1 Loss of Von Hippel-Lindau (VHL) increases systemic cholesterol

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Transcript of 1 Loss of Von Hippel-Lindau (VHL) increases systemic cholesterol

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    Loss of Von Hippel-Lindau (VHL) increases systemic cholesterol level through 1 targeting HIF-2 and regulation of bile acid homeostasis 2

    3 Sadeesh K. Ramakrishnan1, Matthew Taylor1, Aijuan Qu4, Sung-Hoon Ahn4, 4 Madathilparambil V Suresh3, Krishnan Raghavendran3, Frank J. Gonzalez4, and 5 Yatrik M. Shah1,2 6 1Department of Molecular & Integrative Physiology, 2Department of Internal 7 Medicine, Division of Gastroenterology, and 3Department of Surgery, University of 8 Michigan Medical School, Ann Arbor MI. 4Laboratory of Metabolism, Center for 9 Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 10 MD. 11 12 Running Title: HIF-2 regulates cholesterol homeostasis. 13 14 Key words: Systemic hypoxia, cholesterol homeostasis, bile acid homeostasis, 15 Cyp7a1, hypoxia-inducible factor. 16 17 Correspondence: Yatrik M. Shah, Department of Molecular & Integrative 18 Physiology, Department of Internal medicine, Division of Gastroenterology, 19 University of Michigan Medical School, Ann Arbor, MI 48109. Email: 20 shahy@umich.edu 21 22

    MCB Accepts, published online ahead of print on 13 January 2014Mol. Cell. Biol. doi:10.1128/MCB.01441-13Copyright 2014, American Society for Microbiology. All Rights Reserved.

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    Abbreviations: ARNT, aryl hydrocarbon receptor nuclear translocator; Baat, bile 23 acid CoA: amino acid N-acyltransferase; BSEP, bile salt export protein; CIH, chronic 24 intermittent hypoxia; FXR, farsenoid x receptor; HIF, hypoxia-inducible factor; 25 HMGCS, HMG-CoA synthase; Hmgr, HMG-CoA reductase; NTCP, Sodium 26 taurocholate cotransport protein; SHP, small heterodimer partner; Srebp2, sterol 27 regulatory element binding protein 2; Vhl, Von Hippel- Lindau tumor suppressor 28 protein. 29 30 Conflict of interests: The authors declare no conflict of interest. 31

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    Abstract 32 Cholesterol synthesis is a highly oxygen dependent process. Paradoxically, hypoxia is 33 correlated with an increase in cellular and systemic cholesterol levels and risk of 34 cardiovascular diseases. The mechanism for the increase in cholesterol during 35 hypoxia is unclear. Hypoxia signaling is mediated through hypoxia-inducible factor 36 (HIF)-1 and HIF-2. The present study demonstrates that activation of HIF 37 signaling in the liver increases hepatic and systemic cholesterol levels due to a 38 decrease in the expression of cholesterol hydroxylases CYP7A1 and other enzymes 39 involved in bile acid synthesis. Specifically, activation of hepatic HIF-2 (but not 40 HIF-1) led to hypercholesterolemia. HIF-2 repressed the circadian expression of 41 Rev-erb resulting in increased expression of E4BP4, a negative regulator of Cyp7a1. 42 To understand if HIF-mediated decrease in bile acid synthesis is a physiological 43 relevant pathway by which hypoxia maintains or increases systemic cholesterol 44 levels, two hypoxic mouse models were assessed; an acute lung injury model and 45 mice exposed to 10% O2 for three weeks. In both the models cholesterol levels 46 increased with a concomitant decrease in expression of genes involved in bile acid 47 synthesis. The present study demonstrates that hypoxic activation of hepatic HIF-2 48 leads to an adaptive increase in cholesterol levels through inhibition of bile acid 49 synthesis. 50

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    Introduction 51 Cholesterol is an essential nutrient important in many metabolic processes and its 52 levels are tightly regulated. Cholesterol synthesis is a highly oxygen dependent 53 process. The final step from lanosterol to cholesterol conversion requires nine 54 molecules of dioxygen (53). However, systemic hypoxia is associated with an 55 increase in cholesterol levels. Individuals living at high altitude are at high risk for 56 coronary heart disease due to elevated low-density lipoprotein and total cholesterol 57 levels caused by high-altitude hypoxia (51). In chronic hypoxic diseases, such as in 58 patients with obstructive sleep apnea, no change or an increase in serum and liver 59 cholesterol levels are observed, which can be reversed by continuous positive airway 60 pressure (33, 42, 45). Moreover, mice exposed to chronic intermittent hypoxia (CIH) 61 have a significant increase in total cholesterol levels (14, 22). These studies suggest 62 that hypoxia-signaling pathways activate an adaptive response important in 63 cholesterol homeostasis. Several mechanisms were proposed to account for hypoxia-64 induced dyslipidemia (9, 37). However, the mechanisms by which hypoxia regulates 65 cholesterol homeostasis are not clear. Hypoxic signaling is mediated by an oxygen 66 sensitive transcription factor, hypoxia-inducible factor (HIF), consisting of two 67 isoforms, HIF-1 and HIF-2 (56). Activation of HIF-1 induces glycolytic genes 68 whereas activation of HIF-2 regulates the expression of genes that are involved in 69 fatty acid synthesis, fatty acid uptake, inflammation, fibrosis and vascular tumors (17, 70 24, 37, 40, 41). 71 72

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    In this study, liver hypoxia-induced dyslipidemia was shown to be associated with 73 spontaneous hypercholesterolemia. Consistent with previous reports, the increase in 74 hepatic cholesterol by hypoxia was not due to alterations in cholesterol synthesis, 75 intestinal absorptive genes, or mobilization from fat stores (1, 34). Disruption of Vhl 76 led to a HIF-2 dependent decrease in cholesterol hydroxylases involved in the 77 conversion of cholesterol to bile acids. To confirm that the decrease in cholesterol 78 hydroxylase is an important mechanism to maintain cholesterol levels during 79 hypoxia, two systemic hypoxic mouse models were used; mice exposed to 80 normobaric 10% O2 for three weeks and a mouse model of lung contusion (38). Both 81 models exhibited significantly elevated levels of serum cholesterol with a parallel 82 decrease in expression of genes involved in bile acid synthesis. Taken together, this 83 study demonstrates a mechanistic basis for the adaptive maintenance of cholesterol 84 levels during hypoxia. Thus, inhibition of HIF-2 may be relevant and novel 85 therapeutic target to ameliorate hypercholesterolemia during hypoxia. 86 on A

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    Material and Methods 87 Animals and diets 88 VhlF/F, VhlLivKO, VhlInt, Vhl/ArntLivKO, Vhl/Hif1LivKO Vhl/Hif2LivKO, VhlM, 89 VhlIntKO and ODD-luc mice were described previously (17, 44, 49, 50, 55, 58). Vhl90 M were generated by crossing LysM cre mice with VhlF/F mice. All mice were fed 91 ad-libitum and kept in a 12-hour dark/light cycle. Mice were fed with either regular 92 chow diet or atherogenic (35 kcal% fat, 1% cholesterol, 0.5% cholic acid; Research 93 diets, New Brunswick, NJ), high fat (~45 kcal% derived from fat; Research diets) or 94 ethanol diet (6% lieber-decarli diet, Bio-serv, Frenchtown, NJ). For systemic hypoxia 95 experiments, C57BL/6 mice in standard cages were placed in a normobaric hypoxia 96 chamber with 10% O2 for three weeks. Lung contusion (C57BL/6 mice) and in vivo 97 imaging (ODD-luc) were performed as described previously (2, 38). All the mice 98 were sacrificed at 7PM unless mentioned in the figure legend. All animal studies were 99 carried out in accordance with Association for Assessment and Accreditation of 100 Laboratory Animal Care International guidelines and approved by the University 101 Committee on the Use and Care of Animals at the University of Michigan. 102 103 Western blot analysis 104 Microsome and whole cell extracts were prepared as previously described (55). 105 Proteins were separated by SDS-PAGE. Microsomes were used for detection of 106 CYP7A1 (Abcam, Cambridge, MA) and normalized to total protein by coomassie 107 blue staining. 108 109

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    Primary hepatocyte isolation 110 For primary hepatocytes isolation, livers were perfused continuously with 10 ml of 111 EBSS containing 0.5mM EGTA, followed by serum-free L-15 media containing 112 collagenase type II (Worthington, Invitrogen). Hepatocytes were then harvested by 113 gentle teasing and filtered through 100 micron filter. Viable hepatocytes were plated 114 in 6 well plates at a cell density of 4 X 105 in Williams E media containing 10% FBS. 115 5 hours after plating, dead cells were removed and media refreshed. For adenovirus 116 treatment, desired amount of Ad-Cyp7a1, AD-HNF4, and AD-LRH-1 virus was 117 added after 5 hours of plating and then incubated until further analysis. 118 119 Cholesterol assay 120 For cholesterol extraction, 50 mg of liver or white adipose tissue was homogenized in 121 1 ml of 2:1 chloroform to methanol ratio. Primary hepatocytes were collected in 122 300l methanol and then 600l of chloroform added and vortexed. The homogenates 123 were then centrifuged and 200l of water was added to the supernatant and vortexed. 124 The organic phase separated by centrifugation was dried overnight and resuspended 125 in isopropanol. Cholesterol measurements were performed using the Cholesterol E kit 126 (Wako Diagnostics, VA). Serum cholesterol was measured in 20l of serum and 127 lipoprotein profiles were assessed by fast performance liquid chromatography as 128 previously described (36). 129 130 Bile acid assay 131