2,3,7,8-TETRACHLORODIBENZO--DIOXIN (TCDD)ELICITED STEATOSIS: THE ROLE OF ARYL HYDROCARBON RECEPTOR (AHR) IN LIPID UPTAKE, METABOLISM, AND TRANSPORT IN SCD1+/+, SCD1-/-, AND C57BL/6 MICE By Michelle Manente Angrish A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics - Environmental Toxicology 2012 ABSTRACT TCDD-ELICITED STEATOSIS: THE ROLE OF AHR IN LIPID UPTAKE, METABOLISM AND TRANSPORT IN SCD1+/+, SCD1-/-, AND C57BL/6 MICE By Michelle Manente Angrish Metabolic syndrome (MetS) and its associated disorders such as obesity, type II diabetes, non-alcoholic fatty liver disease (NAFLD), and hypertension are epidemic in Western countries including the United States. Conventional thought holds excess energy consumption accountable for MetS phenotypes, and although Western diet and culture is characterized by too many calories consumed and too few calories burned, environmental endocrine disrupting chemicals (EDCs) have emerged into the spotlight for their role in positive energy balance. Dioxin-like compounds (DLCs) including 2,3,7,8-tetrachlorodibenzo--dioxin (TCDD) are environmentally ubiquitous and persistent EDCs that alter energy balance and lipid metabolism in animals and humans. TCDD elicited hepatic steatosis involves aryl hydrocarbon receptor (AhR) activation and is marked by increased triglycerides, free fatty acids, inflammatory cell infiltration, and increased serum alanine aminotransferase levels. Hepatic steatosis in the absence of alcohol consumption is a cryptic, yet significant manifestation of MetS and its associated diseases and may precede cirrhosis as well as other extrahepatic effects. Stearoyl-CoA desaturase 1 (Scd1) catalyzes the rate-limiting step in monounsaturated fatty acid (MUFA) biosynthesis. Its deficiency protects mice from diet-induced steatosis, and the enzyme is a target for the treatment of metabolic related disorders. In this report the role of AhR regulation of lipid uptake, metabolism, and transport in TCDD-elicited steatosis was characterized using Scd1 null mice, diet, and 14 C-lipid uptake studies. Collectively, these studies showed that 1) AhR regulation of Scd1 contributes to the hepatotoxicity of TCDD, 2) dietary fat is the primary source of lipid in TCDD-elicited steatosis, 3) TCDD increases the uptake of dietary lipids, and 4) AhR mediates not only altered hepatic lipid composition, but also systemic lipid composition. This work indicates that AhR activation results in a systemic response that involves coordinated interactions between the digestive tract, circulatory system, and liver, with important health implications for individuals at risk for metabolic disease. FAMILY It is impossible to express in words my gratitude and love for you and your unyielding, everlasting, and unconditional support and love. iv ACKNOWLEDGMENTS Dr. Anna Kopec, a friend and colleague, you were essential to my success in grad school. You uniquely, unyieldingly, understandingly, (and maybe even unknowingly) offered me a pillar of support throughout my graduate career that I am forever grateful. Dr. Barb Sears, thank you for believing, comforting, and mentoring me. I wouldn’t be where I am today without you! Drs. Cindy and Dennis Arvidson same goes for you, I wouldn’t be at this place in life without you either. Dr. Olson, Dr. McCabe, Dr. Arnosti, and Dr. Cheng (my Committee) Thank you for challenging me. Even though I may not have appreciated it at the time, a humbling experience goes a long way. Thank you for accepting a place on my committee and for pushing me to think as an independent scientist. To my advisor, Tim Zacharewski, thank you for offering me a place in your lab and your understanding for family matters. Dr. Jack Harkema, thank you for always making time for me and treating me with respect. Dr. Ed Dere, I missed you these last two years. Bryan Mets, wherever you are and whatever you are doing, I wish you well. Friends, quality matters. Nathan, Christina, Lisa, Tyler, Steve, Alexis, Jenna, Nosh, Julie, Eric, Shanti, and Tony, you are all the best. Thanks to Girls’ night, an essential unwinding occasion. I would also like to thank Claudia Dominici, Michelle D’Souza, and Dan Wright for technical support and Dr. Anna Kopec, Dr. Edward Dere, Rance Nault, and Agnes Forgacs for critical review. v TABLE OF CONTENTS LIST OF TABLES ..................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... ix LIST OF ABBREVIATIONS ..................................................................................................... xi CHAPTER 1 Review of the Literature: Dioxins, AhR, Liver, and Scd1 .................................................. 16 Introduction ........................................................................................................................... 16 Dioxin and Dioxin-like Compounds: Sources and Toxicity ................................................. 18 Aryl Hydrocarbon Receptor (AhR): Molecular Mechanism and Physiological Function ... 20 Liver: A Metabolic Organ and Dioxin Target ...................................................................... 25 Stearoyl-CoA Desaturase 1 (Scd1): Role in Lipid Metabolism and Metabolic Disease ...... 27 Conclusion ............................................................................................................................ 30 References ............................................................................................................................. 32 CHAPTER 2 ................................................................................................................................ 45 Rationale, Hypothesis and Specific Aims.............................................................................. 45 Rationale ............................................................................................................................... 45 Hypothesis............................................................................................................................. 45 Specific Aims ........................................................................................................................ 45 References ............................................................................................................................. 48 CHAPTER 3 ……………………………………………………………………………………51 Aryl Hydrocarbon Receptor-Mediated Induction of Stearoyl-CoA Desaturase 1 Alters Hepatic Fatty Acid Composition in TCDD-Elicited Steatosis ............................................ 51 Abstract ................................................................................................................................. 51 Introduction ........................................................................................................................... 52 Materials & Methods ............................................................................................................ 53 Results ................................................................................................................................... 59 Discussion ............................................................................................................................. 78 Appendix A ........................................................................................................................... 84 References ............................................................................................................................. 93 CHAPTER 4 .............................................................................................................................. 101 Dietary Fat is a Lipid Source in 2,3,7,8-Tetrachlorodibenzo--dioxin (TCDD)-Elicited, Hepatic Steatosis in C57BL/6 Mice ..................................................................................... 101 Abstract ............................................................................................................................... 101 Introduction ......................................................................................................................... 102 Materials and Methods ........................................................................................................ 103 Results ................................................................................................................................. 107 Discussion ........................................................................................................................... 119 vi Appendix B ......................................................................................................................... 127 References ........................................................................................................................... 137 CHAPTER 5 .............................................................................................................................. 143 TCDD-Elicited Effects on Liver, Serum, and Adipose Lipid Composition in C57BL/6 Mice ........................................................................................................................................ 143 Abstract ............................................................................................................................... 143 Introduction ......................................................................................................................... 144 MATERIALS AND METHODS ........................................................................................ 145 Results ................................................................................................................................. 147 Discussion ........................................................................................................................... 158 References ........................................................................................................................... 165 CHAPTER 6 .............................................................................................................................. 172 Conclusions and Future Research ....................................................................................... 172 References ........................................................................................................................... 176 vii LIST OF TABLES Table 1.1. Summary of AhR Alleles Across Mouse Strains ........................................................ 24 Table 3.1. Total Hepatic Lipid Composition (mol/g) in Scd1+/+ and Scd1-/- Mice 168 h Post 30 g/kg TCDD Dose. ............................................................................................................... 65 Table A.1. Putative DRE Sequences ............................................................................................ 84 Table A.2. Terminal Body Weight, Body Weight Gain, Absolute Liver Weight and Relative Liver Weight for Scd1+/+ and Scd1-/- Mice ........................................................................... 85 Table A.3. TCDD Effects on Liver Histopathology in Scd1 Wild-Type and Null Mice............. 86 Table 4.1. Nutrient and Energy Composition of Custom Diets ................................................. 104 Table 4.2. Hepatic Lipid Levels in Mice Fed Fat or Carbohydrate Adjusted Diets 168 h Post 30 g/kg TCDD Dose .............................................................................................................. 108 Table 4.3. DREs and Regions of AhR Enrichment in TCDD Responsive Genes Associated with Lipid and Carbohydrate Transport and Metabolism. .......................................................... 116 Table B.1. Terminal Body Weight, Body Weight Gain, Absolute Liver Weight, and Relative Liver Weight ....................................................................................................................... 127 Table B.2. Hepatic Lipid Levels (mol/g) in Fat Adjusted Diet-Fed Mice 168 h Post 30 g/kg TCDD Dose ........................................................................................................................ 129 Table B.3. Lipid Composition (mol) in Fat Adjusted (A) and Carbohydrate Adjusted (B) Diets ............................................................................................................................................. 131 Table B.4. Hepatic Lipid Levels (mol/g) in Carbohydrate Adjusted Diet-Fed Mice 168 h Post 30 g/kg TCDD Dose ......................................................................................................... 133 Table 5.1. Effect of TCDD on Body, Liver, and Parametrial Fat Pad (PFP) Weights 24, 72, and 168 h Post-Dose .................................................................................................................. 148 Table 5.2. Liver, Parametrial Fat Pad, and Serum Total Lipid Content..................................... 150 viii LIST OF FIGURES Figure 1.1. Chemical Structures of TCDD and dioxin-like chemicals. ....................................... 17 Figure 1.2. General schematic representation of AhR (top) and nuclear receptors (bottom) domain organization.............................................................................................................. 21 Figure 1.3. The ligand-activated AhR signaling pathway. .......................................................... 23 Figure 1.4. The desaturation of fatty acids by stearoyl-CoA desaturase 1 (Scd1). ...................... 28 Figure 3.1. Hepatic lipid composition at 168 h in vehicle and 30 µg/kg TCDD treated Scd1+/+ (Panel A) and Scd1-/- (Panel B) mice. ................................................................................... 61 Figure 3.2. Principal component analysis (PCA) of GC-MS lipid profiles from TCDD (T) or Vehicle (V) treated Scd1+/+, Scd1+/-, and Scd1-/- mice 24, 72 and 168 h post-dose. ............ 68 Figure 3.3. Scd1 activity (desaturation) index and Correlation with Hepatic Triglyceride Levels. ............................................................................................................................................... 69 Figure 3.4. QRTPCR of hepatic lipid transport, modification, and biosynthesis genes in Scd1+/+ (+/+) and Scd1-/- (-/-) mice gavaged with 30 g/kg TCDD or sesame oil vehicle for 24 h. . 70 Figure 3.5. Scd1 activity, mRNA, and protein levels in Scd1+/+ and Scd1-/- mice treated with 30 g/kg TCDD (T) or sesame seed oil (V) 24 h post-dose. ..................................................... 73 Figure 3.6. Dioxin response element (DRE) distribution and TCDD-inducible AhR enrichment within Scd loci, and band shift assays. .................................................................................. 75 Figure A.1. Serum alanine aminotransferase levels in Scd1+/+ and Scd1-/- mice treated 168 h with either vehicle or 30 g/kg TCDD. ................................................................................ 87 Figure A.2. QRTPCR of the TCDD-inducible genes Cyp1a1, Nqo1, and Tiparp in Scd1+/+ (+/+) and Scd1-/- (-/-) mice gavaged with 30 g/kg TCDD or sesame oil vehicle for 24 h. .......... 88 Figure A.3. QRTPCR of Elovl5 mRNA (A), Elovl5 band shift assays with putative, functional DREs (B), and DRE distribution and TCDD-inducible AhR enrichment within the Elovl5 locus (C). ........................................................................................................................................ 89 Figure 4.1. Hepatic absolute and essential fatty levels in mice fed increasing fat or carbohydrate diets treated with sesame oil vehicle (V) or 30 g/kg TCDD (T) for 168 h. ...................... 110 Figure 4.2. Hepatic lipid levels in Scd1 wild-type (wt) and null mice 120 h post-dose with 30 g/kg TCDD dose. .............................................................................................................. 114 ix Figure 4.3. AhR-mediated increase in dietary lipid in TCDD elicited hepatic steatosis. .......... 120 Figure B.1. 14C levels in Scd1 wild-type (wt) and null mice gavaged with 30 g/kg TCDD for 120 h (A-C) or 168 h (D). ................................................................................................... 135 Figure 5.1. Total fatty acid composition in liver (A), parametrial fat pad (PFP, B), and serum (C) of mice 168 h post 30 µg/kg TCDD or sesame oil vehicle dose. ....................................... 152 Figure 5.2. The 18:1n9/18:0 desaturation index in liver, serum, and parametrial fat pad (PFP) tissue of mice treated with 30 µg/kg TCDD or sesame oil vehicle 168 h post-dose. ......... 155 Figure 5.3. Serum cholesterol and apolipoprotein b (Apob) 100 and Apob48 levels in Scd1 wildtype mice treated with 30 µg/kg TCDD or sesame oil vehicle. .......................................... 157 Figure 5.4. Differential expression of hepatic genes involved in cholesterol metabolism (A-E) and parametrial fat pad genes involved in lipid metabolism and transport (F-J) in mice gavaged with 30 g/kg TCDD or sesame oil vehicle for 24 h. .......................................... 159 x LIST OF ABBREVIATIONS AA Arachidonic acid AhR Aryl hydrocarbon receptor ALT Alanine aminotransferase ANOVA Analysis of variance ARNT Aryl hydrocarbon receptor nuclear translocator bHLH Basic-helix-loop-helix BW Body weight cDNA Complementary deoxyribonucleic acid CHOL Cholesterol ChREBP Carbohydrate response element binding protein CM Chylomicron COUP-TF Chicken ovalbumin upstream promoter transcription factor DAG Diacylglycerol DHA Docosahexaenoic acid DBD DNA binding domain DLC Dioxin like chemical DRE Dioxin response element EDC Endocrine disrupting compound EPA Eicosapentaenoic acid ER Estrogen receptor FA Fatty acid xi FFA Free fatty acid FAME Fatty acid methyl ester Foxo1 Forkheadbox transcription factor 1 GC-MS Gas chromatography-mass spectrometry HDL High-density lipoprotein HFD High fat diet HLH Helix-loop-helix HNF4 Hepatocyte nuclear factor alpha LDL Low-density lipoprotein LBD Ligand binding domain LXR Liver X receptor MCD Methionine choline deficient MetS Metabolic syndrome MSS Matrix similarity score MUFA Monounsaturated fatty acid NAFLD Non-alcoholic fatty liver disease NASH Non-alcoholic steatohepatitis N/A Not available NC No change ND Not detected NEFA Non-esterified fatty acids NLS Nuclear localization signal NR Nuclear receptor xii OVX Ovariectomized PAH Polyaromatic hydrocarbon PAS Per-Arnt-Sim PBDF Polybrominated dibenzofuran PCA Principal component analysis PCB Polychlorinated biphenyl PCDD Polychlorinated dibenzo-p-dioxin PCDF Polychlorinated dibenzofuran Per Period PGC1 Peroxisome proliferator activated receptor  coactivator 1  PND Postnatal day PPAR Peroxisome proliferator activated receptor alpha PUFA Polyunsaturated fatty acid PWM Position weight matrix PVDF Polyvinylidene flouride PXR Pregnane X receptor QRTPCR Quantitative real time polymerase chain reaction RAR Retinoic acid receptor RLW Relative liver weight RNA Ribonucleic acid ROS Reactive oxygen species RXR Retinoid X receptor Scd1 Stearoyl-CoA desaturase 1 xiii SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SD Standard deviation SE Standard error SFA Saturated fatty acid Sim Single-minded TAD Transcriptional activation domain TAG Triglyceride TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin TEF Toxic equivalency factor TEQ Toxic equivalents TFA Total fatty acid TiPARP TCDD-inducible poly(ADP-ribose) polymerase 7, PARP7 TLC Thin layer chromatography TR Thyroid receptor VLDL Very low-density lipoprotein xiv CHAPTER 1 15 CHAPTER 1 REVIEW OF THE LITERATURE: DIOXINS, AHR, LIVER, AND SCD1 INTRODUCTION The AhR is a basic helix-loop-helix (bHLH) Period (Per)/ aryl hydrocarbon receptor nuclear translocator (ARNT)/ single minded (Sim) (PAS) transcription factor family member that mediates a wide-range of species- and tissue-specific effects in response to ligand activation [1]. Polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) are the best-characterized high-affinity AhR ligands that are ubiquitously distributed throughout the environment. HAHs describe a family of polychlorinated dibenzo--dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polybrominated dibenzofurans (PBDFs) and polychlorinated biphenyls (PCBs). PCDD/Fs and PBDFs with chlorine or bromine substitutions in the 2, 3, 7, and 8 positions and PCBs that have four or more coplanar chlorine atoms are termed “dioxin-like chemicals” (DLCs) due to their structural and chemical similarity (Figure 1.1). DLCs elicit common, species- and tissue-specific toxic effects including wasting syndrome, immunotoxicity, reproductive abnormalities, tumor promotion, induction of gene expression, hepatotoxicity, and endocrine disruption [2-4]. Due to their resistance to degradation and lipophilicity, DLCs bioaccumulate in animal fat stores, persist in the environment, and pose a risk to wildlife and human health [5]. Persistent AhR activation significantly alters hepatic function with the potential to negatively affect metabolism and influence the development of metabolic disease. AhR 16 Figure 1.1. Chemical Structures of TCDD and dioxin-like chemicals. For interpretation of the references to color in this and all other figures the reader is referred to the electronic version of this dissertation. 