1. Lipids up-regulate uncoupling protein 2 expression in rat hepatocytes
Helena Cortez–Pinto*, Hui Zhi Lin*, Shi Qi Yang*, Shelly Odwin da Costa‡, Anna Mae Diehl* 
Gastroenterology 
Volume 116, Issue 5, Pages (May 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Effect of Intralipid on hepatocyte expression of UCP-2 mRNA. Hepatocytes were isolated from normal rats and cultured for 24 hours in serum-free medium with different concentrations of Intralipid (final concentration, 2%, 4%, or 6%). Total RNA was isolated for Northern blot analysis. (A) Representative blot illustrating UCP-2 mRNA in untreated (control) cultures and in Intralipid-treated cultures at the end of 24-hour treatment. The bottom panel demonstrates 18S RNA expression on the same blot. (B) Phosphoimager analysis of data from 3 different experiments. For each sample, UCP-2 signal was normalized to the expression of 18S RNA in that sample on the same membrane and expressed as a percentage of the untreated control on the same blot. Data are means ± SEM. *P < 0.05 vs. control.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Changes in expression of UCP-2 protein during 24-hour incubation with 6% Intralipid. Primary rat hepatocytes were cultured in serum-free medium without or with 6% Intralipid for 24 hours, fixed in 4% paraformaldehyde, and incubated with antisera to UCP-2 in the (A and B) absence or (C and D) presence of UCP-2 peptide. UCP-2 antigen was demonstrated by Cy3-conjugated, affinity-purified secondary antibodies. All hepatocyte nuclei are stained with Hoechst's dye. Cy3 staining increased in Intralipid-treated cells (B). Preincubation with UCP-2 peptide virtually abolished Cy3 staining in both (C) control and (D) Intralipid-treated cells, confirming assay specificity for UCP-2 protein.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Changes in expression of UCP-2 mRNA during 24-hour incubation with 6% Intralipid. Hepatocytes were isolated from normal rats and cultured for varying times (0, 0.5, 3, 6, or 24 hours) in serum-free medium with Intralipid (final concentration, 6%). Total RNA was isolated for Northern blot analysis. (A) Representative blot illustrating UCP-2 mRNA in untreated (time 0 control) cultures and in Intralipid-treated cultures at 0.5, 3, 6, and 24 hours. The bottom panel shows 18S RNA expression on the same blot. (B) Phosphoimager analysis of data from 2 different experiments. For each sample, UCP-2 signal intensity was normalized to the expression of 18S RNA in that sample on the same membrane and expressed as a percentage of the time 0, untreated control on the same blot. *P < 0.05 vs. untreated time 0 control cultures.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Effect of linoleic and oleic acid on the expression of UCP-2 mRNA. Hepatocytes were isolated from normal rats and cultured for 24 hours in serum-free medium with 150–450 μmol/L linoleic or oleic acid. Total RNA was isolated for Northern blot analysis. (A) The top panel shows a representative blot of UCP-2 mRNA after treatment with 150–450 μmol/L linoleic or oleic acid for 24 hours. The bottom panel shows 18S RNA expression on the same membrane. (B) Phosphoimager analysis of data from 3 different experiments. In each sample, UCP-2 signal intensity was normalized to the expression of 18S RNA in that sample on the same membrane and expressed as a percentage of the untreated control on the same blot. *P < 0.04 for all cultures treated with linoleic or oleic acid vs. untreated control cultures (0).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Time course of UCP-2 induction after exposure to linoleic acid. Hepatocyte cultures were incubated in serum-free medium alone (0) or in the same medium with linoleic acid (450 μmol/L final concentration) for 3, 6, or 24 hours (n = 2 dishes/time point). (A) The top panel shows a representative Northern blot of UCP-2 mRNA levels in each dish at each time point. The bottom panel shows the 18S RNA expression on the same membrane. (B) Phosphoimager analysis of duplicate experiments. On each blot, the UCP-2 signal intensity in each sample was normalized to the 18S RNA expression in the same sample and expressed as a percentage of the time 0 (untreated) culture. *P < 0.05 vs. time 0 cultures.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 6
