 oligonucleotide. Right panels: supershift analysis using antibodies to different NF-κB or AP-1 proteins. Arrows show the positions of supershifted bands. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of individual FAEEs on NF-κB and AP-1 activation in pancreatic acinar cells. (A) Representative EMSAs for NF-κB and AP-1 performed on nuclear extracts from pancreatic acini incubated for 3 hours without (control) or with 3 mmol/L ethyl palmitate (PAEE), oleate (OAEE) or linoleate (LAEE). (B) The extent of NF-κB and AP-1 activation in acini treated with PAEE, OAEE, or LAEE was determined by densitometry of NF-κB and AP-1 bands in the PhosphorImager, and is given as mean ± SE (n = 3) relative to control (open bars). *P < 0.05 compared with control. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of acetaldehyde on NF-κB and AP-1 activation in pancreatic acinar cells. Pancreatic acini were incubated for 6 hours without (control) or with acetaldehyde at indicated concentrations. Left panels: representative EMSAs for NF-κB and AP-1. Right panels: the extent of NF-κB and AP-1 activation was determined by densitometry of NF-κB and AP-1 bands in the PhosphorImager, and is given as mean ± SE (n = 3-4) relative to control. NS, nonspecific. *P < 0.05 compared with control. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Time-dependent decrease in concentration of exogenous acetaldehyde in pancreatic acinar cell suspension. Acetaldehyde was added at 1 mmol/L into a suspension of freshly isolated pancreatic acini in 199 medium, and the incubation proceeded for indicated times. The concentration of acetaldehyde retained in the cell suspension was measured by gas chromatography–mass spectrometry as described in the Materials and Methods section. Values are means ± SE from 2 separate experiments performed in duplicates. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of ethanol on NF-κB and AP-1 activation in pancreatic acinar cells. Isolated pancreatic acini were incubated for indicated times without and with 100 mmol/L ethanol. Nuclear extracts from these cells were subjected to EMSAs for NF-κB or AP-1. (A) Left panels: representative EMSAs for NF-κB and AP-1. Right panels: the extent of activation was determined by densitometry of NF-κB and AP-1 bands in the PhosphorImager, and is given as mean ± SE (n = 4-5) relative to control. *P < 0.05 compared with cells incubated for 1 hour. (B) Subunit composition of NF-κB and AP-1 complexes in acinar cells incubated for 1 hour with 100 mmol/L ethanol was determined in supershift experiments using antibodies to different NF-κB or AP-1 proteins. Arrows show the positions of supershifted bands. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Effect of cyanamide on NF-κB activation in ethanol-treated pancreatic acinar cells. Isolated pancreatic acini were incubated for 3 hours without and with 100 mmol/L ethanol in the presence or absence of 1 mmol/L cyanamide, an ALDH inhibitor. Nuclear extracts from these cells were subjected to EMSA for NF-κB. The extent of NF-κB activation was determined by densitometry in the Phosphor-Imager, and is given as mean ± SE (n = 4) relative to cells incubated without ethanol (bottom panels). *P < 0.05 compared with cells incubated without ethanol. #P < 0.05 compared with cells incubated with ethanol alone. Gastroenterology 2002 122, 106-118DOI: (10.1053/gast.2002.30302) Copyright © 2002 American Gastroenterological Association Terms and Conditions