title: Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.
abstract: Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis.
  
  
  
  We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction.
  
  
  
  Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic conditions, cultured hepatocytes increased expression of Hif1a, Hmox1, and Vegfa messenger RNAs (mRNAs), and down-regulated expression of AQP8 mRNA and protein; AQP8 down-regulation was not observed in cells with knockdown of HIF1A. iH-HIFKO mice had reduced inflammation and mucin deposition in the gallbladder compared with control mice. Liver tissues from patients with NAFLD with gallstones had increased levels of HIF1A, HMOX1, and VEGFA mRNAs, compared with livers from patients with NAFLD without gallstones.
  
  
  
  In steatotic livers of mice, hypoxia up-regulates expression of HIF1A, which reduces expression of AQP8 and concentrates biliary lipids via suppression of water secretion from hepatocytes. This promotes cholesterol gallstone formation. Livers from patients with NAFLD and gallstones express higher levels of HIF1A than livers from patients with NAFLD without gallstones.
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Asai
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan. Electronic address: yamatetsu-tky@umin.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yamada
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Sohei
authors.initials: S
authors.lastname: Tsukita
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Maekawa
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Honma
authors.affiliation: Department of Molecular Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Ikeda
authors.affiliation: Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Keigo
authors.initials: K
authors.lastname: Murakami
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Munakata
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Shirai
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Shinjiro
authors.initials: S
authors.lastname: Kodama
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sugisawa
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Chiba
authors.affiliation: Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Kondo
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Keizo
authors.initials: K
authors.lastname: Kaneko
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Uno
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Shojiro
authors.initials: S
authors.lastname: Sawada
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Junta
authors.initials: J
authors.lastname: Imai
authors.affiliation: Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Yamaguchi
authors.affiliation: Department of Molecular Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
authors.forename: Kozo
authors.initials: K
authors.lastname: Tanaka
authors.affiliation: Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Sasano
authors.affiliation: Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.
authors.forename: Nariyasu
authors.initials: N
authors.lastname: Mano
authors.affiliation: Department of Gastroenterology, Yamagata University Faculty of Medicine, Yamagata, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Ueno
authors.affiliation: Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Tooru
authors.initials: T
authors.lastname: Shimosegawa
authors.affiliation: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan; Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology (CREST), Tokyo, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Katagiri
mesh_terms: D000818:Animals; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism; D001646:Bile / Q000378:metabolism; D001647:Bile Acids and Salts / Q000378:metabolism; D020355:Cholates / Q000008:administration & dosage; D002784:Cholesterol / Q000378:metabolism*; D002791:Cholesterol, Dietary / Q000008:administration & dosage / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D005260:Female; D005704:Gallbladder / Q000473:pathology; D042882:Gallstones / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D051547:Heme Oxygenase-1 / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism*; D007249:Inflammation / Q000209:etiology; D008099:Liver / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D009077:Mucins / Q000378:metabolism; D065626:Non-alcoholic Fatty Liver Disease / Q000150:complications / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D014867:Water / Q000378:metabolism
chemical_list: D020346:Aquaporins; D001647:Bile Acids and Salts; D020355:Cholates; D002791:Cholesterol, Dietary; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; D009077:Mucins; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C108330:aquaporin 8; C467480:vascular endothelial growth factor A, mouse; D014867:Water; D002784:Cholesterol; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse
keywords: Aquaporin-8; Gallstone; Hypoxia; NAFLD