17 ligands elicit lipid abnormalities such as fatty liver (steatosis) [6-9] that is also the hepatic manifestation of MetS [10, 11], a term used to describe a group of multi-factorial risk factors that include dyslipidemia, obesity, insulin resistance or type II diabetes, and hypertension [12]. Examination of ligand-activated, AhR-mediated alterations in global lipid uptake, metabolism, and transport can significantly expand our current understanding of AhR physiological function, as well as etiology of metabolic disease. DIOXIN AND DIOXIN-LIKE COMPOUNDS: SOURCES AND TOXICITY DLCs represent a class of toxic chemicals predominantly produced as by-products of combustion, chemical processes involving chlorine, metals smelting, refining, and process sources, environmental reservoirs, and natural sources such as forest fires and volcanoes [13]. The term dioxin is often used to describe not only TCDD, the most widely studied HAH, but also complex mixtures of TCDD and related chemicals. TCDD is one of the most toxic dioxins with a toxic equivalency factor (TEF) = 1 and therefore used as the reference compound for this class of chemicals. The TEF approach [14, 15] was developed to simplify risk assessment and extrapolate the toxicity of complex dioxin mixtures into a single value expressed as toxic equivalents (TEQs). For the TEF approach, each dioxin-like congener is assigned a TEF by scientific experts based on available data relative to TCDD, ranging from 0.000001 to 1 [16]. The TEFs for each congener in a mixture are multiplied by their respective mass concentration to identify a TEQ representative of a mixture’s toxicity. According to a 2003 draft report, the U.S. Environmental Protection Agency estimated average U.S. adult TEQ intake at 66 pg/day, well above the 2012 EPA non-cancer reassessment reference dose of 0.7 pg/kg/day or 49 pg/day for an average 70 kg adult. Human exposure to 18 dioxin is primarily through the food supply, mainly consumption of animal fats including meat, fish, and dairy. Dioxins bioaccumulate in animal fat depots and in humans, persist with a halflife of ~7 years [17]. Therefore, although strict industrial regulations have led to a ~90% reduction in nationwide emission over the past 40 years, food safety issues have become an increasing public concern [18]. Numerous human effects documented from direct dioxin contamination incidents have emphasized the clinical impact of dioxin exposure. Cross-sectional and longitudinal studies have evaluated 1) U.S. chemical workers exposed to dioxin after a TCP reactor explosion in West Virginia [19], 2) German BASF employees exposed to TCDD after an uncontrolled reactor decomposition reaction [20], 3) U.S. Air Force Ranch Hand personnel exposed to the herbicide Agent Orange during the Vietnam war [21], 4) residents of communities in Missouri exposed to dioxin contaminated waste oil [22], and 5) residents of Seveso, Italy exposed to industrial emissions caused by an explosion of a trichlorophenol reactor [23, 24]. These studies have linked exposure in adults and children to chloracne [25, 26], impaired immune function [20, 22], impaired nervous system and reproductive development [27], altered liver function [20-22, 28], incidences of dyslipidemia [20, 25, 26, 28], and Type II diabetes [29-31]. The fetus and newborn infants, with rapidly developing organ systems, are subgroups particularly sensitive to dioxin exposure. In particular, Dutch postnatal studies identified an association between maternal exposure and elevated TSH and T4 levels in nursing infants [32] that suggest dioxin exposure modulates the hypothalamic-pituitary-thyroid axis, an essential metabolic regulator. Recently, environmental contaminants have received increasing attention for their roles in disrupting energy balance and homeostasis [33-36]. In humans, dioxin’s metabolic disruptive effects are further highlighted by epidemiological studies that frequently link dioxin exposure to 19 dyslipidemia, insulin resistance, and cardiovascular abnormalities [30, 37-44]. In rodents, dioxin elicits similar effects [45] including reduced gluconeogenesis [46], inhibited adipogenesis [47], and decreased glucose transport activity in vitro [48, 49]. Collectively, these studies emphasize the potential for dioxin exposure to adversely affect human health and strongly imply endocrinedisruption. Considering that the AhR mediates most, if not all effects elicited by dioxin, the receptor has been extensively studied and will be discussed below. ARYL HYDROCARBON RECEPTOR (AHR): MOLECULAR MECHANISM AND PHYSIOLOGICAL FUNCTION The AhR is a bHLH-PAS transcription factor that mediates most, if not all, effects elicited by dioxins. The AhR bHLH domain is strictly conserved, allows contact with DNA, and functions as the primary heterodimerization surface [50] (Figure 1.2). The PAS domain serves as a secondary heterodimerization surface that promotes interactions with other PAS protein family members and also functions as a ligand binding domain (LBD) [50-52]. The C-terminal transcriptional activation domain (TAD) is essential for AhR mediated gene transcription and recruits transcriptional co-regulators and other basal transcription machinery [50]. PAS family transcription factors, such as AhR, are structurally and functionally distinct from nuclear receptors (NR). All NR are grouped into a large superfamily of homologous proteins based on their conserved DNA binding domain (DBD) that consists of two highly conserved zinc finger motifs [53]. NRs are classified as either Type I or Type II depending on their ability to homo- or heterodimerize and bind to inverted or direct repeat DNA half sites, respectively. Estrogen receptor (ER) is an example of a Type I NR. Peroxisome proliferator activated receptor (PPAR), retinoid X receptor (RXR), retinoic acid receptor (RAR) and thyroid 20 Figure 1.2. General schematic representation of AhR (top) and nuclear receptors (bottom) domain organization. 21 receptor (TR) are all Type II receptors [53]. The C-terminal conserved LBD contains a liganddependent activation domain, which interacts with chaperone proteins and transcriptional coregulators. The N-terminus also contains a transcriptional activation surface that recruits coregulators [53]. Unlike nuclear receptors, the AhR resides in the cytoplasm. The classical mechanism of AhR activation involves ligand diffusion into the cytoplasm where it is bound by the AhR (Figure 1.3). Ligand binding is presumed to induce a conformational change exposing the AhR nuclear localization signal (NLS) and induce dissociation of chaperone proteins hsp90, p23, and XAP2 [54, 55]. The liganded AhR translocates to the nucleus where it forms a heterodimer with ARNT, another member of the bHLH-PAS family. This high-affinity AhR:ARNT heterodimer binds specific DNA recognition sites with the invariant core sequence GCGTG that are termed dioxin response elements (DREs) [56, 57]. AhR:ARNT DNA binding leads to chromatin remodeling, transcriptional co-activator recruitment, and altered rates of gene transcription [54, 58]. Cytochrome P450 1a1 and 1a2 are the best characterized and prototypical AhR activated genes whose transcriptional activation is often used as litmus test for AhR induction and ligand potency [3, 59-62]. AhR:ARNT-dependent gene transcription terminates after heterodimer dissociation from the DRE followed by ubiquitin-mediated 26S proteasome pathway AhR degradation [63]. Accumulating evidence indicate a polymorphic cytochrome P450 response to TCDD toxicity across species that is dependent on AhR binding affinities [64]. Specifically, inbred mouse strains have inter-individual genetic susceptibility to dioxin toxicity due to four distinct Ahr alleles (Ahrb1, Ahrb2, Ahrb3, and Ahrd, Table 1.1) with 10-20-fold differences in ligand binding affinity that renders them either responsive (b alleles, Kd ≤ 1 nM) or unresponsive d 22 Figure 1.3. The ligand-activated AhR signaling pathway. Ligand passively diffuses into the cell and is bound by the aryl hydrocarbon receptor (AhR). Ligand binding induces a conformational change leading to dissociation of chaperone proteins (hsp90, p23, and XAP2) and exposing the AhR nuclear localization signal. The liganded AhR translocates to the nucleus and heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT). The AhR:ARNT heterodimer binds to dioxin response elements (DRE) located in the promoters of target genes to activate gene transcription. 23 Table 1.1. Summary of AhR Alleles Across Mouse Strains d b1 b2 b3 AhR Allele AhR Affinity for TCDD Low High High High --------- AKR/J MA/MyJ A/J MOLF/Ei SM/J DBA/2J C57BL/6J BALB/cByJ SPRETUS/Ei WSB/EiJ I/LnJ C57BLKS/J C3H/HeJ RIIIS/J LG/J C57BR/cdJ CBA/J FVB/NJ LP/J C57L/J PERA/EiJ NOD/LtJ NZB/BlNJ C58/J PL/J NON/LtJ 129S1/SvImJ SEA/GnJ PWD/PhJ SWR/J BUB/BnJ SJL/J CE/J AhR AhR AhR Unknown Strain CAST/Ei Bold – ancestral wild-type derived strains. Data were summarized from Thomas et al., 2002 [66] and the mouse phenome database (http://phenome.jax.org). 24 Allele (Kd = 16 nM) [65-67]. Studies using transgenic mice harboring the b allele have highlighted the importance of the AhR nuclear signaling pathway in mediating the toxicities of dioxins, with a particular focus on TCDD [68, 69]. Transgenic mice carrying mutations in the NLS or DBD are resistant to TCDD toxicity, including hepatic steatosis. However, TCDDelicited DRE-independent and/or ARNT-independent effects have also been reported [70-72]. Despite generation of the AhR knock-out [73, 74] and transgenic animal models [69, 75], the physiological function of AhR, beyond xenobiotic metabolism and hepatic growth and development, remains elusive. AhR conservation among all vertebrates [76] as well as similar cytochrome P450 up-regulation in fish, rodents, and birds [77, 78] suggests a fundamental role for the AhR. Yet it is unlikely that ancient exposure to POPs (that have been introduced into the environment along with the industrial revolution [79]) provided selective pressure on AhR during vertebrate evolution. No high affinity endogenous AhR ligand has been identified although the AhR is activated by diverse lipophilic endobiotics (indoles, tetrapyrroles, and arachidonic acid metabolites) and naturally derived molecules (vegetable-, fruit-, and tea-derived indoles and flavonoid metabolites) [1, 80]. Collectively, the developmental phenotypes in AhR null mice and AhR evolutionary conservation suggest a role for AhR in cellular physiology other than metabolism of foreign chemicals, although it remains to be elucidated. Nonetheless, AhR ablation is accompanied by several liver defects, a metabolic organ coincidentally targeted by TCDD toxicity. LIVER: A METABOLIC ORGAN AND DIOXIN TARGET The liver is a primary target of TCDD with steatosis, hepatomegaly, and elevated liver enzymes (alanine aminotransferase and glutathione S-transferase) representing significant 25 hepatotoxic endpoints [45, 81-84]. Strategically situated between the portal and systemic circulation, the liver, which has first access to absorbed nutrients as well as other exogenous substances, is the site for primary metabolism. The principal function of the liver is to metabolize, detoxify, inactivate, and convert both endogenous and exogenous substance that are either excreted into bile or returned to the systemic circulation [85]. Another important liver function is synthesis and degradation of protein, carbohydrates, and lipids for distribution to extrahepatic tissues depending on energy needs. Finally, the liver regulates whole body cholesterol balance via biliary excretion of cholesterol (CHOL), CHOL conversion to bile acids, and by regulating CHOL synthesis [85]. Consequently, the liver is an essential regulator of whole body metabolism and energy homeostasis that is disrupted by TCDD. Studies suggest that TCDD exposure in rodents and humans disrupts hepatic energy balance by altering normal anabolic and catabolic pathways. For example, one of the most important metabolic functions performed by the liver is maintenance of blood glucose levels between meals. When insulin levels are high and glucagon levels are low (after a meal), the liver takes-up glucose and either stores it as glycogen or breaks it down into pyruvate. Conversely, when glucose and insulin levels are low and glucagon levels are high, hepatic gluconeogenesis and glycogenolysis prevail [85]. During periods of energy excess, the liver can convert glucose into fatty acids (FAs) that are esterified into triglycerides (TAGs). TAGs are either stored or packaged with cholesterol and apolipoprotein b100 into very low-density lipoproteins (VLDL) for export into the blood. TAGs within circulating VLDL are hydrolyzed by circulating lipases that release FA for peripheral tissue (primarily muscle and adipose) uptake [86]. During periods of negative energy balance, FAs metabolized by -oxidation generate acetyl CoA and ketone bodies. Dioxin exposure disrupts liver metabolism by decreasing gluconeogenesis [87], FA and 26 CHOL synthesis [88, 89], and mitochondrial beta-oxidation [9, 90], while promoting hepatic steatosis [6, 7, 91-93] regardless of the hepatic energy state. These effects collectively net altered energy balance with implications for global metabolic disruption. Importantly, TCDD-elicited steatosis and lipid abnormalities are also observed in humans [8]. Hepatic steatosis in the absence of alcohol consumption constitutes NAFLD, the hepatic manifestation of MetS, with insulin resistance as the primary pathogenic route [11]. Epidemiological studies have not only linked human dioxin exposure to incidences of steatosis, but also other MetS associated risk factors including dyslipidemia, insulin resistance/diabetes [8, 38, 39, 94, 95], and hyperglycemia [96]. Collectively, these studies in combination with TCDDdependent alterations in hepatic lipid and glucose metabolism, further suggest TCDD and similar compounds may underlie AhR-mediated alterations in global lipid metabolism and the etiology of metabolic disease. STEAROYL COA DESATURASE 1 (SCD1): ROLE IN LIPID METABOLISM AND METABOLIC DISEASE Interestingly, several toxicities elicited by AhR activation, including steatosis and insulin resistance, are common effects observed in Scd1 mouse models [97-99]. Scd1 is an endoplasmic reticulum bound trans-membrane 9 desaturase that catalyzes the rate-limiting step in MUFAs synthesis. MUFAs are critical components of complex lipids including membrane lipids, phospholipids, TAGs, and CHOL esters [100]. Scd1 together with cofactors NADH, b5 reductase, cytochrome b5, and oxygen introduce a double bond into fatty acyl-coenzyme A [101] (Figure 1.4). Palmitate (16:0) and 27 stearate (18:0) are the Figure 1.4. The desaturation of fatty acids by stearoyl-CoA desaturase 1 (Scd1). The oxidative reaction catalyzed by Scd1 introduces a double bond into the ∆9 position between carbons 9 and 10. Electrons flow from NADPH to cytochrome b5 reductase to cytochrome b5 to activate O2 that is reduced to H2O. Steatate is the preferred substrate for Scd1 and desaturated to oleate, a major constituent of monounsaturated fatty acids, triglycerides, and membrane lipids. 28 preferred substrates for SCD and are desaturated into palmitoleate (16:1n7) and oleate (18:1n9), respectively. Multiple SCD isoforms have been identified in mice (Scd1-Scd4) [102-105] and humans (SCD1 and SCD5) [106-108] and these genes exhibit tissue-specific expression. In mice, Scd1 and Scd2 are primarily expressed in liver and adipose, but also other lipogenic tissues. Scd2 is expressed in neonates, while Scd1 is the predominant form expressed in adults. Unlike other Scd isoforms that confer activity towards stearoyl-CoA [99], Scd3 exhibits substrate preference for palimtoyl-CoA [109] and is primarily expressed in skin, Harderian gland and preputial gland [105]. Scd4 is exclusively expressed in heart [102]. Human SCD1 is primarily expressed in adipose and liver and shares ~85% amino acid similarity with Scd1-Scd4. SCD5 is unique to primates and abundantly expressed in brain and pancreas [108]. Scd1 is transcriptionally regulated by a variety of nutrients (glucose, fructose, CHOL, polyunsaturated fatty acids (PUFAs)), hormones (insulin, glucagon, thyroid hormone, estrogen) and environmental factors (temperature, light, cadmium) [99, 110, 111] and their cognate transcription factors. Specifically, nutrient sensing by liver X receptor (LXR) [112], sterol regulatory element binding protein-1c (SREBP-1c) [113, 114], carbohydrate response-element binding protein (ChREBP) [115, 116], pregnane -X receptor (PXR) [117, 118] and PPARs [119] positively regulate Scd1 transcription. Scd1 activity is also controlled by post-translational ubiquitin-pathway mediated degradation [120] that is inhibited by PPAR activation [121]. Evidence from knockout and transgenic studies suggests that Scd1, required for TAG, phospholipid, CHOL ester, and VLDL synthesis [122, 123], is important for the development of diet-induced metabolic disorders. Specifically, mice with targeted disruption of the Scd1 gene are protected from high-fat and high-carbohydrate diet-induced steatosis and obesity [113, 124-126], 29 and exhibit improved insulin sensitivity [97, 127]. In humans, increased Scd1 expression is associated with insulin resistance [128] and an increased 18:1n9/18:0 plasma ratio (an indirect measure of Scd1 activity) that positively correlates with increased TAGs [129]. Furthermore, several single nucleotide polymorphisms located within intron, exon, and 3’UTR SCD1 genic regions are associated with body fat distribution and insulin sensitivity [130]. Consequently, Scd1 has become a pharmacological target for the treatment of MetS risk factors including obesity and diabetes [97]. CONCLUSION TCDD and similar chemicals bioaccumulate and persist in the food chain to elicit adverse health effects in humans and animals. Accumulating evidence suggests these chemical effects superimpose upon a Western lifestyle to initiate or exacerbate disruption of energy metabolism [131]. Hepatic steatosis is the consequence of TCDD-mediated AhR activation and also linked with metabolic disease. 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Curr Opin Gastroenterol 2010,,26, 202-208. 