Volume 147, Issue 4, Pages 860-869 (October 2014) Inhibition of CYP4A Reduces Hepatic Endoplasmic Reticulum Stress and Features of Diabetes in Mice  Edmond Changkyun Park, Seung Il Kim, Yeonhee Hong, Jeong Won Hwang, Gun-Sik Cho, Hye-Na Cha, Jin-Kwan Han, Chul-Ho Yun, So-Young Park, Ik-Soon Jang, Zee-Won Lee, Jong-Soon Choi, Soohyun Kim, Gun-Hwa Kim  Gastroenterology  Volume 147, Issue 4, Pages 860-869 (October 2014) DOI: 10.1053/j.gastro.2014.06.039 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 Effect of the CYP4A inhibitor HET0016 in the diabetes of db/db mice. Eight-week-old db/db mice were injected with either HET0016 (5 mg/kg/day) or dimethyl sulfoxide intraperitoneally for 2 weeks. (A) Glucose tolerance test. (B) Fasting blood glucose level. (C) Fasting serum insulin concentration. (D) Photomicrographs taken after paraffin sectioning and H&E staining of the indicated mouse liver. (E) Hepatic TG level was measured in lipid extracts from the liver tissues. (F) Malondialdehyde (MDA) formation, a measure of lipid peroxidation, was estimated by thiobarbituric acid reactive substances (TBARS) assays performed on liver tissues. Data are shown as means ± SD. P values were determined by the Student t test. *P < .05, **P < .01, and ***P < .001 for C57 vs db/db and db/db vs db/db + HET0016. Gastroenterology 2014 147, 860-869DOI: (10.1053/j.gastro.2014.06.039) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 Effect of the CYP4A inhibitor HET0016 on hepatic function, ER stress, insulin resistance, and apoptosis in db/db mice. (A) Expression of gluconeogenesis (G6Pase, PEPCK, and PGC-1) and lipogenesis (DGAT2, FAS, lipin1, and SCD1) markers were examined by Western blotting in the indicated mouse liver. (B) Expression of ER stress markers, such as PERK, phospho-eIF2α, CHOP, and phospho-JNK, was monitored by Western blotting. ER-localized proteins, such as CYP4A and PERK, were analyzed from microsomal fractions of liver tissues. (C) In vivo insulin signaling was examined by investigating phosphorylation of the insulin receptor (IR) and Akt, and apoptosis was monitored by the expression of cleaved caspase-3 and caspase-9, Bax, and Bcl-2 in the liver tissues. Gastroenterology 2014 147, 860-869DOI: (10.1053/j.gastro.2014.06.039) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 Effect of the CYP4A inhibitor HET0016 on diabetic pathophysiology in HFD mice. C57BL/6J mice were fed with an ND or an HFD and injected with either dimethyl sulfoxide or HET0016 (5 mg/kg/day) intraperitoneally for 12 weeks. (A) The changes in body weight of the indicated groups of mice. (B) Glucose tolerance test. (C) Insulin tolerance test. (D) Isolated liver tissues and photomicrographs taken after paraffin sectioning and H&E staining. (E) Hepatic TG level. (F) MDA formation, a measure of lipid peroxidation, was estimated by TBARS assays performed on liver tissues. Data are shown as means ± SD. P values were determined by the Student t test. *P < .05, **P < .01, and ***P < .001 for ND vs HFD and HFD vs HFD + HET0016. Gastroenterology 2014 147, 860-869DOI: (10.1053/j.gastro.2014.06.039) Copyright © 2014 AGA Institute Terms and Conditions

Figure 4 Effect of the CYP4A inhibitor HET0016 on hepatic function, ER stress, insulin resistance, and apoptosis in HFD mice. (A) Expression of gluconeogenesis and lipogenesis markers in the indicated mice livers. (B) Expression of ER stress markers. ER-localized proteins, such as PERK, IRE1, and ATF6, were analyzed from microsomal fractions of liver tissues. (C) In vivo insulin signaling and apoptosis were monitored in the liver tissues. Gastroenterology 2014 147, 860-869DOI: (10.1053/j.gastro.2014.06.039) Copyright © 2014 AGA Institute Terms and Conditions

Figure 5 Effect of hepatic Cyp4a knockdown on diabetes of db/db mice. Cyp4a-specific shRNA lentivirus were introduced into the liver of 8-week-old db/db mice for 2 weeks. (A) Body weight. (B) Fasting blood glucose level. (C) Glucose tolerance test. (D) Insulin tolerance test. (E) Hyperinsulinemic–euglycemic clamp. The steady-state glucose infusion rate was obtained from averaged rates of 90–120 minutes of clamp. Insulin suppression of hepatic glucose production was determined during clamp. (F) Expression of ER stress markers in the liver. ER-localized proteins, such as CYP4A and PERK, were analyzed from microsomal fractions of liver tissues. Gastroenterology 2014 147, 860-869DOI: (10.1053/j.gastro.2014.06.039) Copyright © 2014 AGA Institute Terms and Conditions

Figure 6 Effect of CYP4A inhibition on ER stress in HepG2 cells. HepG2 human hepatoma cells were treated with (A) high glucose, (B) tunicamycin, or (C) thapsigargin with or without pretreatment with HET0016 or siRNAs against Cyp4a. Expression of ER stress markers was determined by Western blotting using the indicated antibodies. Gastroenterology 2014 147, 860-869DOI: (10.1053/j.gastro.2014.06.039) Copyright © 2014 AGA Institute Terms and Conditions