NF-κB–binding activity in primary rat hepatocytes cultured for 24 hours in the absence or presence of 6% Intralipid. As detailed in Materials and Methods, nuclear proteins were isolated from hepatocytes that had been cultured for 24 hours in serum-free medium either without or with 6% Intralipid. Gel mobility shift assays were performed using 10 μg nuclear protein/reaction plus an annealed 32P-labeled oligonucleotide fragment containing a κB binding site. To characterize the NF-κB isoforms that participated in complex formation with this probe, 1 μL of either nonimmune sera (−) or antisera (Ab) to NF-κB p50 or NF-κB p65 was added to reaction mixtures. Extracts were separated by electrophoresis on 5% polyacrylamide gels; gels were dried, evaluated by phosphoimager, and photographed. A representative autoradiograph is shown. Lane 1, extract from control cells cultured without Intralipid. Two bands are shown, and the lower band is more intense than the upper. Lane 2, same as lane 1, except antisera to p50 was added instead of nonimmune sera. This attenuates the bottom band and produces a new “supershifted” band, indicating that the bottom band (and the new supershifted band) contains p50. Lane 3, same as lane 1 except antisera to p65 was added instead of nonimmune sera. This attenuates the upper band and generates a new “supershifted” band that migrates near the top of the gel, indicating that both of these bands contain p65. Lane 4, extract from cells that had been cultured with Intralipid for 24 hours. Notice that the upper band is more intense than the upper band in lane 1 and the lower band is less intense than the lower band in lane 1. Lane 5, same as lane 4 except antisera to p50 was added instead of nonimmune sera. Supershift analysis shows that the lower band contains p50. Lane 6, same as lane 4 except antisera to p65 was added instead of nonimmune sera. Supershift analysis shows that the upper band contains p65.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Effect of TBHP (with and without GSH) on cell death and expression of UCP-2 mRNA. Hepatocytes were isolated from normal rats and cultured for 24 hours in serum-free medium, with 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L). Total RNA was isolated for Northern blot analysis. Lactate dehydrogenase concentration was measured in the culture medium. (A) The effect of 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L), on hepatocyte death, as evaluated by lactate dehydrogenase release into the medium. Lactate dehydrogenase data are expressed as mean ± SEM of 3–5 plates for each TBHP concentration. *P < 0.05 for 900 μmol/L TBHP with GSH vs. 900 μmol/L TBHP without GSH and for both 900 and 1800 μmol/L TBHP with GSH vs. cultures that had been incubated for 24 hours with GSH but without TBHP (□ 0[24 h]); **P < 0.05– for 300–1800 μmol/L TBHP without GSH vs. cultures incubated 24 hours without GSH or TBHP (■, 0[24 h]). (B) UCP-2 mRNA expression after 24-hour incubation with 0–90 μmol/L TBHP. Phosphoimager analysis of data from 3 experiments. *P = vs. untreated control (0); **P < 0.02 vs. untreated control (0); ***P < vs. untreated control (0). (C) UCP-2 mRNA expression after 0, 1, 3, 6, and 24 hours of incubation with 30 μmol/L TBHP. Phosphoimager analysis of data from 3 experiments. *P = vs. time 0 cultures. (D) UCP-2 mRNA expression in cultures incubated for 24 hours with 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L). The middle panel shows 18S RNA expression on the same blot. The paucity (900 μmol/L TBHP without GSH and 1800 μmol/L TBHP with GSH) or absence (1800 μmol/L TBHP without GSH) of an 18S RNA band in these samples reflects RNA degradation because of increased hepatocyte death. The bottom panel shows phosphoimager analysis of data from 2 different experiments. For each sample, UCP-2 signal intensity was normalized to the expression of 18S RNA in that sample and expressed as a percentage of the untreated control on the same blot. UCP-2 expression in all samples was greater than in untreated control cultures on the same blots (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig. 7
Effect of TBHP (with and without GSH) on cell death and expression of UCP-2 mRNA. Hepatocytes were isolated from normal rats and cultured for 24 hours in serum-free medium, with 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L). Total RNA was isolated for Northern blot analysis. Lactate dehydrogenase concentration was measured in the culture medium. (A) The effect of 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L), on hepatocyte death, as evaluated by lactate dehydrogenase release into the medium. Lactate dehydrogenase data are expressed as mean ± SEM of 3–5 plates for each TBHP concentration. *P < 0.05 for 900 μmol/L TBHP with GSH vs. 900 μmol/L TBHP without GSH and for both 900 and 1800 μmol/L TBHP with GSH vs. cultures that had been incubated for 24 hours with GSH but without TBHP (□ 0[24 h]); **P < 0.05– for 300–1800 μmol/L TBHP without GSH vs. cultures incubated 24 hours without GSH or TBHP (■, 0[24 h]). (B) UCP-2 mRNA expression after 24-hour incubation with 0–90 μmol/L TBHP. Phosphoimager analysis of data from 3 experiments. *P = vs. untreated control (0); **P < 0.02 vs. untreated control (0); ***P < vs. untreated control (0). (C) UCP-2 mRNA expression after 0, 1, 3, 6, and 24 hours of incubation with 30 μmol/L TBHP. Phosphoimager analysis of data from 3 experiments. *P = vs. time 0 cultures. (D) UCP-2 mRNA expression in cultures incubated for 24 hours with 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L). The middle panel shows 18S RNA expression on the same blot. The paucity (900 μmol/L TBHP without GSH and 1800 μmol/L TBHP with GSH) or absence (1800 μmol/L TBHP without GSH) of an 18S RNA band in these samples reflects RNA degradation because of increased hepatocyte death. The bottom panel shows phosphoimager analysis of data from 2 different experiments. For each sample, UCP-2 signal intensity was normalized to the expression of 18S RNA in that sample and expressed as a percentage of the untreated control on the same blot. UCP-2 expression in all samples was greater than in untreated control cultures on the same blots (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