43 CHAPTER 2 44 CHAPTER 2 RATIONALE, HYPOTHESIS AND SPECIFIC AIMS RATIONALE Hepatic steatosis in the absence of alcohol consumption is considered the hepatic manifestation of MetS [1]. Human exposure to TCDD and related compounds have been implicated in the development of MetS [2, 3] and its associated risk factors including insulin resistance [4, 5], hypertriglyceridemia [6-8], and steatosis [9]. In rodents Scd1 performs a critical role in lipid homeostasis and when deleted, protects mice from diet-induced hepatic lipid accumulation and improves insulin sensitivity [10]. TCDD and DLC exposure in mice elicits hepatotoxicity marked by the differential expression of genes involved in lipid metabolism, including Scd1 [11], and increased hepatic vacuolization due to lipid accumulation [11-14]. In summary, these results suggest that AhR-mediated effects on Scd1 regulation, lipid metabolism, and transport may underlie DLC-induced steatosis and other related pathophysiologies. HYPOTHESIS TCDD-elicited, AhR mediated steatosis involves Scd1 dependent and independent alterations in lipid uptake, metabolism, and transport. SPECIFIC AIMS Specific aim 1 will test the subhypothesis that Scd1 deficiency protects from TCDD-elicited steatosis. AhR regulation of Scd1 and TCDD-induced steatosis in Scd1 wild-type and null mice will be characterized. 45 Specific aim 2 will test the subhypothesis that diet is the primary source of lipid in TCDDelicited steatosis. Dietary fat, carbohydrate, and 14 C-oleate will be examined as hepatic lipid sources in TCDD-elicited steatosis. Specific aim 3 will test the subhypothesis that TCDD not only alters liver lipid composition, but also parametrial adipose and serum lipid composition. TCDD effects on liver, parametrial fat pad (PFP), and serum fatty acid levels and composition will be examined by gas chromatographymass spectrometry (GC-MS). In addition serum lipid profiles (total CHOL, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TAG) and VLDL) will be examined. 46 REFERENCES 47 REFERENCES 1. Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, Fruchart JC, James WP, Loria CM, Smith SC, Jr.: Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 2009, 120:16401645. 2. 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Boverhof DR, Burgoon LD, Tashiro C, Sharratt B, Chittim B, Harkema JR, Mendrick DL, Zacharewski TR: Comparative toxicogenomic analysis of the hepatotoxic effects of TCDD in Sprague Dawley rats and C57BL/6 mice. Toxicol Sci 2006, 94:398-416. 12. Kopec AK, Boverhof DR, Burgoon LD, Ibrahim-Aibo D, Harkema JR, Tashiro C, Chittim B, Zacharewski TR: Comparative toxicogenomic examination of the hepatic effects of PCB126 and TCDD in immature, ovariectomized C57BL/6 mice. Toxicol Sci 2008, 102:61-75. 13. Kopec AK, Burgoon LD, Ibrahim-Aibo D, Mets BD, Tashiro C, Potter D, Sharratt B, Harkema JR, Zacharewski TR: PCB153-elicited hepatic responses in the immature, ovariectomized C57BL/6 mice: comparative toxicogenomic effects of dioxin and non-dioxin-like ligands. Toxicol Appl Pharmacol 2010, 243:359-371. 14. Kopec AK, D'Souza ML, Mets BD, Burgoon LD, Reese SE, Archer KJ, Potter D, Tashiro C, Sharratt B, Harkema JR, Zacharewski TR: Non-additive hepatic gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,2',4,4',5,5'hexachlorobiphenyl (PCB153) co-treatment in C57BL/6 mice. Toxicol Appl Pharmacol 2011. 49 CHAPTER 3 Angrish MM, Jones AD, Harkema JR, Zacharewski TR: Aryl Hydrocarbon ReceptorMediated Induction of Stearoyl-CoA Desaturase 1 Alters Hepatic Fatty Acid Composition in TCDD-Elicited Steatosis. Toxicol Sci 2011, 124:299-310. 50 CHAPTER 3 ARYL HYDROCARBON RECEPTOR-MEDIATED INDUCTION OF STEAROYL-COA DESATURASE 1 ALTERS HEPATIC FATTY ACID COMPOSITION IN TCDD-ELICITED STEATOSIS ABSTRACT TCDD induces hepatic dyslipidemia mediated by the AhR. Scd1 performs the ratelimiting step in MUFA synthesis, desaturating 16:0 and 18:0 into 16:1n7 and 18:1n9, respectively. To further examine the role of Scd1 in TCDD-induced hepatotoxicity, comparative +/+ studies were performed in Scd1 and Scd1 -/- mice treated with 30 µg/kg TCDD. TCDD induced Scd1 activity, protein, and mRNA levels ~2-fold. In Scd1 +/+ mice, hepatic effects were marked by increased vacuolization and inflammation, and a 3.5-fold increase in serum ALT levels. Hepatic TAGs were induced 3.9-fold and lipid profiling by GC-MS measured a 1.9-fold increase in FA levels, consistent with the induction of lipid transport genes. Induction of Scd1 altered FA composition by decreasing saturated fatty acid (SFA) molar ratios 8% and increasing MUFA molar ratios 9%. Furthermore, ChIP-chip analysis revealed AhR enrichment (up to 5.7fold) and computational analysis identified 16 putative functional DREs within Scd1 genomic loci. Band shift assays confirmed AhR binding with select DREs. In Scd1 -/- mice, TCDD induced minimal hepatic vacuolization and inflammation, while serum ALT levels remained unchanged. Although Scd1 deficiency attenuated TCDD induced TAG accumulation, overall FA 51 levels remained unchanged compared to Scd1 +/+ mice. In Scd1 -/- mice, TCDD induced SFA ratios 8%, reduced MUFA ratios 13%, and induced polyunsaturated fatty acid ratios 5% relative +/+ to treated Scd1 mice. Collectively, these results suggest AhR regulation of Scd1 not only alters lipid composition, but also contributes to the hepatotoxicity of TCDD. INTRODUCTION TCDD and related compounds elicit a broad spectrum of biological responses ranging from effects on development to pathologies affecting specific organ functions as well as the immune and nervous system. These effects are mediated by the AhR a cytosolic ligand-activated bHLH PAS family transcription factor [1, 2]. The proposed mechanism involves ligand binding to the cytosolic AhR, leading to dissociation of chaperone proteins and translocation of the AhR to the nucleus [3]. The activated AhR then heterodimerizes with the ARNT [4] to bind DREs located within regulatory regions of target genes [5] recruiting chromatin remodeling complexes and transcriptional co-regulators that modulate gene transcription [6]. Although the range of endogenous functions regulated by the AhR remains uncertain, studies in null mice show it is necessary for proper liver development and mediates the toxicity of TCDD [7-9]. TCDD elicits hepatomegaly [2] and liver pathologies that include hepatocellular neoplasms [10], inflammation, necrosis, steatosis, and the differential expression of lipid metabolism and transport genes in mice [11-14]. DNA binding by AhR is compulsory for TCDD-induced hepatic steatosis [15]. Scd1 catalyzes the rate-limiting step MUFA biosynthesis and is a target for the treatment of metabolic related disorders [16, 17]. Scd1 desaturates palmitate (16:0) and stearate (18:0) into 52 palmitoleate (16:1n7) and oleate (18:1n9), respectively, which can be metabolized further to other MUFAs and PUFAs, the primary constituents of membrane lipids, triglycerides, phospholipids and cholesterol esters [18, 19]. Dietary, hormonal and environmental factors regulate Scd1 mRNA expression and protein stability, emphasizing the importance of Scd1 in lipid metabolism [18, 20]. Furthermore, Scd1 -/- mice are resistant to diet-induced steatosis and exhibit impaired triglyceride synthesis [19, 21]. In humans, the Scd1 activity index (serum ratios of 16:1/16:0 or 18:1/18:0) correlates with hypertriglyceridemia and insulin resistance [22, 23] and is predictive of metabolic syndrome [24]. Furthermore, single nucleotide polymorphisms have been identified in human Scd1 that are associated with obesity and insulin sensitivity [25]. In this study, we examined AhR regulation of Scd1 and TCDD elicited steatosis in +/+ Scd1 -/- and Scd1 mice. Hepatic fatty acid profiling with complementary histopathology, enzyme activity assays, and gene expression were assessed in immature Scd1 +/+ and Scd1 -/- female mice and integrated with AhR ChIP-chip, band shift, and computational data. Our results suggest that the regulation of hepatic lipid transport and metabolism genes by the AhR, including Scd1, is involved in TCDD-induced steatosis in the mouse. MATERIALS & METHODS ANIMAL BREEDING AND GENOTYPING B6.129-Scd1 tm1Myz /J heterozygous mice were obtained from the Jackson Laboratory (Ben Harbor, Maine) and bred at the Michigan State University Laboratory Animal Care facility. Mice were maintained on a 12h light/dark cycle and housed in autoclaved polycarbonate cages with microisolator lids containing aspen woodchips and nesting material. Animals were allowed free access to Harlan Teklad irradiated F6 rodent diet 7964 (Madison, WI) and autoclaved 53 deionized water throughout the study. On postnatal day (PND) 21 mice were genotyped by ear punch and weaned. All procedures were carried out with the approval of the Michigan State University Institutional Animal Care and Use Committee. IN VIVO TREATMENT On PND 28 mice were gavaged with 0.1 ml of sesame oil for a nominal dose of 0 (vehicle control) or 30 g TCDD per kg body weight. The immature mouse was used to facilitate comparisons with other data sets as well as avoid potential interactions with estrogens produced by developed ovaries. Doses were chosen to elicit moderate hepatic effects while avoiding overt toxicity in long term studies. Litters were combined with no more than five animals per cage. Animals were sacrificed at 24, 72 and 168 h post dose. Mice were weighed and blood was collected via submandibular vein puncture before sacrifice. Tissue samples were removed, weighed, flash frozen in liquid nitrogen and stored at -80C. The right lobe of the liver was fixed in 10% neutral buffer formalin for histological analysis. HISTOPATHOLOGY AND CLINICAL CHEMISTRY Fixed liver tissues were processed as previously described [14] at the Michigan State University Investigative Histopathology Laboratory, Division of Human Pathology, using a modified version of previously published procedures [26]. Serum alanine aminotransferase levels (ALT) were measured by the Michigan State University Diagnostic Center for Population and Animal Health Clinical Laboratory. HEPATIC TRIGLYCERIDE LEVELS Frozen liver samples (~100 mg) were homogenized (Polytron PT2100, Kinematica) in 1 ml of 1.15% KCl. Triglycerides were extracted from 200 µl of hepatic homogenate with 800 µl of isopropyl alcohol by vortex mixing for 10 min. The samples were centrifuged for 5 min at 54 800xg at room temperature and the supernatant was collected into separate vials. The concentration of hepatic triglycerides was determined by spectrophotometry from 20 µl supernatant with a commercial L-Type Triglyceride M kit (Wako Diagnostics, Richmond, VA) with Multi-Calibrator Lipids as a standard (Wako Diagnostics) according to manufacturer's protocol. Final results were normalized to the initial weight of the liver sample. GC-MS FATTY ACID METHYL ESTER (FAMES) HEPATIC LIPID PROFILING Liver samples (~100 mg, n=5/group) were homogenized in 40% methanol and acidified with concentrated HCl (~34L). Lipids were extracted with chloroform: methanol (2:1) containing 1 mM 2,6-di-tert-butyl-4-methylphenol. The organic phase was removed and protein and aqueous phases were re-extracted with chloroform. The organic phases were pooled and solvents evaporated under nitrogen. The samples were resuspended in 3 N non-aqueous methanolic HCl and held at 60ºC overnight. The next day samples were cooled to room temperature and 0.9% (w/v) NaCl and hexane was added. The organic phase was separated by centrifugation, collected, dried under nitrogen and resuspended in hexane. Samples were separated and analyzed with an Agilent 6890N GC with a DB23 column (30 meter length, 0.25 mm id, 0.25 m film thickness) interfaced to an Agilent 5973 MS. 19:1n9 free fatty acid (FFA) and 19:0 TAG were added as extraction efficiency controls and 17:1n1 fatty acid methyl ester was spiked in as a loading control (Nu-chek, Elysian, MN). GC/MS data files were converted to Waters MassLynx file format and were analyzed with MassLynx software and are reported as mol/g liver tissue or mol%. Fatty acid levels are based upon peak areas from total ion chromatograms and mol/g is obtained from a linear calculation of a calibration curve normalized to sample weight. Principal component analysis of fatty acid abundance was performed in R V2.6.0. 55 SCD1 ACTIVITY ASSAY Scd1 activity assays were performed as previously described [27]. Microsomal fractions were isolated by differential centrifugation, and protein was quantified by using the Bradford assay (BioRad). Assays were performed at 37C for 30 min with 100 g microsomal protein, 0.03 Ci 14 C-stearoyl-CoA (ARC 0756, 50-60 mCi/mmol, American Radiolabeled Chemicals, St. Louis, MO) 2 mM NADH, 0.03 mM cold stearoyl-CoA in 0.1 M phosphate buffer at pH 6.8. The reaction was quenched with 2.5 M KOH and saponified at 80C for 45 min. Fatty acids were acidified with formic acid, extracted with hexane and dried under nitrogen. FAs were resuspended in 50 L hexane and separated by 100 g/L AgNO3 impregnated TLC using CHCl3:MeOH:acetic acid:water (90:8:1:0.8 v/v) as a developing solvent. TLC plates were dried and exposed to autoradiography film, bands scraped into scintillation fluid, and measured by liquid scintillation counting. Scd1 activity is expressed as nmol/(mg protein*min). WESTERN BLOTTING Microsomal fractions isolated for activity assays (10 g each) were separated in a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene flouride (PVDF) membrane (Millipore Billerica, MA). Scd1 protein was immunoblotted with an Scd1 antibody (SC-14719; Santa Cruz Biotechnology Santa Cruz, CA). Conventional western blot loading controls such as Gapdh and Actb are cytosolic proteins, and therefore removed during microsome purification. Epoxide hydrolase (Epxh1, ab76226; Abcam, MA) is a microsomal protein and was used as a reference for loading control. Immunoreactive bands were visualized by chemiluminescnce with the Pierce ECL Western blotting substrate (Thermo Scientific Rockford, IL) [21]. Immunoreactive bands were quantified by densitometry (ImageJ) and normalized to the loading control. 56 AHR CHIP-CHIP AND DRE MOTIF COMPARISONS The genomic locations of AhR enrichment were previously determined by ChIP-chip from hepatic tissue of immature female ovariectomized C57BL/6 mice orally gavaged with 30 μg/kg TCDD [28]. The genomic locations of the 5’-GCGTG-3’ DRE core sequences were previously determined in mouse [29]. The DRE core along with the flanking upstream and downstream 7 bp were compared to a position weight matrix and matrix similarity scores (MSS) were calculated [29]. Regions of AhR enrichment were compared against DRE core sequences across Scd1, Scd2, Scd3, and Scd4 loci. Associated mouse genomic annotation (mm9) was downloaded from the UCSC Genome Browser [30]. BAND SHIFT ASSAYS Putative DREs with a MSS > 0.8 located from -10 kb upstream from the Scd1 transcriptional start site (TSS) were targeted for band shift assays that were performed as previously described [31]. Equimolar complimentary DNA oligonucleotides (Table A.1) were combined and annealed by heating at 95C for 5 min and cooling to room temperature. The double-stranded DNA oligonucleotide was labeled with T4 polynucleotide kinase (M0201S; New England Biolabs) and [y-32P]ATP (0135001; MP Biomedicals, OH). Each reaction contained 3 g hepatic guinea pig cytosol extract* mixed with either 20 nM TCDD or an equivalent volume of DMSO and was incubated for 1 hr at room temperature. Guinea pig AhR is readily transformed in vitro and binds DREs with high affinity [32]. The activated cytosols were combined with HEDG buffer (25 mM Hepes, pH 7.7/1 mM EDTA/1 mM dithiothreitol/10% glycerol) and 1.7 g polyd(I-C) (Invitrogen) additional 15 minutes. and incubated at room temperature for an 32 P-labeled double-stranded DNA oligonucleotide (10,000 cpm, 0.1-0.3 ng in HEDG buffer) was added to the pre-incubation mixture and incubated for an additional 15 57 minutes. For supershift assays, either 1 g AhR (ab2769; Abcam, MA) or ARNT (SC-5580; Santa Cruz Biotechnology Santa Cruz, CA) antibody was added to the pre-incubation mixture. Samples were resolved by non-denaturing polyacrylamide gel electrophoresis and imaged by autoradiography. In competition experiments unlabeled competitor DNA was added to the preincubation mixture at 100-fold molar excess. RNA ISOLATION RNA was isolated from frozen liver samples with 1.3 mL TRIzol (Invitrogen) according to the manufacturer’s protocol and an additional acid phenol:chloroform extraction as previously described [11]. Total RNA was resuspended in RNA storage solution, quantified by spectrophotometery at A260 and quality assessed by gel electrophoresis. QRTPCR Quantitative real-time PCR (QRTPCR) of Scd1, Scd2, Scd3, Scd4, Cyp1a1, Nqo1, Tiparp, Elovl5, Cd36, LdlR, Fabp4, Vldlr, Acaca, and Fasn expression was performed as previously described [11]. The copy number of each sample was standardized to the geometric mean of Gapdh, Hprt, and Actb to control for differences in RNA loading, quality, and cDNA synthesis [33]. Data are reported as the fold change of standardized treated over standardized vehicle. STATISTICAL ANALYSIS Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test in SAS, unless otherwise stated. Differences between treatment groups were considered significant when p<0.05. 58 RESULTS TCDD EFFECTS ON BODY WEIGHTS, LIVER HISTOPATHOLOGY, AND CLINICAL CHEMISTRY Consistent with previously reported changes in body weights and liver histopathology +/+ changes in mice exposed to TCDD [11], Scd1 and Scd1 -/- intact female mice gavaged with 30 g/kg TCDD had increased relative liver weight (RLW) with the greatest increases observed at 168 h (Table A.3). No significant alterations in body weight or body weight gain were detected throughout the study. Histopathological changes were marked by cytoplasmic vacuolization in the periportal and midzonal regions that decreased with time (not shown). In Scd1 wild-type mice, hepatic vacuolization was accompanied by cellular inflammation that increased by 168 h, while minimal cellular inflammation was observed at only 168 h in Scd1 null mice. Further analysis of serum ALT levels, a marker of liver damage, identified a 3.5-fold increase in treated wild-type mice at 168 h (Figure A.1), while ALT levels remained unchanged in Scd1 nulls. Increased ALT levels suggest liver damage in treated wild-types only, however longer term exposure may be necessary to differentiate histopathological differences between the two strains. +/+ TCDD EFFECTS ON HEPATIC LIPID CONTENT IN SCD1 +/+ In Scd1 MICE mice, TCDD induced hepatic TAGs and hepatic total fatty acid (TFA) content 3.9- and 1.9-fold, respectively, compared to vehicle controls (Figure 3.1, panel A). Analysis of fatty acid composition by GC-MS identified TCDD-induced alterations in the SFA/MUFA/PUFA molar proportions (Figure 3.1, panel A). Overall, TCDD reduced SFA by 8%, increased MUFA by 9%, and had no effect on PUFA proportions. Further analysis of individual fatty acid species revealed that palmitate (16:0), stearate (18:0), and lignoceric acids 59 (24:0) represented more than 95% of the total SFA content (Table 3.1). Interestingly, palmitate (67%) and stearate (16%) are the major SFA constituents of the rodent chow diet (Harlan Teklad rodent diet 7964). Increases in absolute hepatic levels of MUFAs were primarily due to a 3-fold increase of oleic acid (18:1n9), representing >85% of all MUFAs (Table 3.1). Increases in accumulation of palmitoleic acid (16:1n7, increased 1.9-fold), eicosenoic acid (20:1n9, increased 7.2-fold), erucic acid (22:1n9, increased 2-fold), and nervonic acid (24:1n9, increased 6-fold) accounted for the remaining 15% of MUFAs. , Although TCDD did not alter PUFA levels expressed as mol%, absolute hepatic levels were increased 1.8-fold by TCDD (Table 3.1). In general, TCDD treatment increased all PUFAs (absolute hepatic levels) examined except for timnodonic acid (20:5n3). Linoleic acid (18:2n6) and arachidonic acid (20:4n6, AA) were the dominant PUFAs, representing ~60% and ~25% of PUFAs, respectively. The n-3 and n-6 PUFAs exhibit anti- and pro-inflammatory effects, respectively [34]. The n3/n6 ratio was induced by TCDD and accompanied by the depletion of AA (Figure 3.1, panel A). -/- TCDD EFFECTS ON HEPATIC LIPID CONTENT IN SCD1 MICE Scd1 performs the rate-limiting step in MUFA synthesis and its deficiency has been - reported to impair triglyceride synthesis and protect from diet-induced hepatic steatosis. In Scd1 /- mice, TCDD increased TAGs 2.5-fold and TFA content 1.6-fold compared to vehicle controls. However, TAG levels were 42% lower in Scd1 -/- mice when compared to treated wild-type mice, yet there was no difference in TFA levels between genotypes (Figure 3.1, panel B). 60 Figure 3.1. Hepatic lipid composition at 168 h in vehicle and 30 µg/kg TCDD treated +/+ -/Scd1 (Panel A) and Scd1 (Panel B) mice. Total triglycerides (TAGs) were extracted from mouse liver and quantified using a commercial L-Type Triglyceride M kit (Wako Diagnostics). Data are reported as mg/dL TAG per gram of +/+ -/liver tissue. Absolute total hepatic fat was extracted from Scd1 and Scd1 mouse liver and analyzed by GC-MS and are reported as mol/g tissue. Hepatic fat compositions (saturated fatty acid (SFA)/ monounsaturated fatty acid (MUFA)/ polyunsaturated fatty acid (PUFA) ratios) are reported as mol%. SFA (dark grey)/ MUFA (light grey)/ PUFA (medium grey) proportions are represented. n3/n6 fatty acid ratios are reported as mol %. Arachidonic acid (20:4n6) levels are reported as mol%. Data were analyzed by factorial ANOVA followed by Tukey’s post hoc test. Bars represent mean ± SEM, n=5 biological replicates, (*) represents p<0.05 for TCDD compared -/+/+ to Vehicle within a genotype, (**) represents p<0.05 for Scd1 TCDD compared to Scd1 -/- TCDD, and (***) represents p<0.05 for Scd1 Vehicle compared to Scd1 61 +/+ Vehicle. Figure 3.1 (cont’d) 62 Figure 3.1 (cont’d) 63 Examination of the SFA/MUFA/PUFA ratios in Scd1 -/- mouse liver revealed that TCDD reduced SFA proportions and increased relative MUFA and PUFA levels compared to vehicle controls. Scd1 deficiency also induced SFA by 8%, reduced MUFA by 13%, and induced PUFA by 5% compared to treated wild-type mice, consistent with loss of Scd1 activity (Figure 3.1, panel B). Further analysis of individual fatty acid species identified increases of 18:0, while 16:1n7, 18:1n9, and 20:1n9 were reduced in treated null mouse livers compared to treated wildtype mice (Table 3.1). Surprisingly, MUFA increases in Scd1 null mice were primarily due to a 3.2-fold induction of 18:1n9. Similar to Scd1 TCDD in Scd1 -/- +/+ mice, overall PUFA levels were induced by mice. In contrast, the n3/n6 ratios were not altered and the molar ratio of AA [(nmol/g AA)/(nmol/g total FA detected)] was higher compared to treated wild-type mice (Figure 3.1, panel B). PRINCIPAL COMPONENT ANALYSIS Principal component analysis (PCA) was performed to characterize the trends exhibited by GC/MS-FAMES data. PCA of hepatic TFAs indicated a clear time-, treatment-, and Scd1 dose-dependent effect on hepatic fatty acid composition (Figure 3.2). PC1 and PC2 accounted for 95% of the cumulative proportion of the variance with vehicles clustering along PC1, treated groups separating along PC1, and genotype separating along PC2. All treated animals exhibited a similar temporal trajectory. 64 +/+ -/- Table 3.1. Total Hepatic Lipid Composition (mol/g) in Scd1 and Scd1 Mice 168 h Post 30 g/kg TCDD Dose. +/+ +/+ -/-/Lipid Scd1 Vehicle Scd1 TCDD Scd1 Vehicle Scd1 TCDD 81.4 + 10.4 153.6 + 26.7* 87.6 + 7.3 137.8 + 11.1* Total fatty acids Saturated fatty 40.6 + 6.5 64.6 + 11.1* 52.3 + 5.3 68.8 + 7.1* acid Palmitic acid 19.9 + 2.6 29.2 + 3.7* 19.8 + 2.6 27.8 + 2.8* (16:0) Stearic acid (18:0) 17.0 + 3.1 20.2 + 2.2*, ** 19.6 + 2.1 27.1 + 3.2* Arachidic acid 0.064 + 0.006 0.066 + 0.010 0.117 + 0.076 0.093 + 0.011 (20:0) Behenic acid 0.269 + 0.044*** 0.175 + 0.017 0.425 + 0.140 0.292 + 0.056 (22:0) Lignoceric acid 3.33 + 1.01*** 14.9 + 7.3* 12.4 + 1.9 13.4 + 1.9 (24:0) Monounsaturated 12.7 + 1.5*** 36.8 + 7.8*, ** 5.1 + 0.5 15.8 + 0.01* fatty acid Palmitoleic acid 1.24 + 0.26*** 2.39 + 0.64*, ** 0.20 + 0.03 0.46 + 0.08 (16:1n7) Oleic acid 11.2 + 1.4*** 32.9 + 6.9*, ** 4.68 + 0.44 14.7 + 1.8* (18:1n9) Eicosenoic acid 0.10 + 0.015*** 0.72 + 0.22*, ** 0.040 + 0.008 0.27 + 0.056* (20:1n9) Erucic acid 0.028 + 0.005 0.056 + 0.009* 0.031 + 0.005 0.064 + 0.009* (22:1n9) Nervonic acid 0.135 + 0.043 0.802 + 0.353*, ** 0.189 + 0.047 0.277 + 0.040 (24:1n9) Polyunsaturated 28.1 + 3.4 52.2 + 10.1* 30.1 + 2.4 53.2 + 5.3* fatty acid Total n3 fatty 1.30 + 0.37 4.23 + 1.56* 1.85 + 0.49 3.70 + 0.36* acids Total n6 fatty 26.7 + 3.09 47.6 + 8.5* 28.2 + 2.0 49.2 + 4.9* acids 65 Table 3.1 (cont’d) +/+ +/+ -/-/Lipids Scd1 Vehicle Scd1 TCDD Scd1 Vehicle Scd1 TCDD Linoleic acid 14.8 + 1.83 31.7 + 5.67* 16.7 + 1.65 31.7 + 3.53* (18:2n6) Eicosadienoic acid 0.31 + 0.042 1.26 + 0.26* 0.30 + 0.040 1.20 + 0.25* (20:2n6) Dihomo-ϒlinolenic acid 0.95 + 0.10 2.60 + 0.69* 0.786 + 0.111 2.52 + 0.37* (20:3n6) Arachidonic acid 10.6 + 1.38 12.0 + 2.66 10.4 + 0.88 13.7 + 1.06* (20:4n6) Docosapentaenoic 0.015 + 0.007 0.035 + 0.012* 0.018 + 0.004 0.042 + 0.008* acid (22:2n6) Eicosatetraenoic 0.033 + 0.005 0.162 + 0.057* 0.032 + 0.006 0.153 + 0.046* acid (20:4n3) α-Linolenic acid 0.458 + 0.101 1.36 + 0.30* 0.045 + 0.088 1.09 + 0.25* (18:3n3) Timnodonic acid 0.614 + 0.240 0.593 + 0.231 0.629 + 0.234 0.694 + 0.146 (20:5n3) Docosapentaenoic 0.193 + 0.078 2.12 + 1.04* 0.743 + 0.232 1.76 + 0.24* acid (22:5n3) Data were analyzed by factorial ANOVA followed by Tukey's post hoc test, n=5 biological replicates. (a) p<0.05 for TCDD vs. Vehicle within the same genotype; (b) p<0.05 for +/+ TCDD vs. -/- TCDD; (c) p<0.05 for +/+ Vehicle vs. -/- Vehicle 66 TCDD EFFECTS ON THE SCD1 DESATURATION INDEX Scd1 is the primary hepatic ∆9 desaturase, metabolizing 16:0 and 18:0 into 16:1n7 and 18:1n9, respectively. TCDD induced the hepatic 18:1n9/18:0 ratio in Scd1 +/+ mice compared to vehicles (Figure 3.3A). In humans, positive correlations between the Scd1 desaturation index and triglyceride levels have been reported [35]. Similarly, TAG levels increased with the 18:1n9/18:0 ratio in TCDD treated Scd1 +/+ mice (Pearson’s r = 0.825, p = 0.0003) (Figure 3.3B). TCDD EFFECTS ON HEPATIC LIPID METABOLISM GENE EXPRESSION +/+ To further examine TCDD’s effects on hepatic lipid composition in Scd1 and Scd1 -/- mice, QRTPCR was used to quantify mRNA levels for genes involved in lipid metabolism. Consistent with TCDD-induced increases in hepatic lipid levels, the fatty acid transport genes Cd36, Ldlr, Vldlr and Fabp4, were induced 3-5-fold in both genotypes (Figure 3.4A-D). Furthermore, Cd36, Vldlr and Fabp4 were significantly increased in treated Scd1 compared to Scd1 +/+ -/- mice mice and may explain increases in 18:1n9 levels, the primary MUFA (81.3% of all MUFAs) in Harlan Teklad rodent diet 7964, in null mice. Four isoforms, Scd1-4, exist in the mouse and may underlie increases in 18:1n9. However, Scd3 is reported to be expressed by sebocytes in skin, the preputual gland, and the Harderian gland [36] while Scd4 expression is limited to the heart [37]. Neither Scd3 or 4 were detected in the liver of either TCDD-treated genotype. Scd2 is primarily expressed in adipose tissue, but also in the neonatal mouse liver. Scd2 was expressed in wild-type mice, but not induced by TCDD (Figure 3.4E). Surprisingly, Scd2 mRNA levels were scarce (100-fold lower) in null mice compared to wild-types. The lack of Scd2 mRNA expression in nulls suggests Scd1 deletion also affected Scd2 expression. 67 Figure 3.2. Principal component analysis (PCA) of GC-MS lipid profiles from TCDD (T) or +/+ +/-/Vehicle (V) treated Scd1 , Scd1 , and Scd1 mice 24, 72 and 168 h post-dose. PCA was performed in R as described in the Materials and Methods. PC1 and PC2 accounted for 95% of the cumulative proportion of the variance with vehicles clustering along PC1, treated -/groups separating along PC1 and genotype separating along PC2. Dashed lines (Scd1 ) dotted lines (Scd1 +/- ) solid lines (Scd1 +/+ ), n=5 biological replicates. 68 Figure 3.3. Scd1 activity (desaturation) index and Correlation with Hepatic Triglyceride Levels. +/+ -/(A) Scd1 desaturation index in female TCDD (T) or vehicle (V) treated Scd1 and Scd1 mice. The desaturation index is the ratio of palmitoleic (16:1n7) or oleic acid (18:1n9) to the precursors palmitic (16:0) or stearic acids (18:0), respectively. Bars represent mean ± SEM, n=5 biological replicates, * represents p<0.05 for TCDD compared to vehicle within a genotype, ** represents p<0.05 -/+/+ -/+/+ for Scd1 TCDD compared to Scd1 TCDD, and *** represents p<0.05 for Scd1 vehicle compared to Scd1 vehicle. Data were analyzed by factorial ANOVA followed by Tukey’s post hoc test. (B) Correlation analysis was performed in Graphpad Prism +/+ 5.0a between the hepatic triglyceride (TAG) levels and the 18:1n9/18:0 desaturation index in Scd1 mice gavaged with 30g/kg with TCDD or vehicle for 168 h. 69 Figure 3.4. QRTPCR of hepatic lipid transport, modification, and biosynthesis genes in Scd1+/+ (+/+) and Scd1-/- (-/-) mice gavaged with 30 g/kg TCDD or sesame oil vehicle for 24 h. The gene expression ratio is the total quantity normalized to the geometric mean of Hprt, Actb, and Gapdh. Genes are indicated by official gene symbols. Error bars represent the standard error mean (SEM), n=5, * represents p<0.05 for TCDD compared to vehicle within a genotype, ** +/+ -/represents p<0.05 for Scd1 TCDD compared to Scd1 TCDD and *** represents p<0.05 for +/+ -/- Scd1 vehicle compared to Scd1 Tukey’s post hoc test. vehicle. Data were analyzed by factorial ANOVA followed by 70 Figure 3.4 (cont’d) 71 TCDD-mediated increases in the PUFAs eicosatetraenoic acid (20:4n3) and docosapentaenoic acid (22:5n3) were consistent with the 2-fold induction of Elovl5 (Figure 3.4F) an elongase that catalyzes the elongatation very long chain PUFAs [38]. The lipogenic gene Acaca, which provides malonyl-CoA for fatty acid biosynthesis, was not altered by TCDD (Figure 3.4G). Fasn, the long-chain fatty acid synthetase was reduced 2.5-3-fold by TCDD in both genotypes (Figure 3.4H), suggesting that increases in hepatic lipid content are not due to de novo lipogenesis. Finally, the well-characterized TCDD-inducible genes Cyp1a1, Nqo1, and Tiparp exhibited comparable induction in wild-type and null mice (Figure A.2). TCDD EFFECTS ON SCD1 ACTIVITY, MRNA AND PROTEIN The effects of TCDD on Scd1, activity, mRNA, and protein levels were examined. Scd1 enzyme activity was measured by following the conversion of 14 C18:0 to 14 C18:1n9. Consistent with increases in the 18:1n9/18:0 desaturation index, TCDD induced Scd1 activity 2-fold in wild-type mice compared to vehicles at 24 h (Figure 3.5A). QRTPCR also showed that TCDD induced Scd1 mRNA levels 1.5-fold in Scd1 +/+ mice (Figure 3.5B). Furthermore, western blots showed an increase in Scd1 immunoreactivity in treated Scd1 +/+ microsomal fractions (Figure 3.5C and 3.5D). Induction in Scd1 activity, mRNA, and protein were not detected in control or -/- TCDD-treated Scd1 mice. PUTATIVE DRE DISTRIBUTION AND AHR ENRICHMENT AT SCD1 LOCI To further examine AhR regulation of Scd1, Figure 3.6A summarizes the ChIP-chip analysis of AhR enrichment at Scd1-4 genomic loci induced by TCDD [28]. The moving average (MA) value visualizes the enriched genomic regions within the Scd1-4 loci while the log2 72 +/+ -/- Figure 3.5. Scd1 activity, mRNA, and protein levels in Scd1 and Scd1 mice treated with 30 g/kg TCDD (T) or sesame seed oil (V) 24 h post-dose. (A) Scd1 activity. Hepatic microsomes (100 g, n=5) isolated from mice were incubated with 14 14 0.03 Ci C-stearoyl-CoA (14C 18:0), NADH and stearoyl-CoA. C 18:0 was separated from 14 C 18:1 by silver ion chromatography and radioactivity measured by scintillation counting. 14 Scd1 activity is expressed as nmol 14C 18:0 converted to C 18:1 per mg Scd1 protein per min. (B) QRTPCR of Scd1 mRNA (n=5). Expression is represented as a ratio of the total quantity of Scd1 normalized to the geometric mean of Hprt, Actb, and Gapdh. (C) Scd1 Western blot. Hepatic Scd1 protein (n=3) was detected in 10 g of microsomes. Epoxide hydrolase (Epxh1) was used as a microsomal protein reference for loading control. (D) Densitometry. Densitometry was determined with ImageJ from Scd1 bands and normalized to Ephx1 bands. ‡ for p = 0.08. For A, B, and D bars represent mean ± SEM, * indicates p<0.05 for T compared to V within a genotype. Data were analyzed by factorial ANOVA followed by Tukey’s post hoc test. 73 enrichment illustrates the fold change for each Affymetrix probe. Six AhR enriched regions (up to 5.7-fold, FDR < 0.01, red bar) ranging from 70 to 4200 bp were associated with Scd1 (Figure 3.6A). AhR enrichment was located within 10 kb upstream region through the 3’-UTR. In contrast, the adjacent ~150 kb genomic region spanning Scd2, Scd3, and Scd4 exhibited only two, AhR-enriched regions (2- to 2.7-fold). AhR is proposed to regulate gene expression through DNA binding at DREs containing the core sequence 5’-GCGTG-3’. DREs with matrix similarity scores (MSS) ranging from 0.69 to 0.93 were identified within the genomic region spanning Scd genomic loci [29]. Of the 39 DREs possessing a putative functional (high scoring) MSS (> 0.8; track 4, horizontal line), 16 were located 10 kb upstream of the Scd1 TSS through the Scd1 3’-UTR (Figure 3.6B). Five of these 16 high scoring Scd1 DREs overlapped with regions of AhR enrichment (track 4, yellow shading). Notably, 7 DREs that did not overlap with regions of significant AhR enrichment coincided with regions lacking tiling probes (track 4, purple shading). One DRE core fell within a 2-fold AhR enriched region that failed to meet the FDR cut-off of 0.01 (track 4, green shading). Moreover, three AhR enriched regions lacked any DRE cores (track 5, orange shading) consistent with promoter- and genome-wide ChIP-chip studies reporting 50% overlap between DRE cores and AhR enrichment as well as other studies suggesting AhR interaction with DNA independent of ARNT [28, 29, 39-41]. BAND SHIFT ASSAYS Two DRE cores (Figure 3.6B, track 4, indicated by*) within 10 kb upstream of the Scd1 TSS and with a MSS > 0.8 exhibited band shifts with TCDD-activated guinea pig cytosol (Figure 3.6C). The addition of AhR or ARNT antibodies to the incubation resulted in a supershift, 74 Figure 3.6. Dioxin response element (DRE) distribution and TCDD-inducible AhR enrichment within Scd loci, and band shift assays. (A) Genomic region spanning Scd1, Scd2, Scd3, and Scd4. (B) Scd1 genomic region only. Genomic DRE distributions and regions of AhR enrichment induced by TCDD were previously determined [28, 29]. Track 1: scale and chromosome position. Track 2: probe tiling across the Affymetrix 2.0R mouse array. Track 3: gene organization including transcriptional start site (TSS) (closed arrow), exons (closed boxes), introns and direction of transcription (solid arrowhead line). Track 4: location of DRE cores (5’-GCGTG-3’). Height of vertical bars indicate matrix similarity score (MSS) for the 19 bp DRE sequence [29]. The horizontal line indicates the MSS = 0.8. MSSs greater than 0.8 are considered putative functional DREs. The asterisk (*) denotes 19 bp DRE sequences that bound TCDD-activated AhR in band shift assays. Track 5: regions of significant (FDR<0.01) AhR enrichment in genome-wide ChIP-chip assays. Tracks 6 and 7: histograms depicting the signal intensities for the moving average (MA; blue, track 6) and log2 fold enrichment (green, track 7) values for regions exhibiting AhR enrichment in genome-wide ChIP-chip assays. The above tracks were modified from the UCSC genome browser. (B) Scd1 genomic region only. Track 4: yellow shading, putative DRE-AhR enrichment overlap; purple shading, putative DREs lacking affymetrix tiling probes; grey shading, putative DREs that do not overlap with AhR enrichment; green shading, putative DRE in an AhR region that failed to meet the FDR cut-off of 0.01. Track 5: orange shading, AhR enriched regions that do not overlap with putative DREs. (C) Representative band shift assays with putative, functional DREs (track 4, asterisks (*)). The DRE position is numerically indicated relative to the Scd1 TSS. Solid arrows indicate a TCDDinducible band shift. Hatched or dashed arrows indicate an AhR or ARNT supershift, respectively. 75 Figure 3.6 (cont’d) 76 Figure 3.6 (cont’d) 77 confirming AhR binding to these putative DREs. Nucleotides flanking the DRE core sequence binding affinity and co-activator recruitment and may underlie differences in the banding pattern between the two DREs [42-45]. These results, in addition to mRNA, protein and activity levels, support AhR recruitment to and regulation of Scd1 by TCDD. DISCUSSION Previous studies examining the role of Scd1 in hepatic diseases have focused on dietary or genetic modulation of lipid metabolism [25, 35, 46-48], but not the effects of environmental contaminants. In this study, AhR mediated induction of Scd1 by TCDD and subsequent effects on hepatic FA composition were examined in Scd1 +/+ and Scd1 -/- mice. Here we report that TCDD induced hepatic TRG and FA accumulation, elicited differential gene expression of lipid metabolism and transport, and increased Scd1 mRNA, protein, and activity, as a result of AhR recruitment to Scd1 genic regions. Collectively, these studies suggest that AhR-regulation of Scd1 alters hepatic fatty acid composition, which influences TCDD-induced hepatotoxicity. TCDD altered hepatic gene expression associated with lipid transport, partitioning and metabolism in mice [11, 14, 49]. For example, TCDD induced the lipolytic genes lipoprotein lipase (Lpl), phospholipase A2, group XIIA (Pla2g12a), monoglyceride lipase (Mgll) and pancreatic lipase-related protein (Pnliprp1) that hydrolyze hepatocellular TRG stores into FFAs and monoglycerides [50]. It also induced low-density lipoprotein receptor (Ldlr), very lowdensity lipoprotein receptor (Vldlr), Cd36 antigen (Cd36), and fatty acid binding protein (Fabp4) +/+ in Scd1 and Scd1 -/- mice. Ldlr and Cd36 are membrane-associated proteins that facilitate the uptake of chylomicron and VLDL remnants as well as long-chain fatty acids [51, 52]. Cd36 has been implicated in the etiology of obesity and diabetes, and Cd36 null mice exhibit minimal 78 TCDD-elicited steatosis [53-56]. Fabps are cytosolic, high-affinity long-chain fatty acid binding proteins that target lipids to intracellular compartments [57]. Fabp4, which mediates lipid trafficking to the nucleus and may provide ligands for PPARs [58, 59], was also induced. Furthermore, inhibition of VLDL secretion has been reported in mice following TCDD treatment [56]. These changes likely underlie effects that contribute to TCDD-elicited hepatic fatty acid accumulation. ChIP-chip analysis identified six Scd1 genic regions with AhR enrichment. Two proximal AhR enriched regions were within 2 kb of the TSS [28]. The 3’ regions also exhibited AhR enrichment and high-scoring DREs, suggesting AhR regulation by distal enhancer sites. Although distal regulation remains largely uninvestigated, studies suggest transcription factor binding at these sites promotes chromatin looping and structural modifications that facilitate gene expression [60-62]. Two 5’ AhR enriched regions at distal sites lacked DRE cores, providing further evidence of DRE-independent AhR-DNA interactions, which may involve tethering to other DNA interacting transcription factors [63]. However, the arrays do not have uniform tiling across the genome and several genomic regions lack probe coverage in areas containing high-scoring DREs that may confound mapping AhR enrichment to regions containing DREs. AhR enrichment at Scd1 genic regions and the induction of Scd1 mRNA, protein, and activity by TCDD provided compelling evidence for AhR regulation of Scd1. The induction of Scd1 activity increased MUFA levels, decreased SFA levels and increased MUFA:SFA and PUFA:SFA ratios. Scd1 deficiency did not affect TCDD induced hepatic lipid accumulation (as measured by GC-MS analysis of FAMES). However, null mice had fewer TAGs, reduced MUFA levels and exhibited less hepatic injury relative to treated wild-type mice. More 79 -/- specifically, Scd1 mice exhibited no increase in serum ALT, less hepatic vacuolization and reduced immune cell infiltration compared to Scd1 +/+ mice suggesting lower overall TCDD elicited hepatotoxicity. In addition to Scd1, hepatic FA profiles indicate that AhR regulates other lipid modifying enzymes in addition to Scd1. Increases in n-6 and n-3 pathway intermediates (e.g. 20:2n6, 20:3n6, 20:4n3, 22:5n3) suggests the induction of Elovl5 activity [64]. QRTPCR, band shift and ChIP-chip assays (Appendix A Figure 3A-C) verified AhR mediated induction of Elovl5 by TCDD. The n-3 and n-6 PUFAs, particularly eicosapentanoic acid (EPA, 20:5n3) and arachidonic acid (AA, 20:4n6) metabolites, exhibit anti- and pro-inflammatory activities, respectively [34, 65-67]. EPA is an Elovl5 substrate, therefore excess 20:5n3 may be elongated into 22:5n3, which is increased in both genotypes. AA is not an Elovl5 substrate, but is rapidly metabolized by TCDD-inducible prostaglandin-endoperoxide synthase 1 (Ptgs1), arachidonate 12-lipoxygenase (Alox12) [14, 28], and glutathione transferases [68] into pro-inflammatory ecosanoids. Additionally, AA is liberated from membrane phospholipids by Pla2g12a, another +/+ TCDD-inducible gene. AA levels were lower in treated Scd1 mice compared to vehicles and treated nulls, and, although we cannot rule-out their conversion into inflammatory ecosanoids, are consistent with the increased level of inflammation in wild-type mice compared to nulls. +/+ Inflammation in Scd1 mice may also be due to the induction of Scd1, which would sequester cytochrome b5, uncouple P450 monoxygenases, and increase superoxide and ROS formation [69, 70]. However, P450 monoxygenase and xanthine dehydrogenase induction by TCDD are the primary ROS contributors [71, 72]. 80 Our results differ from studies examining the protective effects of Scd1 deficiency from steatosis and exacerbated steatohepatitis elicited using in vivo dietary-induced models of liver -/- injury. For example, high fat diets (HFD) induce steatosis in mice. Scd1 mice fed HFD exhibit no evidence of hepatomegaly or histological changes [73], yet hepatomegaly is observed in all animals exposed to TCDD (Poland and Knutson, 1982). Methionine choline deficient (MCD) diets induce steatohepatitis, but in contrast to TCDD, decrease Scd1 expression and induce Cyp4A expression, an enzyme involved in lipid peroxidation (Li et al., 2009, Yamaguchi et al., 2007, and Anstee & Goldin, 2006). Cyp4a is a peroxisome proliferator activated receptor (PPAR) target and administration of a PPAR agonist to MCD fed mice decreases liver damage (Nagasawa et al., 2006), suggesting a role for PPAR rather than AhR in MCD-elicited liver injury. Furthermore, our results are consistent with increased MUFA:SFA ratios in lipotoxic mechanisms of liver injury (Larter et al. 2008). TCDD-induced steatosis is a significant hepatotoxic effect and AhR-mediated induction of Scd1 exacerbates TCDD hepatotoxicity by altering the composition of accumulated lipids. Scd1-mediated increases in unsaturated fatty acid levels may alter membrane fluidity, increase lipid peroxidation and reactive oxygen species formation, as well as propagate inflammatory responses through TRAIL-mediated cytotoxicity [74, 75]. Steatosis followed by a progressive inflammatory response poses a significant risk of progression to cirrhosis and may contribute to hepatocellular carcinoma development in rodents. The induction of hepatic lipid accumulation in mice is consistent with the occurrence of dyslipidemia in humans following TCDD exposure at high doses [76]. Interestingly, the hepatic fatty acid composition induced by TCDD is similar to serum and lipid profiles (e.g. increase in TAGs, and 16:1n7 and 18:1n9 levels) described for human NAFLD patients [77, 78]. NAFLD is 81 considered the hepatic manifestation of metabolic syndrome, and precedes non-alcoholic steatosis, and cirrhosis. This report suggests that AhR regulation of lipid transport, metabolism, and modifying enzymes, including Scd1, alters lipid composition that contributes to the hepatotoxicity of TCDD. FOOTNOTES 1 Guinea pig cytosol was a gift from Dr. Michael Denison, Department of Environmental Toxicology, Meyer Hall, University of California, Davis, CA 95616, USA. FUNDING This work was supported by the National Institute of Environmental Health Sciences Superfund Basic Research Program (P42ES04911). 82 APPENDIX A 83 APPENDIX A Table A.1. Putative DRE Sequences Gene MSS Position Sequence Scd1 0.812732 -2849 TCTCTCTGCGTGCCTTTAT AGAGAGACGCACGGAAATA 0.803264 -1116 TGGCTAAGCGTGACCACAG ACCGATTCGCACTGGTGTC Matrix Similarity Score (MSS), Position is relative to the transcriptional start site. 84 Table A.2. Terminal Body Weight, Body Weight Gain, Absolute Liver Weight and Relative Liver Weight for Scd1 +/+ and -/- Scd1 Mice Time Genotype Treatment (h) Body Weight (g) Body Weight Gain (g) Liver Weight (g) Relative Liver Weight 24 -/- +/+ 72 -/- +/+ 168 -/- Vehicle 14.7 ± 1.0 1.4 ± 0.9 0.844 ± 0.064 0.057 ± 0.004 TCDD 14.2 ± 0.5 0.8 ± 0.5 0.914 ± 0.079 0.064 ± 0.004* Vehicle 13.1 ± 1.3 1.2 ± 0.4 0.771 ± 0.116 0.059 ± 0.005 TCDD 13.2 ± 1.3 0.6 ± 0.5 0.905 ± 0.123 0.068 ± 0.003* Vehicle 13.2 ± 2.5 1.4 ± 0.9 0.728 ± 0.204 0.054 ± 0.008 TCDD 13.0 ± 1.2 1.9 ± 0.7 0.818 ± 0.116 0.063 ± 0.006 Vehicle 12.6 ± 1.5 2.2 ± 0.5 0.756 ± 0.095 0.060 ± 0.003 TCDD 12.8 ± 1.6 1.9 ± 0.5 0.891 ± 0.102 0.070 ± 0.010* Vehicle +/+ 15.5 ± 1.2 2.6 ± 1.8 0.778 ± 0.135 0.050 ± 0.006 TCDD 15.0 ± 1.3 2.0 ± 0.8 0.959 ± 0.114* 0.064 ± 0.003*, ** Vehicle 15.8 ± 1.8 3.5 ± 0.7 0.926 ± 0.097 0.059 ± 0.004 15.3 ± 1.5 3.3 ± 0.7 1.080 ± 0.111 0.071 ± 0.005* TCDD Data were analyzed by factorial ANOVA followed by Tukey's post hoc test, n=8 biological replicates. * p<0.05 for TCDD vs. Vehicle within the same genotype, ** p<0.05 for +/+ TCDD vs. -/- TCDD 85 Table A.3. TCDD Effects on Liver Histopathology in Scd1 Wild-Type and Null Mice Time (hr) 24 Genotype 72 168 Hypertrophy +/+ -/- +/+ -/- V 0.5 0.2 0.3 0.3 0.1 0.0 3.0 2.3 2.3 1.5 0.9 1.6 V 0.3 0.0 0.0 0.0 0.4 0.0 T 0.4 0.0 0.4 0.0 1.5 0.4 V Inflammation -/- T Vacuolization +/+ 0.0 0.0 0.0 0.0 0.0 0.0 T 1.4 0.0 0.8 0.0 1.3 1.0 The severity of vacuolization, inflammation and hypertrophy are scored as 1-minimal, 2-mild, 3moderate, 4-marked. N=8, TCDD (T), Vehicle (V). 86 Figure A.1. Serum alanine aminotransferase levels in Scd1+/+ and Scd1-/- mice treated 168 h with either vehicle or 30 g/kg TCDD. Serum alanine amino transferase (ALT) levels in Scd1+/+ and Scd1-/- mice treated with 30 g/kg TCDD or sesame seed oil vehicle 24 h post-dose. Error bars represent the standard error mean (SEM), n=3, (*) represents p<0.05 for TCDD compared to vehicle within a genotype. Data were analyzed by Dunnett’s t-test. 87 Figure A.2. QRTPCR of the TCDD-inducible genes Cyp1a1, Nqo1, and Tiparp in Scd1 -/- +/+ (+/+) and Scd1 (-/-) mice gavaged with 30 g/kg TCDD or sesame oil vehicle for 24 h. The gene expression ratio is the total quantity normalized to the geometric mean of Hprt, Actb, and Gapdh. Genes are indicated by official gene symbols. Error bars represent the standard error mean (SEM), n=5, * represents p<0.05 for TCDD compared to vehicle within a genotype and ** +/+ -/represents p<0.05 for Scd1 TCDD compared to Scd1 TCDD. Data were analyzed by factorial ANOVA followed by Tukey’s post hoc test. 88 Figure A.3. QRTPCR of Elovl5 mRNA (A), Elovl5 band shift assays with putative, functional DREs (B), and DRE distribution and TCDD-inducible AhR enrichment within the Elovl5 locus (C). (A) Elovl5 mRNA expression is represented as a ratio of the total quantity of Elovl5 normalized to the geometric mean of Hprt, Actb, and Gapdh. Bars represent mean ± SEM, * indicates p<0.05 -/for T compared to V within a genotype and ** represents p<0.05 for Scd1 T compared to +/+ Scd1 T within a time point. Data were analyzed by factorial ANOVA followed by Tukey’s post hoc test, n=5. (B) The DRE position is numerically indicated relative to the Elovl5 TSS. Solid arrows indicate a TCDD-inducible band shift. Hatched or dashed arrows indicate an AhR or ARNT supershift, respectively. (C) DRE distributions and regions of AhR enrichment induced by TCDD were previously determined [28, 29]. Track 1: scale and chromosome position. Track 2: probe tiling across the Affymetrix 2.0R mouse array. Track 3: gene organization including transcriptional start site (TSS) (closed arrow), exons (closed boxes), introns and direction of transcription (solid arrowhead line). Track 4: location of DRE cores (5’-GCGTG-3’). Height of vertical bars indicate matrix similarity score (MSS) for the 19 bp DRE sequence [29]. The horizontal line indicates the MSS = 0.8. MSSs greater than 0.8 are considered putative functional DREs. The asterisk (*) denotes 19 bp DRE sequences that bound TCDD-activated AhR in band shift assays. Track 5: regions of significant (FDR<0.01) AhR enrichment in genome-wide ChIPchip assays. Tracks 6 and 7: histograms depicting the signal intensities for the moving average (MA; blue, track 6) and log2 fold enrichment (green, track 7) values for regions exhibiting AhR enrichment in genome-wide ChIP-chip assays. The above tracks were modified from the UCSC genome browser. 89 Figure A.3 (cont’d) 90 Figure A.3 (cont’d) 91 REFERENCES 92 REFERENCES 1. Croutch CR, Lebofsky M, Schramm KW, Terranova PF, Rozman KK: 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) alter body weight by decreasing insulin-like growth factor I (IGF-I) signaling. Toxicol Sci 2005, 85:560-571. 2. 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(accepted, Toxicol Sci). 100 CHAPTER 4 DIETARY FAT IS A LIPID SOURCE IN 2,3,7,8-TETRACHLORODIBENZO- -DIOXIN (TCDD)-ELICITED, HEPATIC STEATOSIS IN C57BL/6 MICE ABSTRACT TCDD increases FA transport and FA levels, resulting in hepatic steatosis in mice. Diet as a source of lipids was investigated using customized diets, Scd1 null mice, and 14 C-oleate (18:1n9) uptake. C57BL/6 mice fed 5%, 10%, or 15% fat or 50%, 60%, or 70% carbohydrate diets exhibited increased relative liver weight following gavage with 30 µg/kg TCDD for 168 h. Hepatic lipid extract analysis from mice fed 5%, 10% and 15% fat diets identified a dosedependent increase in total FAs induced by TCDD. Fat-diet fed mice also exhibited a dosedependent increase in the dietary essential linoleic (18:2n6) and α-linolenic (18:3n3) acids. No dose-dependent FA increase was detected on carbohydrate diets, suggesting dietary fat as a source of lipids in TCDD-induced steatosis as opposed to de novo lipogenesis. TCDD also induced oleate levels 3-fold in Scd1 null mice that are incapable of desaturating stearate (18:0). This finding is consistent with oleate representing >90% of all MUFAs in rodent chow. Moreover, TCDD increased hepatic 14 C-oleate levels 2-fold in wild-type and 2.4-fold in Scd1 null mice concurrent with the induction of intestinal and hepatic lipid transport genes (Slc27a, Fabp, Ldlr, Cd36, and Apob). In addition, computational scanning identified putative DREs and in vivo ChIP-chip analysis revealed regions of AhR enrichment proximal to lipid transport genes 101 differentially regulated by TCDD. Collectively, these results suggest the AhR mediates increased uptake of dietary fats that contribute to TCDD-elicited hepatic steatosis. INTRODUCTION Activation of the AhR, a bHLH PAS transcription factor, elicits a broad spectrum of species-specific effects [1]. Epidemiological and rodent studies have linked exposure to TCDD and related compounds to dyslipidemia and disrupted energy balance [2, 3]. Briefly, ligands bind to the cytosolic AhR causing a conformational change, dissociation of chaperone proteins, and translocation to the nucleus where it heterodimerizes with ARNT [4, 5]. The complex binds DREs to modulate gene transcription, although DRE-independent binding to DNA has also been reported [6, 7]. The role of the AhR in hepatotoxicity and lipid metabolism has not been fully elucidated. TCDD induced hepatic steatosis is characterized by increases in TFAs, TAGs, vacuolization, inflammatory cell infiltration, and serum ALT levels [8, 9] that are absent in AhR, null mice The AhR also mediates mobilization of peripheral fat [10-12], inhibition of FA oxidation [10, 13], repression of VLDL secretion [13], and hepatic lipid and composition alterations [14]. In this report, we examine diet as a lipid source in TCDD-elicited hepatic steatosis. Dosedependent increases in hepatic fat accumulation, including essential dietary FAs, suggest dietary fat rather than carbohydrate is an important lipid source in TCDD-elicited steatosis. Increases in hepatic MUFA levels in Scd1 null mice, and hepatic 14 C levels, provide further evidence that AhR activation increases dietary fat processing that contributes to TCDD-elicited hepatic steatosis. Complementary gene expression analysis integrated with computational DRE search [15] and ChIP-chip [6] data indicate the AhR mediates intestinal and hepatic responses that enhanced dietary fat processing and transport, resulting in hepatic steatosis. Collectively, these 102 results suggest continuous AhR activation may contribute to diseases associated with hepatic steatosis. MATERIALS AND METHODS ANIMAL HANDLING C57BL/6 ovariectomized (ovx) female mice were obtained from Charles Rivers Laboratories (Portage, MI) on postnatal day (PND) 25 with body weights within 10% of the mean body weight upon arrival. B6.129-Scd1 tm1Myz /J heterozygous mice (Jackson Laboratory, Ben Harbor, ME) had free access to chow or custom diets and water upon arrival and throughout the study. On PND 21 mice were genotyped and weaned. Mice were maintained on a 12 h light/dark cycle, housed in standard cages containing aspen woodchips. B6.129-Scd1 tm1Myz /J heterozygous mice were fed Harlan Teklad 7964 F6 Rodent diet (chow). All procedures were carried out with All-University Committee on Animal Use and Care approval. DIET COMPOSITIONS Custom diets consisted (by weight) of 19.6% protein, 5%, 10%, and 15% fat with decreasing carbohydrate (68%, 56%, and 44%) for an isocaloric intake of 3.7 kcal/g (Table 4.1). Carbohydrate adjusted diets consisted (by weight) of constant fat and protein (6.6% and 19.6%, respectively) with increasing total carbohydrate content of 50%, 60% and 70% for total caloric intakes of 3.3, 3.7, and 4.1 kcal/g, respectively (Table 4.1). Custom diets use different ingredients compared to standard chow, which confounds comparisons to other studies. Specifically, custom 103 Table 4.1. Nutrient and Energy Composition of Custom Diets Constant Fat, Carbohydrate Chow Isocaloric, Fat Adjusted Diets Adjusted Diets % Fat % Carbohydrate 5% 10% 15% 50% 60% 70% 3.1 3.7 3.7 3.7 3.3 3.7 4.1 Fat 6.4 5 10 15 6.6 6.6 6.6 Protein 24.3 18.3 18.3 18.3 18.3 18.3 18.3 Carbohydrate 38.7 63.6 52.4 41.1 48.6 59 68.6 Fiber 11.3 3 10.5 18 18 7 --- Metabolizable Energy (kcal/g) % by Weight 104 diet formulations use purified ingredients. In contrast, chow (31% protein, 19% fat, and 50% carbohydrate calories by weight) consists of a proprietary blend of soybean meal, ground corn, wheat, fishmeal, soybean oil, whey, brewers yeast, and other vitamins and minerals. DIET STUDY IN VIVO TREATMENT On PND 28, mice fed custom diets (n=5) were gavaged with 0.1 ml of sesame oil (vehicle control) or 30 g TCDD (Dow Chemical Company, Midland, MI) per kg body weight. Immature ovx mice were used to facilitate comparisons with other data sets, as well as minimize potential interactions with estrogens from maturing ovaries. The dose was chosen to elicit moderate hepatic effects while avoiding overt toxicity. Animals were sacrificed at 24 and 168 h post dose, weighed, and blood was collected via submandibular vein puncture before sacrifice. Tissue samples were removed, weighed, flash frozen in liquid nitrogen, and stored at -80C. GC-MS FATTY ACID METHYL ESTER (FAMES) HEPATIC LIPID PROFILING Hepatic lipid analysis was performed as previously described [14]. Briefly, liver lipids were extracted by Folch method, dried down under nitrogen, converted to methyl esters and resuspended in hexane. Samples were separated and analyzed by GC-MS. 19:1n9 FFA and 19:0 TAG were added as extraction efficiency controls and 17:1n1 FAME (Nu-chek, Elysian, MN) was spiked in as a loading control. Data were analyzed with QuanLynx software and reported as mol/g liver tissue. FA levels are based on peak areas from total ion chromatograms and mol/g is obtained from a linear calculation of a calibration curve normalized to sample weight. 14 C-OLEATE STUDIES On PND 28, Scd1 wild-type and null mice (n=5) were gavaged with sesame oil or 30 g/kg TCDD. 4 h before sacrifice at 120 h, mice were gavaged with 2 Ci 14 C-oleate (0.1 mL of 20 Ci/mL in sesame oil; ARC 0297; American Radiolabeled Chemicals, St. Louis, MO). 105 Blood was collected from the saphenous vein at 0.5, 1 and 2 h after 14 C-oleate gavage or the submandibular vein at 4 h. Tissues were harvested, weighed, flash frozen in liquid nitrogen, and stored at -80C. Duodenum (~3.5 cm) and jejunum (~6 cm) sections were collected, flushed with phosphate buffered saline, and cut longitudinally. Intestinal epithelium were scraped into vials containing ~1.0 ml of TRIzol (Invitrogen, Carlsbad, CA), snap-frozen in liquid nitrogen, and stored at -80°C. For fecal pellet analysis, mice were gavaged with 2 Ci TCDD dose and all fecal matter was collected until 48 h post 14 14 C-oleate 120 h post C-oleate gavage. Liver, parametrial adipose, and muscle samples were homogenized in Folch solution (2:1 chloroform:methanol), 0.2 mL 40% methanol was added, vortexed, and centrifuged at 10,000 x g for 5 min. The organic phase was dried under nitrogen and resuspended in hexane. Samples were directly added to 10 mL liquid scintillation fluid (Safety-Solve, RPI, Mount Prospect, IL) and 14 C levels counted on a Packard Tri Carb Liquid Scintillation Counter (PerkinElmer; Waltham, MA). Each sample was spiked with 17:1n1 FAME (Nu-chek; Elysian, MN) to control for extraction efficiency and quantified by GC-MS. Liver and adipose samples were normalized to sample weight x whole organ weight. Muscle samples were normalized to sample weight. For 14 C levels in fecal pellets, ~30 g dried pellets were ground to a fine powder with mortar and pestle and added to 10 mL liquid scintillation fluid. Fecal samples were normalized to total dry fecal pellet weight. For 14 C levels in serum, 5 L of serum was added directly to scintillation cocktail. Samples were normalized to the average body weight of each mouse. 106 QUANTITATIVE REAL TIME PCR (QRTPCR) RNA was isolated from frozen liver samples and intestinal scrapings and QRTPCR expression was performed as previously described [8]. The copy number of each sample was standardized to the geometric mean of Gapdh, Hprt, and Actb to control for differences in RNA loading, quality, and cDNA synthesis [16]. Data are reported as the fold change of standardized treated over standardized vehicle values. STATISTICAL ANALYSIS Data were analyzed by ANOVA followed by Tukey’s post hoc test in SAS V9.2 (SAS Institute, Cary, NC). Differences between treatment groups were considered significant when p<0.05. RESULTS BODY AND LIVER WEIGHTS Mice fed fat adjusted diets had increased RLWs (Appendix B, Table 1) at 24 and 168 h following a single oral gavage of 30 g/kg TCDD. In contrast, mice fed carbohydrate adjusted diets exhibited increased RLW at 168 h only. There were no significant alterations in body weight or body weight gain throughout the study, suggesting treatment had no effect on feed consumption. HEPATIC LIPID CONTENT IN MICE FED AN ISOCALORIC, FAT ADJUSTED DIET Vehicle treated mice exhibited a dose-dependent decrease in TFAs primarily due to decreases in MUFAs (Table 4.2A). However, TCDD increased TFAs 1.4-, 1.7-, and 2.0-fold in mice fed 5%, 10%, and 15% fat diets, respectively (Figure 4.1A), compared to diet-matched vehicles. More specifically, absolute levels 107 of SFA MUFA, and PUFA Table 4.2. Hepatic Lipid Levels in Mice Fed Fat or Carbohydrate Adjusted Diets 168 h Post 30 g/kg TCDD Dose A. Adjusted Fat Diet % Fat Treatment Total FA Vehicle TCDD Total SFA Vehicle TCDD Total MUFA Vehicle TCDD Total PUFA Vehicle TCDD 5% 135.6  16.7 195.0  14.0* 40.9  3.3 Total FA Vehicle TCDD Total SFA Vehicle TCDD Total MUFA Vehicle TCDD Total PUFA Vehicle TCDD 113.5  9.5 194.3  16.4* 41.9  3.8 53.9  3.3*, ** 54.1  12.1 92.6  12.0*, ** 53.8  1.4* 31.0  4.8†† 66.0  11.0* 33.2  2.7 47.7  2.8*, **, † 40.6  2.6 74.4  4.4*, ** B. Adjusted Carbohydrate Diet Treatment 10% 15% 96.3  188.0  36.6  47.7  18.2  54.8  41.5  85.5  7.1†† 15.6* 2.0 3.8* 2.8†† 7.3† 2.9 7.2* % Carbohydrate 50% 60% 70% 108.8  24.6 156.8  7.6* 113.0  4.7 148.6  22.3** 135.9  193.0  45.1  7.74 54.3  2.7 46.7  3.2 51.0  5.4** 49.7  64.5  28.4  11.6 50.7  5.5** 31.0  2.6 51.3  12.8 51.2  79.8  35.4  5.6 51.8  2.5* 35.3  3.3 46.3  4.9* 35.0  48.7  9.8 34.8* 4.9 8.7* 6.1 26.0* 2.4 5.0* A. Adjusted Fat Diet : *p<0.05 for TCDD compared with Vehicle within a diet, **p<0.05 for TCDD 15% fat compared with TCDD 5% or 10%, † p<0.05 for TCDD 5% fat compared with TCDD 10%, ††p<0.05 for Vehicle 5% compared with Vehicle 10% or 15%, 108 Table 4.2 (cont’d) n=5. B. Adjusted Carbohydrate Diet: * p<0.05 for TCDD compared with Vehicle within a diet,** p<0.05 for TCDD 70% carbohydrate compared with TCDD 50% or 60% carbohydrate, n=5. 109 Figure 4.1. Hepatic absolute and essential fatty levels in mice fed increasing fat or carbohydrate diets treated with sesame oil vehicle (V) or 30 g/kg TCDD (T) for 168 h. (A-C) 5%, 10%, and 15% fat diet fed mice, (A) total fatty acids (TFA), (B) -linoleic acid (18:2n6), and (C) -linolenic acid (18:3n3) levels. * p<0.05 for TCDD compared with Vehicle within a diet, ** p<0.05 for TCDD 15% fat or 10% fat compared with TCDD 5% fat. (D-F) 50%, 60%, and 70% carbohydrate diet fed mice, (D) TFAs, (E) 18:2n6, (F) 18:3n3. * p<0.05 for TCDD compared with Vehicle within a diet; ** p<0.05 for TCDD 70% carbohydrate compared with TCDD 50% carbohydrate. (A-F) Bars represent mean  standard error of the mean (SEM), n=5. 110 Figure 4.1 (cont’d) 111 increased with palmitic (16:0) and oleic (18:1n9) acids representing 80-90% of all hepatic SFAs and MUFAs, respectively (Table B.2). Note that palmitic and oleic acids represent >66% and >98% of dietary SFAs and MUFAs, respectively, in the fat adjusted diets (Table B.4A), with absorption efficiencies of >90% [17]. Hepatic PUFAs also exhibited a dietary fat dose-dependent increase (Table 4.2A) primarily due to linoleic acid (18:2n6) accumulation (Figure 4.1B). 18:2n6, which represents ~65% of all hepatic PUFAs, is an essential FA that can only be acquired from the diet with a reported absorption efficiency of >95% [17]. -Linolenic acid (18:3n3), another dietary essential FA, exhibited similar hepatic increases with increasing dietary fat content that was further induced by TCDD (Figure 4.1C). 18:2n6 and 18:3n3 represent >99% of the PUFA content in isocaloric, fat adjusted diets (Table B.4A). These results suggest that AhR activation enhances dietary fat processing and/or transport that contributes to TCDD elicited hepatic steatosis. HEPATIC LIPID CONTENT IN MICE FED A CONSTANT FAT, CARBOHYDRATE ADJUSTED DIET Mice fed 50%, 60%, or 70% carbohydrate diets did not exhibit a dose-dependent increase in hepatic TFA following TCDD treatment. TCDD induced TFAs ~1.4-fold across all carbohydrate adjusted diets (Figure 4.1D). Absolute SFA and MUFA levels increased 1.3- and 1.6-fold, respectively, in mice fed the 70% carbohydrate diet compared to controls (Table 4.2B). 16:0, 18:1n9, and 18:2n6 were the predominant hepatic SFA, MUFA, and PUFA species, respectively, similar to the composition in mice fed fat adjusted diets (Table B.4). However, 18:2n6 and 18:3n3 levels, the essential dietary FAs, remained constant across TCDD treated mice on carbohydrate diets (Figure 4.1E-F), suggesting dietary carbohydrate is not a significant contributor to AhR-mediated steatosis. 112 14 C-OLEATE STUDIES Lipid accumulation in the liver was investigated in Scd1 wild-type and null mice gavaged with 2 Ci 14 C-oleate 5 days post-dose with 30 g/kg TCDD. Scd1 performs the rate-limiting step in MUFA synthesis. Therefore, null mice are incapable of desaturating 18:0 to 18:1n9, yet 18:1n9 is still available via the diet (Table B.4). 14 C-levels in hepatic lipid extracts increased 2- and 2.4-fold in TCDD treated wild-type and Scd1 null mice, respectively (Figure 4.2A). This increase is consistent with the ~2-fold oleate increase in mice fed fat or carbohydrate adjusted diets (Appendix B Tables 2 and 4), and the ~3-fold increase in 18:1n9 levels in TCDD-treated Scd1 wild-type and null mice (Figure 4.2B) [14]. The absorption efficiency of oleate is reported to be >95% efficient [17]. Furthermore, oleate represents >90% of total MUFAs in treated mouse livers (Figure 4.2C) [14]. TCDD increased serum (1.4-fold) and muscle (1.2-fold) and decreased adipose (-1.3-fold) and stool (-1.5-fold) 14 C levels, although statistical significance was not achieved (Figure B.1A-D). DIFFERENTIAL INTESTINAL AND HEPATIC GENE EXPRESSION To further examine the effect of AhR activation on dietary lipid uptake gene expression, intestinal and hepatic mRNA was examined. Of the genes examined, >80% contained a putative, functional DRE (matrix similarity score > 0.847) and/or had hepatic AhR enrichment in ChIPchip analysis (Table 4.3) [6, 15]. Dietary FA (>16C) hydrolyzed from TAG by gastric and pancreatic lipases in the intestinal lumen are actively transported into enterocytes before export into the lymphatic and systemic circulation [18]. In the duodenum, TCDD induced Ldlr (2.3-fold), Cd36 (2.7-fold), and Slc27a4 (1.4-fold), as well as Fabp1 (1.4-fold), and Fabp4 (1.4-fold) (Table 4.3). Although implicated in 113 Figure 4.2. Hepatic lipid levels in Scd1 wild-type (wt) and null mice 120 h post-dose with 30 g/kg TCDD dose. 14 (A) C-levels were measured in lipid extracts by liquid scintillation counting after gavage with 14 2 Ci C-oleate 4 h prior to sacrifice. GC-MS analysis of hepatic oleate (18:1n9) (B) and monounsaturated fatty acid (C) levels (expressed in nmol/g) in Scd1 wild-type and null mice 168 h after oral gavage with 30 g/kg TCDD [14]. * p<0.05 for TCDD compared with Vehicle, ** p < 0.05 for Scd1 wt TCDD compared with Scd1 null TCDD. Bars represent mean  SEM, n=5. 114 mitochondrial -oxidation [19, 20] and insulin sensing [21], Slc27a1 expression (2.3-fold in duodenum, -5.8-fold in jejunum) occurs primarily in muscle and adipose tissue making its role in other tissues uncertain. Endothelial lipases hydrolyze serum lipids absorbed by the intestine. The resulting products are taken up by the liver via facilitated transport or receptor-mediated endocytosis. TCDD induced hepatic long-chain FA uptake family members Cd36 (4.4-fold), Slc27a3 (2.7fold), Slc27a4 (3.0-fold), and Ldlr (3.2-fold) (Table 4.3). Hydrolytic cleavage of TAG by cytosolic lipases further adds FAs to the hepatic pool. TCDD induced Lpl (3.2-fold), Clps (2.6fold), Pnlprp1 (3.7-fold), and Mgll (1.4-fold), but repressed Pnpla3 (3.3-fold) (Table 4.3). Interestingly, sequence variations in PNPLA3 are associated with hepatic TAG content and NAFLD in humans [22]. Intracellular FAs are directed to TAG biosynthetic and -oxidation pathways by Fabps. Fabp1 and 4 were induced 2.9- and 1.4-fold, respectively. However, TCDD repressed hepatic mitochondrial acyl-CoA synthetase genes (Acsm1-4 and Acsl1, 3-4, repressed 1.3 to 2.3-fold) that activate FAs for transport into the inner membrane space for subsequent -oxidation (Table 4.3). These gene expression changes are consistent with the reported inhibition of mitochondrial -oxidation by TCDD [10, 13]. Furthermore, TCDD induced the expression of hepatic triglyceride biosynthesis genes (Mogat1, Mogat2, Dgat1, and Dgat2 induced 3.6-, 3.0-, 1.6-, and 1.3-fold, respectively), consistent with hepatic TAG accumulation [9, 14]. Hepatic differential gene expression is also consistent with TCDD elicited disruption of carbohydrate catabolism (Table 4.3). TCDD induced hexokinase (Hk3) 2.2-fold, which catalyzes the irreversible phosphorylation of glucose to glucose-6-phosphate (G6P). In contrast, genes involved in gluconeogenesis (Pcx -1.3-fold, Pck1 -2.0-fold, G6pc -2.7-fold) and glycogen 115 1 2 Table 4.3. DREs and Regions of AhR Enrichment in TCDD Responsive Genes Associated with Lipid and Carbohydrate Transport and Metabolism. # of ChIP Gene 3 3 Gene ID Liver Duodenum Jejunum 1 2 Function Regulated by Symbol DREs peaks 2h Xenobiotic Metabolism 13076 Cyp1a1 5799* 222* 22.7* 7 4 xenobiotic metabolism AhR Fatty Acid and Triglyceride Synthesis 14104 Fasn -2.8* NC NC 4 1 fatty acid synthesis SREBP, TR, LXR, cAMP, AMPK 153674 Acly -2.6** nd nd 0 0 citrate metabolism oxaloacetate, ATP 107476 Acaca -1.4** nd nd 3 5 malonyl CoA acylation glucagon 68393 Mogat1 3.6* NC NC 1 0 233549 Mogat2 3.0* NC 1.4* 4 3 Dgat1 2.0 1.4 1.6* 4 0 PPAR, CEBP 13350 triglyceride synthesis 67800 Dgat2 1.3 1.4 1.6 2 7 Fatty Acid Transport 238055 Apob 2.5* NC 1.8* 2 0 VLDL and chylomicron assembly APOBEC-1 16835 Ldlr 3.2* 2.3* NC 2 4 lipoprotein uptake LXR 116 Table 4.3 (cont’d) Gene Symbol Liver Duodenum Jejunum # of 1 DREs ChIP 2 peaks 2h Function 12491 Cd36 4.4* 2.7* NC 0 1 fatty acid uptake PPAR, CEBP, AMPK 26457 Slc27a1 -2.9* 2.3* -5.8† 2 0 mitochondrial oxidation PPAR, PPAR 26568 Slc27a3 2.7* NC NC 1 0 unknown peroxisomal oxidation, CHOL-ester synthesis Gene ID 26569 Slc27a4 3.0* 1.4† 1.4† 0 0 14080 Fabp1 2.9* 1.4* 1.7* 2 4 11770 Fabp4 1.4† 1.5 2.5 0 0 117147 Acsm1 -2.0** nd nd 3 0 233799 Acsm2 -1.5** nd nd 0 0 20216 Acsm3 -1.6** nd nd 2 0 233801 Acsm4 -1.2** nd nd 0 0 14081 Acsl1 -1.4** nd nd 6 5 74205 Acsl3 -2.3** nd nd 0 0 50790 Acsl4 -1.3** nd nd 0 2 117 3 TAG synthesis, -oxidation chylomicron assembly medium chain fatty acid transport (mitochondrial -oxidation) long chain fatty acid transport (mitochondrial -oxidation) Regulated by 3 PPAR, SREBP1c PPAR, HNF4 cJun, PPAR acetyl-CoA, malonyl-CoA, NADPH, NADH Table 4.3 (cont’d) Gene ID Gene Symbol Liver Duodenum Jejunum # of 1 DREs ChIP 2 peaks 2h Function 3 Regulated by 3 Fatty Acid Metabolism 16956 Lpl 3.2** nd nd 2 0 109791 Clps 2.6** nd nd 0 0 18946 Pnliprp1 3.7** nd nd 0 0 11343 Mgll 1.4** nd nd 9 116939 Pnpla3 -3.3** nd nd TAG metabolism of lipoproteins & chylomicrons Insulin, glucagon, epinephrine 0 monoglyceride metabolism PPAR 0 1 TAG metabolism Glycolysis/ Gluconeogenesis/ Glycogen Synthesis 18534 Pck1 -2.0** nd nd 0 14377 G6pc -2.7** nd nd 1 5 18563 Pcx -1.3** nd nd 5 4 103988 Gck -1.5** nd nd 3 2 212032 Hk3 2.2** nd nd 1 0 232493 Gys2 -1.5** nd nd 2 insulin, glucagon, cAMP 1 4 gluconeogenesis insulin, glucose ATP glucose metabolism G6P, insulin, glucagon * p<0.05 or † p<0.01 for TCDD compared with Vehicle, QRT PCR data at 24h post-dose. QRTPCR data were analyzed by Dunnett’s 1 t-test, n-=5. ** for P1(t)  0.999 for microarray data at 168 h post-dose [15]. DRE distributions were previously determined [15]. 2 3 Only DREs satisfying a matrix similarity score of > 0.85 were included. AhR enrichment was previously determined [6]. Data from [19, 20, 51]. NC, no change; nd, not detected. 118 synthesis (Gck, -1.5-fold, Gys2 -1.5-fold) were repressed. Similarly, genes that provide FA synthesis substrates, such as ATP-citrate lyase (Acly -2.6-fold) and acetyl-CoA carboxylase (Acaca -1.4-fold), and fatty acid synthetase activity (Fasn, -2.8-fold) were repressed (Table 4.3). These changes are consistent with the lack of a dose-dependent increase in hepatic TFAs through de novo lipogenesis in mice fed carbohydrate adjusted diets. Furthermore, reported changes in hepatic gene expression are consistent with hepatic TCDD reported in the same model [8]. DISCUSSION Hepatic steatosis can result from the disruption of multiple processes involved in lipid and carbohydrate uptake, metabolism and efflux. Our studies provided evidence that dietary fat, rather than carbohydrate, is an important lipid source in TCDD-elicited steatosis in mice. Computational DRE search, ChIP-chip, and gene expression [6] data indicate AhR activation results in a coordinated response involving the digestive, circulatory and hepatic systems (Figure 4.3). This suggests any ligand (e.g., chemical, drug, endogenous substance, natural product) capable of activating the AhR may enhance dietary fat processing and transport, although continuous exposure may be required. Increases in hepatic 14 C levels clearly demonstrated diet as a source of lipids in TCDD- elicited hepatic steatosis. Previous studies have implicated the mobilization of peripheral adipose tissue based on increased serum 16:0, 18:1, 18:2, and 18:3 free FA levels and their abundance in adipose tissue [10-12]. However, these FAs also represent the primary lipids in chow (Table B.4). Furthermore, increases in oleate, the primary MUFA in rodent chow, and 119 Figure 4.3. AhR-mediated increase in dietary lipid in TCDD elicited hepatic steatosis. Step 1 - Dietary fat is hydrolyzed in the intestinal lumen by pancreatic lipase, colipase, and bile salts into glycerol and free fatty acids (FFAs). Step 2 - FFA are transported into enterocytes by fatty acid transport proteins and sequestered by cytosolic fatty acid binding proteins (Fabps). Step 3 - Fabps deliver FAs to the endoplasmic reticulum (ER) where they are synthesized into triglycerides (TAGs) with glycerol. Step 4 - TAGs are packaged with apolipoprotein b48, cholesterol, and phospholipids into chylomicrons that are secreted into the systemic circulation. Step 5 - In the blood, endothelial lipases hydrolyze TAGs associated with chylomicrons and lipoproteins into FFAs and remnant lipoprotein products that are actively transported into hepatocytes via fatty acid transport proteins and receptor-mediated endocytosis. Step 6 - Intracellular lipoprotein TAGs are further hydrolyzed by cytosolic lipases into free fatty acids. Step 7 - Fabp binds FFA and transports them to the endoplasmic reticulum for TAG biosynthesis. Step 8 - In the ER, Mogat and Dgat synthesize TAG from FFA and glycerol. Step 9 - TAGs synthesized in the ER are stored in cytosolic lipid droplets and/or packaged with apolipoprotein b100 (Apob100), cholesterol, and phospholipids into very low-density lipoproteins (VLDL). Step 10 TCDD is reported to inhibit VLDL secretion thus contributing to hepatic fat accumulation [25]. Step 11 - Acyl-CoA synthetase is required for FA acylation, transport into the mitochondria, and subsequent -oxidation. Step 12 - Glucose is transported into hepatocytes via the insulin-independent glucose transporter Glut2. Step 13 - Glucose is phosphorylated to glucose-6-phosphate by glucokinase (Gck) or hexokinase (Hk3). Step 14 - The mitochondrial gluconeogenic enzyme pyruvate carboxylase (Pcx) converts pyruvate to oxaloacetate that can enter the TCA cycle for conversion to citrate. Step 15 – Cytosolic citrate is converted by ATP citrate lyase (Acly) to acetyl CoA and oxaloacetate. Step 16 - Oxaloacetate may be converted to glucose via gluconeogenic enzymes phosphoenolpyruvate carboxykinase (Pck1) and glucose 6 phosphatase (G6pc). Step 17 – Acetyl-CoA carboxylase (Acaca) converts acetyl-CoA to malonyl-CoA, a substrate for fatty acid synthesis that is catalyzed by fatty acid synthase (Fasn). Lines with arrowheads, reaction/pathway direction; lines with blunted ends, reaction/pathway inhibition; red boxes, induced gene expression; green boxes, repressed gene expression. 120 Figure 4.3 (cont’d) 121 hepatic 14 C levels in Scd1 null mice provide further evidence of a role for dietary fat in AhR- mediated steatosis. Complementary gene expression analysis is consistent with a role for the AhR in mediating hepatic accumulation of dietary lipids (Figure 4.3). Free FAs hydrolyzed by pancreatic and gastric lipases, colipases and bile salts passively diffuse (FA<16C) and are actively transported (FA>16C) into enterocytes by Cd36 and Slc27a4 that were induced by TCDD (steps 1-3). A role for Cd36 in intestinal lipid clearance and FFA uptake has been demonstrated in null mice [23], while Slc27a4 (Fatp4) is associated with obesity in humans [24]. Once intracellular, fatty acid binding proteins (Fabps) sequester FAs to prevent their transport back into the intestinal lumen and targets them to specific organelles [25]. TCDD inducible Fabp1, unlike other family members, binds two rather than one FA, as well as other small hydrophobic ligands [26], and is involved in intestinal FA processing and chylomicron maturation (step 4) [27, 28]. Although intestinal lipid absorption is highly efficient [17, 29], AhR activation may enhance intestinal lipid processing and efflux, consistent with TCDD induced increases in serum FFAs and TAGs [8]. The concurrent induction of several hepatic genes associated with lipid transport, processing and metabolism further promotes steatosis (Figure 4.3). Ldlr, Cd36, and Slc27a actively transport increased circulating FFAs, chylomicrons and lipoprotein remnants into the liver (step 5). TCDD also induced lipoprotein lipase (Lpl), monoglyceride lipase (Mgll), pancreatic lipase-related protein 1 (Pnliprp1), and pancreatic colipase (Clps) that hydrolyze intracellular lipoprotein remnants to further increase the intracellular FA pool (step 6). Induced Fabp1 binds and sequesters intracellular FAs and targets them for TAG synthesis [30]. Mogat1/2 and Dgat1/2, induced by TCDD, then facilitate hepatic TAG biosynthesis (steps 7-8). TAGs are 122 stored in lipid droplets (step 9), or incorporated into VLDLs (step 10). However, TCDD inhibits VLDL secretion (step 10) [13], consistent with AhR-mediated increases in hepatic TAG and vacuolization [14, 31]. Fabp1 also targets FAs for mitochondrial -oxidation [30]. TCDD inhibits FA oxidation [10, 13] possibly by inhibiting transport into mitochondria, further adding to hepatic FA accumulation. More specifically, TCDD inhibits medium and long chain FA mitochondrial acylCoA synthetase gene expression (Acsm1-4, Ascl1 and 3-4) (step 11) that is required for transport across the mitochondrial matrix via carnitine for subsequent -oxidation. Yet, TCDD induced ketone body accumulation in vitro [10], suggesting an intact carnitine pathway. Nonetheless, plasma ketones do not increase in response to TCDD exposure in vivo [32] and requires additional investigation. The inability of carbohydrate diets to enhance hepatic steatosis appears to involve TCDD dysregulation of carbohydrate metabolism gene expression (step 12). For example, anabolic pathways typically dominate in fed animals, yet TCDD decreased glucokinase (Gck) and glycogen synthase (Gsy2), suggesting suppression of hepatic glycogen synthesis (step 13). Glucokinase is the predominant enzyme regulating hepatic glucose metabolism in response to nutritional states such as refeeding and insulin stimulation [33]. Although TCDD induced hexokinase (Hk3), this enzyme is expressed at low levels in hepatocytes, yet exhibits compensatory induction following glucokinase repression, as found in liver cirrhosis [34]. TCDD also inhibited mitochondrial pyruvate carboxylase (Pcx) that converts pyruvate into oxaloacetate, suggesting flux towards glycerol production to support hepatic TAG production leading to greater sequestration of hepatic FA. Metabolomic studies also report TCDD increases 123 hepatic glycerol levels [35] and that TCDD treatment prevents glycerol ketogenesis without affecting esterification to TAG [10]. TCDD suppressed phosphoenolpyruvate carboxykinase (Pck1) and glucose-6 phosphatase (G6pc) expression, key regulators of gluconeogenesis (steps 14 and 16). These genes are commonly regulated by peroxisome proliferator-activated receptor  (PPAR) coactivator 1 (PGC-1) [36] that is functionally impaired by AhR-mediated induction of TiPARP (TCDD-inducible poly(ADP-ribose) polymerase 7, PARP7) [37]. TCDD also repressed ATP-citrate lyase (Acly), which converts citrate to oxaloacetate and acetyl-CoA (step 15). Acetyl CoA is critical for de novo FA synthesis by Fasn (step 17), which was dose-dependently repressed by TCDD, TCDF, and PCB126 [38]. These gene expression changes are consistent with TCDD-mediated inhibition of gluconeogenesis [39], de novo lipogenesis [40] and FA oxidation, and may be partially explained by AhR interactions with other signaling pathways involved in hepatic glucose and FA metabolism regulation including PPARs (Table 4.3), Pgc1 [37], Forkhead box O1 (Foxo1) [41] and hepatocyte nuclear factor 4, alpha (HNF4) [19]. Evidence suggests AhR and PPAR signaling pathways interact [42-44] to alter PPAR expression [45]. Other studies identified overrepresentation of PPAR and HNF4 binding motifs in ChIP-chip regions of AhR enrichment that lack DRE cores suggesting AhR binding to DNA independent of DREs [6, 7]. For example, AhR interacts with chicken ovalbumin upstream promoter transcription factor (COUP-TF) [46], and COUP-TF is reported to antagonize HNF4-mediated responses by binding to HNF4 response elements [47]. Consequently, AhR-COUP-TF complexes binding to HNF4 response elements may inhibit HNF4-regulated lipid transport and metabolism gene 124 expression and contribute to TCDD-elicited steatosis [6]. Interestingly, hepatic steatosis has been reported in HNF4 null mice [48]. Collectively, our data indicate that TCDD-mediated hepatic steatosis involves enhanced uptake of dietary fat suggesting a novel endogenous role for the AhR. Other ligands including endogenous metabolites (indoles, tetrapyrroles, and arachidonic acid metabolites), and natural products (e.g. vegetable-, fruit-, and tea-derived indole and flavonoid metabolites) [49, 50] also activate the AhR providing a possible selective evolutionary advantage that optimizes fat absorption to maximize energy intake. Interactions with other nuclear receptors and transcription factors can further impact energy homeostasis and lipid metabolism, transport and deposition. However, persistent AhR activation in combination with the consumption of a high fat diet may also have adverse health implications for fatty liver and its associated diseases including NAFLD, metabolic syndrome and diabetes. Preliminary studies indicate AhR-activation increases TFA levels in human primary hepatocytes (data not shown), although further studies are needed to elucidate species-specific differences in AhR-mediated effects including steatosis. FUNDING This work was supported by the National Institute of Environmental Health Sciences Superfund Research Program (P42ES04911). 125 APPENDIX B 126 APPENDIX B Table B.1. Terminal Body Weight, Body Weight Gain, Absolute Liver Weight, and Relative Liver Weight Time (h) % by weight Treatment Terminal Body Weight (g) Body Weight Gain (g) Liver Weight (g) Relative Liver Weight Vehicle 14.26 ± 0.71 0.84 ± 0.57 0.82 ± 0.10 TCDD Vehicle TCDD 14.70 ± 0.78 14.68 ± 0.66 14.68 ± 0.73 1.08 ± 0.41 1.20 ± 0.43 1.24 ± 0.22 1.04 ± 0.07 0.89 ± 0.06 0.99 ± 0.05 0.058 ± 0.071 ± 0.060 ± 0.067 ± Vehicle TCDD 14.92 ± 0.41 14.40 ± 0.60 1.04 ± 0.21 0.70 ± 0.31 0.87 ± 0.06 0.94 ± 0.05 0.058 ± 0.004 0.065 ± 0.003* Vehicle 17.28 ± 0.87 3.50 ± 1.37 1.00 ± 0.09 3.30 2.58 2.02 3.56 3.46 1.29 0.84 1.05 0.90 1.14 0.058 ± 0.077 ± 0.053 ± 0.067 ± 0.052 ± 0.067 ± A. Fat Adjusted Diets 5% 24 10% 15% 5% TCDD 16.78 Vehicle 15.88 168 10% TCDD 15.72 15% Vehicle 17.12 TCDD 17.14 B. Carbohydrate Adjusted Diets 50% 24 60% 70% ± ± ± ± ± 1.13 1.01 0.61 0.90 0.75 ± ± ± ± ± 0.74 0.89 0.82 0.75 0.33 ± ± ± ± ± 0.11 0.05 0.08 0.06 0.07 0.005 0.002* 0.002 0.002* 0.003 0.002* 0.001 0.003* 0.002 0.004* Vehicle TCDD 16.02 ± 1.11 15.92 ± 0.35 0.18 ± 0.13 0.12 ± 0.16 0.86 ± 0.09 0.94 ± 0.02 0.053 ± 0.004 0.059 ± 0.001 Vehicle TCDD Vehicle 16.22 ± 0.32 15.80 ± 0.66 15.90 ± 0.49 0.58 ± 0.37 0.10 ± 0.19 0.52 ± 0.16 0.89 ± 0.21 0.94 ± 0.10 0.96 ± 0.04 0.055 ± 0.012 0.060 ± 0.004 0.060 ± 0.004 127 Appendix B, Table 1 Cont’d Time (h) % by weight Terminal Body Weight (g) Body Weight Gain (g) Liver Weight (g) TCDD 50% Treatment 15.94 ± 0.47 0.56 ± 0.27 1.09 ± 0.12 0.068 ± 0.007 Vehicle 17.40 ± 0.60 1.96 ± 0.71 0.99 ± 0.08 0.057 ± 0.004 1.88 2.30 0.98 2.50 1.54 1.14 1.04 1.14 1.11 1.18 0.066 0.058 0.067 0.060 0.068 TCDD 17.34 Vehicle 18.14 168 60% TCDD 17.06 Vehicle 18.38 70% TCDD 17.24 * p<0.05 for TCDD vs. Vehicle within a diet. ± ± ± ± ± 0.64 1.01 0.56 0.46 0.51 128 ± ± ± ± ± 0.42 0.64 0.64 0.84 0.62 ± ± ± ± ± 0.05 0.08 0.04 0.11 0.12 Relative Liver Weight ± ± ± ± ± 0.002* 0.003 0.001* 0.005 0.006 Table B.2. Hepatic Lipid Levels (mol/g) in Fat Adjusted Diet-Fed Mice 168 h Post 30 g/kg TCDD Dose Treat5% Fat 10% Fat 15% Fat ment Saturated Fatty Acids Palmitic acid (16:0) Vehicle 27.50 + 3.63 21.62 + 1.97† 17.59 + 1.19† TCDD 33.79 + 3.27*, ** 29.79 + 2.51* 24.94 + 3.27* Behenic acid (22:0) Vehicle 0.26 + 0.03 0.28 + 0.03 0.30 + 0.01 TCDD 0.15 + 0.01* 0.22 + 0.02* 0.23 + 0.01* Monounsaturated Fatty Acids 5.69 7.50 47.41 81.90 0.61 2.72 + + + + + + 1.88 1.54** 10.19** 10.29*, **, †† 0.13 0.34*, ** 2.58 5.42 27.71 58.30 0.37 1.87 + + + + + + 0.31† 1.20* 4.37** 9.52*, ** 0.08† 0.35*, †† Vehicle TCDD Vehicle TCDD Vehicle TCDD 0.88 1.65 0.04 0.25 0.06 0.17 + + + + + + 0.21 0.19*, **, †† 0.01 0.03*, **, †† 0.02 0.03**, †† 1.37 3.53 0.06 0.53 0.08 0.42 + + + + + + Vehicle TCDD Vehicle TCDD 0.40 0.30 0.24 0.68 + + + + 0.04 0.03*, †† 0.03 0.04*, **, †† 0.08 0.42 0.41 0.60 + + + + Palmitoleic acid (16:1n7) Vehicle TCDD Oleic acid (18:1n9) Vehicle TCDD Eicosenoic acid Vehicle (20:1n9) TCDD Polyunsaturated Fatty Acids α-Linolenic acid (18:3n3) Eicosatrienoic acid (20:3n3) Eicosatetraenoic acid (20:4n3) Timnodonic acid (20:5n3) Docosapentaenoic acid (22:5n3) 129 1.31 3.91 16.42 48.95 0.23 1.66 + + + + + + 0.36† 1.02* 2.52 6.10* 0.05† 0.25* 0.12† 0.55* 0.01† 0.07* 0.02 0.03* 1.74 4.48 0.07 0.65 0.09 0.61 + + + + + + 0.19* 0.70* 0.01† 0.07* 0.01 0.16* 0.02 0.02* 0.02 0.02* 0.40 0.76 0.31 1.49 + + + + 0.03 0.15* 0.06 0.33* Table B.2 (cont’d) Treatment Vehicle TCDD Vehicle 5% Fat 10% Fat 15% Fat 18.21 + 2.46 25.19 + 1.80† 26.55 + 2.25† 30.50 + 1.90*, **, †† 49.83 + 4.24*, ** 58.89 + 5.18* Eicosadienoic acid 0.45 + 0.02 0.49 + 0.05† 0.49 + 0.04 (20:2n6) TCDD 1.43 + 0.1*, **, †† 2.42 + 0.28*, ** 2.94 + 0.18* Dihomo-ϒ-linolenic Vehicle 1.33 + 0.02 1.08 + 0.14 0.98 + 0.08 acid (20:3n6) TCDD 2.24 + 0.1*, **, †† 3.26 + 0.10*, ** 3.98 + 0.48* Arachidonic acid Vehicle 11.29 + 0.39 11.27 + 0.75 10.59 + 0.75 (20:4n6) TCDD 9.71 + 1.22*, †† 11.61 + 0.71 10.62 + 0.11 The following fatty acids were detected, but not significantly altered by TCDD: adrenic acid (22:4n6), nervonic acid (24:1n9), stearic acid (18:0), and behenic acid (22:0). Data are reported as g fatty acid methyl ester/g liver tissue. N=5, * p<0.05 for TCDD vs. Vehicle within a diet, ** p<0.05 for TCDD 15% fat vs. TCDD 5% or 10%, † p<0.05 for Vehicle 5% fat vs. Vehicle 10% or 15%, †† p<0.05 for TCDD 5% fat vs. TCDD 10%. Linoleic acid (18:2n6) 130 Table B.3. Lipid Composition (mol) in Fat Adjusted (A) and Carbohydrate Adjusted (B) Diets A. Fat Diets % Fat 5% 10% 15% Palmitic Acid (16:0) 23.91 + 3.01 43.92 + 4.44 59.18 + 9.10 Stearic Acid (18:0) 9.18 + 1.09 18.39 + 1.71 26.34 + 4.08 Arachidic Acid (20:0) 0.54 + 0.06 1.72 + 0.12 1.72 + 0.26 Behenic Acid (22:0) 0.50 + 0.05 1.05 + 0.11 1.59 + 0.24 Lignoceric Acid (24:0) 0.11 + 0.02 0.23 + 0.03 0.36 + 0.06 0.19 + 0.02 0.33 + 0.04 0.48 + 0.08 39.17 + 4.76 80.08 + 6.81 113.47 + 16.74 0.24 + 0.03 0.51 + 0.06 0.76 + 0.12 Linoleic Acid (18:2n6) 94.51 + 11.47 180.39 + 16.52 248.93 + 37.16 -Linolenic Acid (18:3n3) 16.95 + 2.10 47.74 + 3.00 47.74 + 7.10 Saturated Fatty Acids Monounsaturated Fatty Acids Palmitoleic Acid (16:1) Oleic Acid (18:1) Gondoic Acid (20:1) Polyunsaturated Fatty Acids 131 Table B.3 (cont’d) B. Carbohydrate Diets % Carbohydrate 50% 60% 70% Palmitic Acid (16:0) 28.33 + 2.48 31.47 + 1.42 26.01 + 5.73 Stearic Acid (18:0) 12.25 + 0.95 11.47 + 0.1 11.47 + 1.91 Saturated Fatty Acids Arachidic Acid (20:0) 0.81 + 0.06 0.76 + 0.04 0.76 + 0.09 Behenic Acid (22:0) 0.78 + 0.05 0.75 + 0.08 0.75 + 0.06 Lignoceric Acid (24:0) 0.22 + 0.03 0.19 + 0.02 0.19 + 0.03 0.25 + 0.03 0.27 + 0.00 0.23 + 0.04 53.01 + 4.22 56.91 + 1.41 46.48 + 1.82 0.34 + 0.03 0.33 + 0.02 0.33 + 0.04 115.13 + 9.59 126.43 + 2.75 106.73 + 20.02 19.86 + 1.63 21.81 + 0.23 18.54 + 3.44 Monounsaturated Fatty Acids Palmitoleic Acid (16:1) Oleic Acid (18:1) Gondoic Acid (20:1) Polyunsaturated Fatty Acids Linoleic Acid (18:2n6) -Linolenic Acid (18:3n3) 1 GC-MS-FAMES hepatic lipid profiling was performed as described in the materials and methods from 100 mg ground rodent diet. 132 Table B.4. Hepatic Lipid Levels (mol/g) in Carbohydrate Adjusted Diet-Fed Mice 168 h Post 30 g/kg TCDD Dose Treatment 50% Carbohydrate 60% Carbohydrate 70% Carbohydrate Saturated Fatty Acids Palmitic acid (16:0) Vehicle Stearic acid (18:0) Vehicle Lignoceric acid (24:0) TCDD TCDD Vehicle TCDD 22.64 + 26.67 + 11.18 14.29 + 17.05 + 1.63 7.80 + 10.20 + 0.90 2.72 + 4.26 + 1.16 24.80 + 44.68 + 10.31 0.75 + 1.18 + 0.16 0.74 + 1.26 + 0.32 0.05 + 0.18 + 0.02 0.89 2.71 + 4.03 + 0.42 27.40 + 45.51 + 2.37 0.43 + 1.24 + 0.05 0.74 + 1.10 + 0.74 0.05 + 0.16 + 0.59* 1.10 7.18 + 8.86 + 1.27 1.86 14.69 + 15.97 + 1.04** 24.46 + 25.83 + 0.05 3.67** 1.49 1.10 26.92 + 35.72 + 3.21 15.04 + 19.14 + 1.20 7.32 + 9.34 + 1.10 4.00 + 6.72 + 0.54 45.80 + 70.95 + 5.50 0.89 + 1.67 + 0.20 0.75 + 1.28 + 0.11 0.05 + 0.13 + 0.01 7.18* 2.24* 0.85* Monounsaturated Fatty Acids Palmitoleic acid (16:1n7) Vehicle Oleic acid (18:1n9) Vehicle Eicosenoic acid (20:1n9) Vehicle TCDD TCDD TCDD 0.62** 4.71** 0.17* 1.23*, ** 11.31** 0.30* 2.15* 23.23* 0.65* Polyunsaturated Fatty Acids α-Linolenic acid (18:3n3) Vehicle Eicosatrienoic acid (20:3n3) Vehicle TCDD TCDD 0.14* 0.03* 133 1.10 0.16* 0.26* 0.02* Table B.4 (cont’d) Treatment Timnodonic acid (20:5n3) Vehicle Linoleic acid (18:2n6) Vehicle Eicosadienoic acid (20:2n6) Vehicle Dihomo-ϒlinolenic acid (20:3n6) Arachidonic Acid (20:4n6) TCDD TCDD TCDD Vehicle TCDD Vehicle TCDD 50% Carbohydrate 0.42 + 0.46 + 0.12* 17.76 + 28.55 + 4.13 0.48 + 1.11 + 0.09 1.23 + 2.46 + 0.21 14.03 + 16.71 + 1.30 60% Carbohydrate 0.33 + 0.36 + 17.79 + 25.47 + 0.13* 0.73* 1.26 13.97 + 14.88 + 0.13* 0.48 1.26 + 2.32 + 2.18* 17.79 0.48 + 1.01 + 0.03 0.33* 13.97 0.36 25.47* 1.01* 2.32* 14.88 70% Carbohydrate 0.34 + 0.42 + 0.02 18.07 + 27.06 + 0.97 0.59 + 0.94 + 0.05 1.49 + 2.43 + 0.13 13.14 + 15.40 + 1.52 0.06* 3.28* 0.14* 0.36* 1.42 The following fatty acids were detected, but not significantly altered by TCDD: adrenic acid (22:4n6), nervonic acid (24:1n9), behenic acid (22:0), docosapentaenoic acid (22:5n3), and eicosatetraenoic acid (20:4n3). 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Boron W, Boulpaep E: Medical Physiology. 2nd edn: Saunders; 2008. 141 CHAPTER 5 Angrish MM, Dominici CY, Jones AD, Zacharewski TR: 2,3,7,8-Tetrachlorodibenzo--dioxin (TCDD)-Elicited Effects on Liver, Serum, and Adipose Lipid Composition in C57BL/6 Mice. 142 CHAPTER 5 TCDD-ELICITED EFFECTS ON LIVER, SERUM, AND ADIPOSE LIPID COMPOSITION IN C57BL/6 MICE ABSTRACT The AhR mediates alterations in hepatic lipid composition elicited by TCDD. In order to further investigate the effects of TCDD, liver, serum and parametrial fat pad (PFP) FAMEs and lipids were examined in fasted 4-week-old female mice orally gavaged with 30 µg/kg TCDD and sacrificed at 24, 72, and 168 h. Mean (µmol/g) hepatic FAME levels (Vehicle/TCDD: 236.7 compared with 392.2) increased, but did not change in PFP or serum. Hepatic levels of SFAs (16:0, 18:0, 20:0, and 22:0) decreased, while TCDD increased MUFAs (16:1n7, 18:1n9, and 20:1n9) consistent with AhR-mediated induction of Scd1. In addition, TCDD induced levels of selected PUFAs (20:2n6, 20:3n6, 18:3n3, and 22:5n3) while others (20:4n6, 22:6n3) decreased. There were modest increases in PFP 20:2n6 and 20:3n6 levels, while 18:0 decreased and 18:1n9 increased in serum. TCDD also lowered total CHOL, low-density lipoprotein (LDL), and highdensity lipoprotein (HDL) by 25% in serum. Decreases in serum CHOL, LDL, and HDL levels were consistent with the differential expression of hepatic CHOL metabolism and transport genes Hmgcs1 (-2.1-fold), Hmgcr (-2.3-fold), Apoa1 (-1.7-fold), Lcat (2.0-fold), and Ldlr (3.6fold). Moreover, TCDD decreased serum Apob100 (4.4-fold) and Apob48 (2.2-fold) levels, consistent with serum lipid clearance and decreased hepatic efflux. Collectively, these results suggest TCDD-elicited, AhR-mediated decreases in circulating lipid levels are consistent with the induction of hepatic steatosis. 143 INTRODUCTION TCDD and similar chemicals bioaccumulate in food supplies, mainly animal fats, and elicit adverse hepatotoxic effects in animals and humans that are mediated by the AhR. The AhR is a ligand activated PAS transcription factor family member [1] that binds structurally diverse environmental contaminants (halogenated aromatic hydrocarbons and non-halogenated polycyclic aromatic hydrocarbons) with high affinity [2]. Although the AhR also binds endogenous endobiotics (indoles, tetrapyrroles, and arachidonic acid metabolites), and natural products (vegetable-, fruit-, and tea-derived indoles and flavonoid metabolites) [3, 4], no known endogenous high affinity ligand has been identified. The classical AhR pathway involves ligand activation, translocation to the nucleus, and heterodimerization with ARNT [5, 6]. The AhR:ARNT heterodimer binds DREs in the promoters of target genes and recruits transcriptional co-activators to positively regulate gene transcription [7, 8]. TCDD-elicited, AhR-mediated disruption of hepatic energy balance results in steatosis accompanied by increased triglycerides, inflammation and the differential expression of hepatic genes involved in lipid metabolism [9-11]. These gene expression changes include induction of hepatic lipid transporters Cd36 [12], Vldlr, Ldlr, Fabp1, and Slc27a3-4 concurrent with decreased Fasn expression [13, 14] and enzyme activity [15]. Diet and 14 C-oleate studies strongly suggest dietary fat as a lipid source in TCDD-elicited steatosis [16] and, collectively with gene expression data, imply hepatic lipid accumulation is due to TCDD enhanced hepatic uptake of dietary lipid rather than de novo synthesis. It is also believed that adipose lipolysis can contribute to ectopic fat accumulation, including hepatic steatosis. However, data regarding the importance of adipose lipolysis in the promotion of TCDD-elicited hepatic steatosis are lacking 144 since conclusions were based on serum FA measurements [17-19]. No experiments have reported TCDD effects on adipose tissue FA composition. Excessive hepatic lipid accumulation frequently results in non-alcoholic steatohepatitis (NASH), a widespread condition linked to insulin resistance [20] and dyslipidemia that is characterized by altered serum lipid profiles [21]. Animal and human epidemiological studies collectively provide conflicting data relating serum lipid levels to TCDD exposure, even from the same lab [22, 23]. However hepatic steatosis and insulin resistance are conditions associated with TCDD exposure in humans [9, 10, 24, 25]. Considering that the liver partitions lipids for hepatocellular storage as well as storage in peripheral adipose tissue via the circulatory system [26], it is appreciated that TCDD effects on hepatic lipids are complex. Therefore, TCDD alterations in FA composition in the liver, PFP, and serum of fasted mice were investigated. Furthermore, serum lipid panels, which are routine tests used to identify alterations in metabolism, were also examined. Collectively, the reported changes in systemic lipid profiles provide insight into hepatic lipid metabolism with potential implications for novel biomarkers of TCDD-elicited hepatotoxicity and steatosis. MATERIALS AND METHODS ANIMAL HUSBANDRY AND IN VIVO TREATMENT Heterozygous B6.129-Scd1tm1Myz/J mice (Jackson Laboratory, Ben Harbor, Maine) were bred, genotyped and weaned as previously described [27]. On PND 28 mice (n=5) were gavaged with 0.1 ml of sesame oil (vehicle control) or 30 g/kg TCDD (Dow Chemical Company, Midland, MI). Wild-type, immature mice were used to facilitate comparisons with other data sets and to minimize potential interactions with estrogens produced by developing ovaries. The dose 145 was chosen to elicit moderate hepatic effects while avoiding overt toxicity. Animals were fasted 4 h prior to sacrifice at 24, 72, and 168 h post-dose. Mice were weighed and blood was collected via submandibular vein puncture before sacrifice. Liver and PFP tissue were weighed and samples flash frozen. All procedures were carried out with Michigan State University Institutional Animal Care and Use Committee approval. SERUM LIPID PANELS Serum total CHOL, free CHOL, LDL, high density HDL, FFA, and TAG were measured according to the manufacturer’s microtiter protocol (CHOL E; Free CHOL E, L-Type LDL, LType HDL, NEFA-HR(2), L-Type TG M, Wako Diagnostics). GC-MS FATTY ACID METHYL ESTER (FAMES) LIPID PROFILING Lipid analysis was performed as previously described [27]. Lipids extracted from liver (~100 mg), PFP (~30 mg), and serum (50 L) were separated and analyzed with an Agilent 6890N GC with a 30mDB23 column interfaced to an Agilent 5973 MS. 19:1n9 FFA and 19:0 TAG were added as extraction efficiency controls and 17:1n1 FAME (Nu-chek, Elysian, MN) was spiked in as a loading control. GC-MS data files were converted to Waters MassLynx file format, analyzed with MassLynx software and reported as mol/g liver or adipose tissue and nmol/mL serum. FA levels are based on peak areas from total ion chromatograms, and mol is obtained from a linear calculation of a calibration curve and normalized to sample weight (liver and PFP) or volume (serum). Mol% is the ratio of a particular FA relative to TFA. WESTERN BLOT Apolipoprotein B (ApoB) Western blot was performed from serum. Briefly, serum was diluted 1:5 in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1mM EDTA). Diluted serum was separated in 5% SDS-PAGE and transferred to a 146 PVDF membrane (Millipore Billerica, MA). ApoB protein was immunoblotted with an ApoB antibody (SC-11795; Santa Cruz Biotechnology Santa Cruz, CA). Immunoreactive bands were visualized by chemiluminescence with the Pierce ECL Western blotting substrate (Thermo Scientific Rockford, IL) and quantified by densitometry (ImageJ). RNA ISOLATION RNA was isolated from frozen liver and PFP samples with ~1.0 mL TRIzol (Invitrogen) according to the manufacturer’s protocol and an additional acid phenol:chloroform extraction as previously described [13]. Total RNA was resuspended in RNA storage solution, quantified by spectrophotometery at A260 and quality assessed by gel electrophoresis. QRTPCR Quantitative real-time PCR (QRTPCR) was performed as previously described [13]. The copy number of each sample was standardized to the geometric mean of Gapdh, Hprt, and Actb to control for differences in RNA loading, quality, and cDNA synthesis [28]. Data are reported as the fold change of standardized treated over standardized vehicle. STATISTICAL ANALYSIS Data were analyzed by ANOVA followed by Tukey’s post hoc test in SAS 9.2 (SAS Institute, Cary, NC), unless otherwise stated. Differences between treatment groups were considered significant when p<0.05. RESULTS TCDD EFFECTS ON BODY, LIVER, AND ADIPOSE WEIGHTS Fasted mice gavaged with 30 g/kg TCDD had increased absolute and relative (whole liver weight normalized to whole body weight at sacrifice) liver weights at 72 and 168 h 147 Table 5.1. Effect of TCDD on Body, Liver, and Parametrial Fat Pad (PFP) Weights 24, 72, and 168 h Post-Dose Time (h) Treatment Body Weight (g) Body Weight Gain (g) Absolute Liver Weight (g) Relative Liver Weight Absolute PFP Weight (g) Relative PFP Weight Vehicle 12.1 ± 1.9 0.5 ± 0.6 0.74 ± 0.103 0.061 ± 0.002 0.04 ± 0.02 0.0035 ± 0.0011 TCDD 12.8 ± 0.9 -0.2 ± 0.8 0.80 ± 0.095 0.063 ± 0.007 0.05 ± 0.01 0.0040 ± 0.0009 Vehicle 14.5 ± 0.9 1.6 ± 0.2 0.79 ± 0.051 0.055 ± 0.001 0.08 ± 0.02 0.0055 ± 0.0013 TCDD 14.0 ± 0.9 1.4 ± 0.1 0.94 ± 0.074* 0.067 ± 0.003* 0.06 ± 0.03 0.0039 ± 0.0019 Vehicle 15.3 ± 0.7 3.3 ± 1.1 0.85 ± 0.070 0.055 ± 0.002 0.06 ± 0.01 0.0041 ± 0.0006 TCDD 15.2 ± 0.6 3.4 ± 1.0 * p<0.05 for TCDD compared with Vehicle. 1.04 ± 0.060* 0.068 ± 0.002* 0.07 ± 0.02 0.0044 ± 0.0010 24 72 168 148 post-dose (Table 5.1). There were no significant alterations in body weight, body weight gain, or adipose weight throughout the study suggesting treatment had no effect on food consumption. TOTAL LIVER, ADIPOSE, AND SERUM LIPID CONTENT We have previously reported TCDD- and Scd1 genotype-dependent alterations on hepatic lipid composition in fed mice [27]. Similar to the livers of fed TCDD treated mice, analysis of FA composition by GC-MS in fasted mice identified increased mean (mol/g) TFAs (1.7-fold), SFAs (1.25-fold), MUFAs (3.1-fold), and PUFAs (1.5-fold) (Table 5.2). In contrast to liver, TFA levels were unaffected in adipose and serum. FAME LEVELS IN LIVER, PFP AND SERUM As reported in fed mice, TCDD decreased hepatic SFA levels relative to vehicle (Figure 5.1A). Palmitate (16:0) and stearate (18:0), precursors for long chain FA synthesis and the Scd1 desaturation reaction, were significantly depleted. However, palmitate (16:1n7), oleate (18:1n9), and eicosanoic acid (20:1n9), downstream products of SFA desaturation and elongation, increased with treatment. The n-3 and n-6 PUFAs are derived from the diet or via modification of the essential FAs linoleic (18:2n6) and -linolenic (18:3n3) acids [29]. Both n-3 and n-6 pathway intermediates exhibited a mixed response to TCDD (Figure 5.1A). Specifically, 18:2n6 levels were not altered, while levels of its derivatives, eicosadienoic acid (20:2n6) and dihomo--linolenic acid (20:3n6), were increased. Arachidonic acid (AA, 20:4n6), another downstream product of linoleic acid that can be further metabolized into bioactive products promoting pro-inflammatory conditions [30], exhibited a significant decrease in TCDD-treated mice. N-3 derivatives are highly regarded for their health benefits, including improved insulin 149 Table 5.2. Liver, Parametrial Fat Pad, and Serum Total Lipid Content Treatment 413 ± 104 392 ± 58* 3928 ± 945.2 411 ± 100 Vehicle 89 ± 6.8 1251 ± 435.3 147 ± 37 113 ± 10* 1476 ± 413.2 137 ± 39 Vehicle 34 ± 4.2 901 ± 338.2 63 ± 17 106 ± 22* 1033 ± 214.6 74 ± 19 Vehicle 114 ± 15 1257 ± 513.0 204 ± 53 173 ± 28* 1420 ± 323.6 200 ± 47 Vehicle 0.8 ± 0.1* 0.022 ± 0.010 0.24 ± 0.04 0.5 ± 0.1 0.027 ± 0.004 0.21 ± 0.05 Vehicle 1.0 ± 0.03* nd TCDD 20:5n3/18:3n3 3408 ± 1280.9 TCDD 20:4n6/18:2n6 237 ± 26 TCDD Total PUFA Vehicle TCDD Total MUFA Serum (nmol/mL) TCDD Total SFA PFP (µmol/g) TCDD Total FA Liver (µmol/g) 0.4 ± 0.1 nd 1 2 nd 1 1 2 nd 2 * p<0.05 for TCDD compared with Vehicle; nd, not detected. 20:5n3 was not detected in PFP. 20:5n3 and 18:3n3 were not detected in serum. 150 resistance and plasma lipid profiles [30]. The precursor for all n-3 FAs, -linolenic acid (18:3n3), was increased by TCDD relative to vehicles (Figure 5.1A). The n-3 intermediate timnodonic acid (20:5n3) was decreased, while docosapentaenoic acid (22:5n3) was induced. Docosahexaenoic acid (DHA, 22:6n3), required for proper infant growth and neurodevelopment [29], was decreased by TCDD. Increased 20:2n6, 20:3n6, and 22:5n3 were consistent with Elovl5 induction [27]. Although lower 20:4n6 and 20:5n3 levels suggest decreased 5 desaturase activity [31], 5 destaturase mRNA levels were unaffected by TCDD (data not shown). Therefore, decreased n-3 and n-6 pathway product/precursor ratios (20:5n3/18:3n3, decreased 57% and 20:4n6/18:2n6, decreased 40%, Figure 5.2) are the possible result of TCDD-mediated metabolic PUFA conversions into anti- and pro-inflammatory signaling molecules [32]. TCDD had minimal effects on PFP FAME levels with no alterations in SFAs or MUFAs (Figure 5.1B). Only 20:2n6 and 20:3n6 PUFAs were increased by treatment. In serum, 16:0, 18:0, 18:1n9, 18:2n6, and 20:4n6 (Figure 5.1C) were detected. These FAs are the predominant species in Harlan Teklad diet F6 rodent diet 7964 as well as lipoprotein glycerolipids, cholesteryl esters, and phospholipids [33]. The SFA 18:0 was decreased, while the MUFA 18:1n9 increased. No alterations in serum PUFAs were detected. TCDD INDUCTION OF THE SCD1 DESATURATION INDEX We previously reported AhR-mediated increases in Scd1 activity [27]. The 18:1n9/18:0 ratio is an indirect measure of Scd1 activity, and its increase is associated with hyperlipidemia in humans [34]. The Scd1 activity index was induced 2.9- and 1.4-fold in TCDD compared with vehicle in liver and serum, respectively (Figure 5.2). The adipose Scd1 activity index was not affected by treatment. 151 Figure 5.1. Total fatty acid composition in liver (A), parametrial fat pad (PFP, B), and serum (C) of mice 168 h post 30 µg/kg TCDD or sesame oil vehicle dose. Total fatty acids were extracted by Folch method and quantitated by GC-MS as described in the materials and methods. * p<0.05 for TCDD compared with vehicle. SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid. Bars represent mean + standard error of the mean (SEM). Liver, n=4; adipose and serum, n=5. 152 Figure 5.1 (cont’d) 153 Figure 5.1 (cont’d) 154 Figure 5.2. The 18:1n9/18:0 desaturation index in liver, serum, and parametrial fat pad (PFP) tissue of mice treated with 30 µg/kg TCDD or sesame oil vehicle 168 h post-dose. The desaturation index is the ratio of oleic acid (18:1n9) to the precursors stearic acids (18:0) and an indirect measure of Scd1 activity. * p<0.05 for TCDD compared with vehicle. Bars represent mean + SEM, n=5. 155 ALTERATIONS IN SERUM LIPIDS A 20% decrease in total CHOL, a measure of free CHOL (unchanged by TCDD, data not shown), LDL CHOL (decreased 30%), and HDL CHOL (decreased 20%) was detected in TCDD mice compared with vehicles (Figure 5.3A). Decreased LDL, a modification product of VLDL [33], suggests decreased VLDL secretion that was further supported by a 4.3-fold decrease in serum Apob100 protein levels (Figure 5.3B-C). Apob100 is the major non-exchangeable lipoprotein found in VLDL and LDL particles produced by the liver. Apob48 is a truncated form of Apob100 and the major scaffold protein of chylomicrons produced exclusively by the small intestine of mammals and also the livers of mice [33]. TCDD decreased serum Apob48 protein levels 2.8-fold compared to vehicles (Figure 5.3B & D). Serum free FA, free CHOL, and triglycerides (TAG) were not affected by treatment in fasted mice (data not shown). DIFFERENTIAL HEPATIC AND ADIPOSE GENE EXPRESSION BY TCDD To further examine the effect of TCDD on lipid metabolism, QRTPCR was performed from liver and adipose mRNA extracts. Decreases in serum CHOL levels suggested TCDDelicited alterations in hepatic CHOL biosynthesis. QRTPCR analysis identified a 2.1- and 2.3fold decrease in hepatic Hmgcs1 and Hmgcr (Figure 5.4A-B) whose gene products catalyze the initial condensation and reduction reactions, respectively, in the cholesterol biosynthetic pathway [33]. Apolipoprotein A1 (Apoa1) is the primary HDL apolipoprotein expressed in the liver, and to a lesser extent in the intestine. Apoa1 is also a cofactor for lecithin cholesterolacyltransferase (Lcat), an enzyme that produces the bulk of plasma CHOL esters. In the liver, TCDD decreased Apoa1 1.7-fold, while Lcat was increased 2-fold (Figure 5.4C-D). The lipoprotein transporter Ldlr, involved in lipoprotein endocytosis and reverse CHOL transport, was increased 3.6-fold by TCDD (Figure 5.4E). Increased Lcat and Ldlr expression are consistent with TCDD-mediated 156 Figure 5.3. Serum cholesterol and apolipoprotein b (Apob) 100 and Apob48 levels in Scd1 wild-type mice treated with 30 µg/kg TCDD or sesame oil vehicle. (A) Total cholesterol (CHOL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels were decreased 25% by TCDD at 168 h post-dose. Serum lipids were measured by commercial assay (WAKO Diagnostics) (n=8) and are presented as mg/dL. (B) Apob100 and Apob48 protein levels were detected by Western blot at 72 h from serum diluted 1:5 (n=3). (CD) Densitometry (determined with ImageJ) identified a TCDD-dependent 4.4- and 2.2-fold decrease in Apob100 (C) and Apob48 (D) bands, respectively. * p<0.05 for TCDD compared with Vehicle. Bars represent mean + SEM. 157 increases in hepatic CHOL levels, while decreased CHOL synthesis gene expression suggests feedback inhibition. In addition to the marginal changes in PFP lipid composition, TCDD altered the expression of several genes involved in FA metabolism and transport. For example, Slc27a1, a fatty acid transporter that translocates to the plasma membrane in response to insulin [35], was induced 7.5-fold, and triglyceride lipase Pnpla3 was repressed 2.4-fold (Figure 5.4F-G). Interestingly, TCDD induced Adipoq 1.6-fold, whose gene product adiponectin exhibits hormonal regulation of lipid and glucose metabolism (Figure 5.4H). Furthermore, TCDD induced the prototypical TCDD-inducible gene Cyp1a1 mRNA levels (Figure 5.4I), indicating adipose tissue is responsive to TCDD treatment. TCDD also induced Scd2, the predominant adipose Δ9 desaturase, 2.1-fold, consistent with TCDD-dependent AhR-mediated regulation of Δ9 desaturase activity (Figure 5.4J) [27]. DISCUSSION The liver’s role in lipid storage and lipid partitioning is a fundamental process regulating whole body energy metabolism [36]. Altered hepatic and systemic lipid homeostasis is a pathophysiological sign of liver disease [21, 37] that is also observed after TCDD exposure. By examining lipogenic tissue FA levels and composition along with serum lipid panels, the current study provides direct insight into the effects of acute TCDD exposure on the state of whole-body FA and CHOL metabolism, with implications for AhR-mediated hepatic steatosis. The current study corroborated existing evidence that TCDD increases hepatic lipid accumulation and alters hepatic FA composition [27, 38, 39]. Specifically, TCDD increased MUFA levels, particularly 18:1n9 consistent with AhR mediated induction in Scd1 activity [27]. 158 Figure 5.4. Differential expression of hepatic genes involved in cholesterol metabolism (AE) and parametrial fat pad genes involved in lipid metabolism and transport (F-J) in mice gavaged with 30 g/kg TCDD or sesame oil vehicle for 24 h. The relative abundance is the total mRNA quantity normalized to the geometric mean of Hprt, Actb, and Gapdh. Genes are indicated by official gene symbols. * Represents p < 0.05 for TCDD compared to vehicle. Bars represent mean + SEM, n=5 biological replicates. 159 Figure 5.4 (cont’d) 160 TCDD also increased PUFA levels marked by increased 20:3n6 and decreased 20:4n6 (AA) and 22:6n3 (DHA), indicators of increased Elovl5 activity [40]. These TCDD-elicited FA changes are common effects observed during excessive hepatic lipid accumulation including NAFLD, a condition considered the hepatic manifestation of MetS risk factors, including obesity and other pathophysiological conditions of hepatic FA overload such as insulin resistance [41, 42]. Insulin resistance is a condition shown to be associated with TCDD exposure in humans [43, 44] as well as adipose tissue fatty acid mobilization [36]. Furthermore, adipose lipolysis is a reported FA source in TCDD-elicited steatosis [17-19]. However, the current study challenges TCDD-mediated adipose lipolysis by the observation that adipose FA levels were not affected by treatment. This was not completely surprising since studies attributed adipose lipolysis to increased serum 16:0, 18:1, 18:2, and 18:3 levels. Not only are these FAs predominant in adipose tissue [45-48], but also in rodent chow [16]. Furthermore, TCDD-treated animals examined from these studies were not fasted and it has been demonstrated that dietary fat is an important lipid source in TCDD-elicited steatosis [16]. The current study further supports existing evidence that diet is a source of lipids in TCDD-elicited steatosis. In the fasted state, serum reflects lipid origins and analysis of serum FAMEs, FFAs, and TAGs from fasted mice identified no change after TCDD treatment. In contrast, FFAs [13, 49] and TAGs [23, 50, 51] were increased by TCDD in fed animals, suggesting diet is necessary for TCDD-elicited hyperlipidemia. In the fasted state, serum lipids also reflect lipid metabolic transformations [26]. Specifically, the 18:1n9/18:0 product/precursor ratio, a functional measure of Scd1 activity increased in the liver, was also increased in serum. Elevated 18:1n9/18:0 ratios are associated with hyperlipidemic humans [34] raising the possibility that 18:1n9/18:0 ratios are a potential biomarker for hepatic steatosis. 161 Alterations in CHOL, lipoproteins, and apolipoproteins provided further insight into TCDD-dependent alterations in lipid metabolism. TCDD decreased serum Apob48 and Apob100 protein levels, the primary apolipoproteins in CM and VLDL particles, suggesting enhanced clearance from serum and/or efflux from liver. However, unlike humans that exclusively synthesize Apob48 in the intestine and Apob100 in liver, post-transcriptional editing in mouse liver produces Apob48 and Apob100 protein [52]. Therefore, Apob levels in treated, fasted mice suggest decreased hepatic efflux and are supported by TCDD-mediated decreases in VLDL secretion [12]. Decreased serum Apob levels may be explained by TCDD-dependent activation of IRS-PI3K-AKT pathway [53] and/or promotion of ER stress that assists Apob degradation [42, 54]. Specifically, microsomal triglyceride transfer protein (Mttp), a lipogenic mediator required for VLDL assembly [54-56], is regulated by Foxo1, a transcription factor inhibited by the IRS-PI3K-AKT signaling [57]. Furthermore, Foxo1 is regulated by PGC1, a transcriptional coactivator deactivated by TCDD induction of TiPARP [58]. However, Mttp is minimally repressed by TCDD (-1.2-fold, [59]) and the exact relationship between intracellular signaling, Mttp function, and Apob production/degradation requires further investigation. Decreased Apob and VLDL levels are also consistent with TCDD-mediated decreases in LDL particles, products of metabolic VLDL transformation in the serum [33], and decreased serum total CHOL and HDL. These changes in conjunction with increased hepatic expression of reverse CHOL transport genes Ldlr and Lcat, decreased hepatic CHOL biosynthesis gene expression, and increased hepatic CHOL levels [14] strongly imply TCDD-dependent dysregulation of liver CHOL metabolism. Increased reverse CHOL transport further adds to the hepatic CHOL pool enhancing feedback inhibition of CHOL biosynthesis. Furthermore, increased hepatic CHOL may initially stimulate bile acid production, that is later feedback 162 inhibited, netting hepatic hypercholesterolaemia and exacerbated hepatic steatosis. These changes are consistent with differential expression of Cyp7a1 (induced at 24 h, repressed at 72168 h post-TCDD dose) the rate-limiting enzyme in bile acid synthesis that is also feedback inhibited by CHOL [59]. Given these data, it would be interesting to further investigate DREindependent AhR-mediated regulation of the CHOL biosynthetic pathway [60] in the absence of direct CHOL regulation. Lipids are not only important biological components, but also potent signaling molecules, metabolic regulators and transcription factor ligands [33] that when altered, effect diverse biological processes. 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Hepatology 2012. 170 CHAPTER 6 171 CHAPTER 6 CONCLUSIONS AND FUTURE RESEARCH The preceding reports examined the role of AhR-mediated regulation of lipid uptake, metabolism, and transport in TCDD-elicited steatosis using Scd1 null mice, diet, 14 C-lipid uptake, and GC-MS lipid analysis. Collectively, these studies showed that AhR regulation of Scd1 contributed to the hepatotoxicity of TCDD (Chapter 3), dietary fat is the primary source of lipid in TCDD-elicited steatosis (Chapter 4), and AhR mediated not only altered hepatic lipid composition, but also systemic lipid composition (Chapter 5). Collectively, this work provides compelling evidence that TCDD activation of the AhR coordinates interactions between the digestive tract, circulatory system and liver to evoke hepatic steatosis, the hepatic manifestation of MetS. Considering that TCDD and related chemicals are ubiquitous environmental contaminants, the data suggest a pathophysiological relationship between DLC exposure and adverse health effects such as metabolic disease. Although the toxicological effects of AhR activation have been extensively studied, this research illustrates a complex and equivocal function for the AhR in biological and physiological processes effecting lipid metabolism. The data generated advances knowledge on how TCDDdependent AhR activation produces steatosis, which is a significant hepatotoxic effect of TCDD. It furthers our understanding of the mode-of-action and events that may be linked to cancer. Specifically, the novel findings that AhR regulation of Scd1 promotes inflammation and increased MUFA levels may be key events linked to cryptic cirrhosis and hepatocellular carcinoma observed in numerous rodent bioassays [1]. Cancer cell growth and proliferation requires energy and the production of new lipids for membrane synthesis. MUFAs are the major 172 fatty acid species in mammals and fundamental constituents of diacylglycerols (DAGs), phospholipids, TAGs, and CHOL esters vital for membrane structure and energy storage [2]. Furthermore, Scd1 performs the rate-limiting step in MUFA formation and its inhibition slows the rate of cell proliferation and decreases cell survival [3-5]. Therefore, the understudied involvement of Scd1 in mechanisms of TCDD-elicited carcinogenesis warrants further examination. However, establishing that Scd1 is not only necessary, but also sufficient for the observed TCDD-elicited increase in inflammation and MUFAs may be an important step prior to conducting carcinogenicity bio-assays. Notably, studies in Scd1 null mice identified increased hepatic MUFAs even though no Scd1 activity was detected (Chapter 3). Diet and 14 C studies demonstrated for the first time that these MUFA increases were due to TCDD enhanced lipid uptake from the diet. Yet, it remains unclear whether increased hepatic lipid content resulted from enhanced intestinal absorption, hepatic clearance from the serum, or both. To clarify, intestinal lipoprotein secretion rate in vivo could be measured [6-8] by tracking 14 C-oleate gavage immediately followed by injection with Triton WR-1339 (500 mg/kg), a detergent that forms micelles around lipid particles preventing their hydrolysis and absorption [9]. Serum 14 C secretion rates would indicate whether TCDD increases dietary lipid absorption with implications for dioxin as an emerging factor in the obesity epidemic [10, 11]. Lipid balance is tightly regulated since its alteration influences cellular functions. GC-MS analysis conducted throughout Chapters 3-5 identified significant treatment effects on FA composition linked to gene expression changes. It is not known, however, whether these TCDDelicited FA changes affect the lipid signatures of free FA, TAG, DAG, CHOL ester, and 173 phospholipids, important complex lipids regulating signaling, membrane integrity, and lipid secretion and storage. Future comprehensive lipidomic analyses coupled with gene expression, ChIP-chip, and computational DRE distribution data [12] should provide the appropriate framework for hypothesis generation and confirmation by focused studies. In conclusion, although several epidemiological [13-20] and rodent [21-24] studies have linked dioxin exposure to lipid abnormalities and increased risk of metabolic disease, a variety of factors obscure their relevance and impact on human health. Exposures rarely occur in isolation and seldom affect a genetically homogenous population. 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