Open Huang KH Dissertation Final.Pdf

Open Huang KH Dissertation Final.Pdf

The Pennsylvania State University The Graduate School The College of Health and Human Development EXOGENOUS LIPIDS REGULATE THE DEVELOPMENT OF HEPATIC STEATOSIS IN A LEAN NAFLD MODEL-FED A HIGH CARBOHYDRATE DIET A Dissertation in Nutritional Sciences by Kuan-Hsun Huang ã 2016 Kuan-Hsun Huang Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2016 The dissertation of Kuan-Hsun Huang was reviewed and approved* by the following: A. Catharine Ross Professor of Nutrition and Physiology Dissertation Advisor Chair of Committee Michael H. Green Professor of Nutrition and Physiology Interim Department Head Connie J. Rogers Associate Professor of Nutrition and Physiology Andrew D. Patterson Associate Professor of Molecular Toxicology Rebecca L. Corwin Professor of Nutritional Neuroscience Professor in Charge, Graduate Program in Nutritional Sciences *Signatures are on file in the Graduate School. ii ABSTRACT Non-alcoholic fatty liver disease (NAFLD), an umbrella term that encompasses hepatic steatosis, steatohepatitis, fibrosis, and cirrhosis, has become the most common chronic liver disease in the developed countries. NAFLD has been shown to be positively associated with obesity, and thus not surprisingly the majority of NAFLD studies have utilized obese models to explore NAFLD causality. However, previous studies indicated that 25% of patients with NAFLD are not obese and that 7.4% of lean adults have steatosis, and they are more likely to be younger and female, suggesting that these people with lean NAFLD were metabolically obese. Additionally, insulin resistance is an independent risk factor to the development of lean NAFLD, which has been shown to be related to cardiovascular disease and diabetes mellitus. Therefore, understanding the etiology of lean NAFLD in the early stage of the development of steatosis becomes an urgent need. On the other hand, lifestyle modification intervention including diet and physical activity is believed to improve NAFLD or even reverse it, but very few studies have focused on the reversal of hepatic steatosis in the lean NAFLD model. Thus, the overall research hypothesis in this dissertation is that the exogenous lipids as a form of lipid emulsion (LE) and physical activity are capable to reverse hepatic triacylglycerol (TG) accumulation in the lean mouse model with preexisting steatosis. Previous work in our laboratory indicated that 13.5% (percent of total energy, en-%) Intralipid® given orally ameliorated TG accumulation in the liver of nonobese mice fed a high carbohydrate diet (HCD) for 5 weeks. Here, in my first study (chapter 3) I examined whether HCD can induce hepatic steatosis in a short period of time (8d) and whether Intralipid® and voluntary exercise can prevent liver triacylglycerol (TG) accumulation by iii regulating the de novo lipogenesis-associated transcripts and the concentrations of total fatty acids, in 8 d, on the development of steatosis in a lean mouse model. The results revealed that hepatic TG contents in the HCD-fed mice were significantly increased, confirmed by Oil Red O staining, suggesting the 8d period of induction of steatosis was sufficient to induce mild steatosis. Supplementation with 13.5% Intralipid®, with or without exercise, also suppressed HCD-induced steatosis. qRT-PCR analysis showed that including 13.5% Intralipid® to the HCD significantly decreased the transcript levels for lipogenesis-associated genes, whereas mice-fed HCD with exercise had less beneficial effect in the early stage of steatosis, as compared to HCD supplemented with 13.5% Intralipid®. Fatty acid profiling also showed a consistency with transcriptional data that the concentration of monounsaturated FA was decreased significantly. As noted in the previous study that HCD is capable to induce mild hepatic steatosis within 8d, I conducted a second study (chapter 4) to test whether the beneficial effect contributed by 13.5% Intralipid® supplementation will be extended to the mouse with preexisting steatosis. To investigate this, the mice were fed a HCD for 2.5 weeks to establish hepatic steatosis, then switched to HCD+13.5%LE for the final 2.5 wk. A combined targeted biochemical and untargeted metabolomics approach was employed, and biochemical analyses revealed that hepatic TG level and the lipogenic genes were significantly decreased in mice fed HCD with 13.5% Intralipid®. Total fatty acids and metabolomic profiles in liver and plasma indicated that the mice fed a HCD supplemented with 13.5% Intralipid® exhibited reduced hepatic lipogenesis, but increases in hepatic Krebs cycle intermediates, and in plasma very-low density lipoprotein. iv Many studies have shown that n-3 and n-9 FA have beneficial effects on lipid metabolism. In a third study (chapter 5), I compared 3 LEs-- Intralipid®, Omegaven® and ClinOleic®-- for their ability to reverse hepatic TG accumulation after the onset of steatosis. At this time, we used biochemical, transcriptomic and metabolomic approaches to capture an overall metabolic change in the lean NAFLD mouse model. The results showed that HCD-induced hepatic steatosis did not develop insulin resistance and that 13.5% Omegaven® had the strongest reversal effect on hepatic TG accumulation, as well as on the genes involved in the hepatic glucose and lipid metabolism, which can further maintain energy homeostasis in the mice with preexisting steatosis. Additionally, lipolysis in the adipose tissue was greatly improved by introduction of Omegaven®; the metabolites, assayed by nuclear magnetic resonance spectroscopy, were consistent with the transcriptomic data, suggesting a plausible mechanism of how n-3 FA-based LE reversed hepatic steatosis. In summary, adding 13.5% Intralipid® to a HCD not only prevents the development of steatosis in the early dynamic stage by reducing de novo lipogenesis, but also mitigates the preexisting hepatic steatosis and energy homeostasis in the liver in a 5-wk study. However, Omegaven® provides beneficial effect on the reversal of preexisting steatosis as well as an improvement on energy homeostasis in both liver and adipose tissue. v TABLE OF CONTENTS List of Figures .................................................................................................................... x List of Tables .................................................................................................................. xiii List of Abbreviations ..................................................................................................... xiv Acknowledgment .......................................................................................................... xviii Chapter 1 Review of the Literature .............................................................................. 1 1.1 Non-alcoholic fatty acid disease (NAFLD) .............................................................. 1 1.1.1 NAFLD Epidemiology ....................................................................................... 1 1.1.2 Diagnosis of NAFLD ......................................................................................... 2 1.1.3 The pathogenesis of NAFLD ............................................................................. 4 1.1.4 The treatment of NALFD ................................................................................... 8 1.2 Lipid metabolism ...................................................................................................... 9 1.2.1 Digestion and absorption of lipid ....................................................................... 9 1.2.2 Lipid transport .................................................................................................. 10 1.2.3 NAFLD and lipid metabolism ......................................................................... 12 1.3 NAFLD and lipid supplementation ......................................................................... 13 1.3.1 NAFLD and n-6 fatty acids .............................................................................. 13 1.3.2 NAFLD and n-3 fatty acids .............................................................................. 14 Chapter 2 Hypotheses and Objectives ........................................................................ 16 2.1 Hypotheses .............................................................................................................. 17 2.2 Specific aims ........................................................................................................... 17 2.2.1 Specific aim to the hypothesis 1 ...................................................................... 17 vi 2.2.2 Specific aim to hypothesis 2 ............................................................................ 17 2.2.3 Specific aim to the hypothesis 3 ...................................................................... 18 Chapter 3 Lipid Emulsion and Voluntary Exercise Reduce Lipogenesis and Ameliorate Early-Stage Hepatic Steatosis in High Carbohydrate Diet-Fed Mice .... 19 3.1 Materials and Methods ............................................................................................ 19 3.1.1 Animal protocol ............................................................................................... 19 3.1.2 Diets and Study Design .................................................................................... 19 3.1.3 Hepatic TG quantification ................................................................................ 22 3.1.4 RNA isolation and quantitative

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