10. Fig. 7
Effect of TBHP (with and without GSH) on cell death and expression of UCP-2 mRNA. Hepatocytes were isolated from normal rats and cultured for 24 hours in serum-free medium, with 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L). Total RNA was isolated for Northern blot analysis. Lactate dehydrogenase concentration was measured in the culture medium. (A) The effect of 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L), on hepatocyte death, as evaluated by lactate dehydrogenase release into the medium. Lactate dehydrogenase data are expressed as mean ± SEM of 3–5 plates for each TBHP concentration. *P < 0.05 for 900 μmol/L TBHP with GSH vs. 900 μmol/L TBHP without GSH and for both 900 and 1800 μmol/L TBHP with GSH vs. cultures that had been incubated for 24 hours with GSH but without TBHP (□ 0[24 h]); **P < 0.05– for 300–1800 μmol/L TBHP without GSH vs. cultures incubated 24 hours without GSH or TBHP (■, 0[24 h]). (B) UCP-2 mRNA expression after 24-hour incubation with 0–90 μmol/L TBHP. Phosphoimager analysis of data from 3 experiments. *P = vs. untreated control (0); **P < 0.02 vs. untreated control (0); ***P < vs. untreated control (0). (C) UCP-2 mRNA expression after 0, 1, 3, 6, and 24 hours of incubation with 30 μmol/L TBHP. Phosphoimager analysis of data from 3 experiments. *P = vs. time 0 cultures. (D) UCP-2 mRNA expression in cultures incubated for 24 hours with 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L). The middle panel shows 18S RNA expression on the same blot. The paucity (900 μmol/L TBHP without GSH and 1800 μmol/L TBHP with GSH) or absence (1800 μmol/L TBHP without GSH) of an 18S RNA band in these samples reflects RNA degradation because of increased hepatocyte death. The bottom panel shows phosphoimager analysis of data from 2 different experiments. For each sample, UCP-2 signal intensity was normalized to the expression of 18S RNA in that sample and expressed as a percentage of the untreated control on the same blot. UCP-2 expression in all samples was greater than in untreated control cultures on the same blots (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

11. Fig. 7
Effect of TBHP (with and without GSH) on cell death and expression of UCP-2 mRNA. Hepatocytes were isolated from normal rats and cultured for 24 hours in serum-free medium, with 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L). Total RNA was isolated for Northern blot analysis. Lactate dehydrogenase concentration was measured in the culture medium. (A) The effect of 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L), on hepatocyte death, as evaluated by lactate dehydrogenase release into the medium. Lactate dehydrogenase data are expressed as mean ± SEM of 3–5 plates for each TBHP concentration. *P < 0.05 for 900 μmol/L TBHP with GSH vs. 900 μmol/L TBHP without GSH and for both 900 and 1800 μmol/L TBHP with GSH vs. cultures that had been incubated for 24 hours with GSH but without TBHP (□ 0[24 h]); **P < 0.05– for 300–1800 μmol/L TBHP without GSH vs. cultures incubated 24 hours without GSH or TBHP (■, 0[24 h]). (B) UCP-2 mRNA expression after 24-hour incubation with 0–90 μmol/L TBHP. Phosphoimager analysis of data from 3 experiments. *P = vs. untreated control (0); **P < 0.02 vs. untreated control (0); ***P < vs. untreated control (0). (C) UCP-2 mRNA expression after 0, 1, 3, 6, and 24 hours of incubation with 30 μmol/L TBHP. Phosphoimager analysis of data from 3 experiments. *P = vs. time 0 cultures. (D) UCP-2 mRNA expression in cultures incubated for 24 hours with 30–1800 μmol/L TBHP, with or without GSH (10 mmol/L). The middle panel shows 18S RNA expression on the same blot. The paucity (900 μmol/L TBHP without GSH and 1800 μmol/L TBHP with GSH) or absence (1800 μmol/L TBHP without GSH) of an 18S RNA band in these samples reflects RNA degradation because of increased hepatocyte death. The bottom panel shows phosphoimager analysis of data from 2 different experiments. For each sample, UCP-2 signal intensity was normalized to the expression of 18S RNA in that sample and expressed as a percentage of the untreated control on the same blot. UCP-2 expression in all samples was greater than in untreated control cultures on the same blots (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions