Indiana University-Purdue University Indianapolis (IUPUI) / This thesis explores a minimally invasive approach of diagnosing Non-Alcoholic Fatty Liver disease (NAFLD) on mice and humans which can be useful for pathologists while performing their diagnosis. NAFLD is a spectrum of diseases going from least severe to most severe – steatosis, steatohepatitis, fibrosis and finally cirrhosis. This disease primarily results from fat deposition in the liver which is unrelated to alcohol or viral causes. In general, it affects individuals having a combination of at least three of the five metabolic syndromes namely, obesity, hypertension, diabetes, hypertriglyceridemia, and hyperlipidemia. Given how common these metabolic syndromes have become, the rate of NAFLD has increased dramatically over the years affecting about three-quarters of all obese individuals including many children, making it one of the most common diseases in United States. Our study focuses on building various computational models which help identify different histological features in a liver biopsy image, thereby analyzing if a person is affected by NAFLD or not. Here, we develop and validate the performance of automated classifiers built using image processing and machine learning methods for detection of macro- and microsteatosis, lobular and portal inflammation and also categorize different types fibrosis in murine and human fatty liver disease and study the correlation of automated quantification of macrosteatosis, lobular and portal inflammation, and fibrosis (amount of collagen) with expert pathologist’s semi-quantitative grades. Our research for macrosteatosis and microsteatosis prediction shows the model’s precision and sensitivity as 94.2%, 95% for macrosteatosis and 79.2%, 77% for microsteatosis. Our models detect lobular and portal inflammation(s) with a precision, sensitivity of 79.6%, 77.1% for lobular inflammation and 86%, 90.4% for portal inflammation. We also present the first study on identification of the six different types of fibrosis having a precision of 85.6% for normal fibrosis and >70% for portal fibrosis, periportal fibrosis, pericellular fibrosis, bridging fibrosis and cirrhosis. We have also quantified the amount of collagen in a liver biopsy and compared it to the pathologist semi-quantitative fibrosis grade.

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Non-alcoholic fatty liver disease (NAFLD) is a liver disorder linked to obesity that is rapidly increasing in incidence worldwide. It is a disorder that ranges in severity; from a benign condition of hepatic steatosis to a potentially deadly one resulting in cirrhosis and hepatocellular carcinoma. It is currently known that NAFLD is strongly associated with various aspects of metabolic syndrome: insulin resistance, elevated triglyceride levels, obesity, and type two diabetes mellitus. The multifactorial pathogenesis of NAFLD is still uncertain and closer attention is needed on the effect of one’s diet on NAFLD. In this study, we directly compare a westernized diet containing high levels of fat and fructose to a diet high in fat and containing cholate using mouse models in order to determine the role of each dietary factor in the incidence and severity of the different stages of NAFLD. We will evaluate the severity of hepatic steatosis and hepatocellular damage via hematoxylin and eosin (H&E) stained liver tissue and the severity of hepatic fibrosis via trichrome-stained liver tissue. Our hypothesis is that mice on the fructose-based diet are expected to have higher levels of hepatic steatosis and hepatocellular damage relative to mice on the cholate-based diet while mice on the cholate-based diet are expected to have higher levels of hepatic fibrosis relative to the fructose-based diet. The results of this study will aid in elucidating and strengthening the connection between one’s diet and the prevalence and severity of NAFLD.

Using a novel, adipose-liver, two-organ, human-on-a-chip system, the metabolic disease non-alcoholic fatty liver disease was modeled. This model was then used to test the effects of the gut microbiome on NAFLD progression. Two products of the gut microbiome, Trimethylamine-n-oxide and butyrate, were selected as representatives of potentially harmful and potentially beneficial compounds. A dose response, adipocyte and hepatocyte monocultures controls, and HoaC systems were run for 14 days. Through this experimentation, it was found that a dysbiosis of the gut microbiome could be influencing NAFLD progression. Additionally, further development and discovery regarding adipose-liver systems was added to the ongoing conversation of HoaC systems and their usages.

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PhD Dissertation / Obesity is a major risk factor for type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), and is attributed to excess energy intake in comparison to energy expenditure. Therapeutics that reduce energy intake in obesity have limited efficacy, with weight loss typically reaching less than 10% of initial body mass, leading to efforts to uncover new therapies that may increase energy expenditure. Unlike lipid-storing white adipose tissue, brown and beige adipose tissues undergo futile cycling, oxidizing lipids and carbohydrates thereby increasing energy expenditure. With obesity, the metabolic activity of brown and beige adipose tissue is reduced, suggesting that restoring adipose tissue thermogenesis may represent a new means to enhance energy expenditure. Previous studies in mice have shown that peripheral serotonin synthesis by the enzyme tryptophan hydroxylase 1 (Tph1) inhibits adipose tissue thermogenesis and contributes to the development of obesity, insulin resistance and NAFLD. However, the primary Tph1 expressing tissue(s) inhibiting adipose tissue futile cycling is not known. In this thesis, we genetically removed Tph1 in mast cells of mice and discovered that this elevated beige adipose tissue activity protecting mice from developing high-fat diet induced obesity, insulin resistance and NAFLD. In contrast to these findings, genetic deletion of Tph1 in adipocytes did not result in protection from obesity, suggesting that mast cells are the primary source of serotonin that inhibits white adipose tissue thermogenesis. Lastly, to determine the importance of adipose tissue thermogenesis in mediating the beneficial metabolic effects of reduced Tph1, mice were housed at thermoneutrality, blocking the requirement for adipose tissue thermogenesis. Under these conditions, mice lacking Tph1 had comparable brown and beige adipose tissue metabolic activity, energy expenditure and adiposity, however, surprisingly, were still protected from insulin resistance and NAFLD. The studies in this dissertation have discovered that mast cell Tph1 is critical for inhibiting adipose tissue thermogenesis and that serotonin plays an important role in promoting NAFLD, independently of its inhibitory effects on adipose tissue thermogenesis. Collectively, these findings further define the roles of serotonin in regulating whole-body energy metabolism, providing critical clues and mechanistic insights for potential therapies to mitigate metabolic diseases. / Dissertation / Doctor of Philosophy (Medical Science) / Obesity, type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD) can develop when caloric intake exceeds expenditure. In contrast to lipid-storing white fat, brown and beige fat burn calories. Serotonin is a hormone that reduces the burning of calories in fat, therefore finding ways to inhibit its effects on fat tissue without altering serotonin in the brain may lead to new therapies for obesity and other related diseases. In this thesis, we examined potential sources of serotonin that might inhibit the burning of calories in adipose tissue of mice. By reducing the synthesis of serotonin in a white blood cell called mast cells, but not fat cells, mice were protected from obesity, pre-diabetes and NAFLD due to increased activity of beige fat. Moreover, when we kept mice in a warm environment, thus reducing the need for mice to burn calories in brown and beige fat, this eliminated the effects of serotonin to promote obesity, but not pre-diabetes and NAFLD. These studies have identified how serotonin generated from mast cells inhibits the burning of calories in adipose tissue, a finding that may lead to new therapies for obesity, T2D and NAFLD.

Nina Varah MSc Thesis / BACKGROUND: Brown adipose tissue (BAT) has emerged as an attractive target to address the dramatic rise in obesity and non-alcoholic fatty liver disease (NAFLD) in adults and children due to its ability to clear lipids through thermogenesis when activated with cold stimulation. Cross-sectional studies have identified an inverse relationship between BAT and NAFLD in adults, although no linking mechanism or relevance in children is known. Metabolomics provides a non-invasive platform to investigate BAT physiology and its relationship with hepatic fat in an effort to identify potential targets for further investigation. PROJECT OBJECTIVES: 1) To explore the associations between the plasma metabolome and BAT in adults and children. 2) To explore the associations between the plasma metabolome and hepatic fat in adults and children. 3) To identify metabolites associated with both BAT and hepatic fat as potential linking mechanisms for further study. METHODOLOGY: We recruited 63 male and female adults aged 18 to 57 years and 25 healthy male children aged 8 to 10 years into this cross-sectional study. Study participants underwent blood work, body composition measurement (dual energy X-ray absorptiometry; DXA) and magnetic resonance imaging (MRI) - proton density fat fraction (PDFF) measurements of whole liver hepatic fat, pre- and post-cold supraclavicular fat. BAT activity was calculated as the percent change between post and pre-cold BAT PDFF with the cold stimulus consisting of a water-perfused suit maintained at 18°C for 3-hours (adult) or 1-hour (pediatric). Targeted liquid-chromatography/mass spectrometry metabolomics of 102 metabolites was conducted on fasted plasma and multivariate linear regression with multiple testing correction was used to examine metabolite predictors of BAT measures and hepatic fat. RESULTS: In the adult cohort (n=63, median age 25.9 years, median body mass index (BMI) 25.4 kg/m2), five metabolites were associated with baseline BAT lipid content, where an elevated lipid content may indicate a whiter adipose tissue-like phenotype. Aconitate and creatine commonly predict increased baseline BAT lipid content (β=0.420, P=0.001 and β=0.408, P=0.001, respectively), and reduced BAT activity (β=-0.462, P=0.002 and (β=-0.402, P=0.002, respectively). Alanine and two acyl-carnitines also predicted reduced BAT activity. Glutamic acid was similarly related to higher baseline BAT (β=0.480, P<0.001) and hepatic lipid content independent of age and sex (β=0.392, P=0.002). Three other metabolites were directly related to hepatic fat, and serine inversely. In children (n=25, median age 9.89 years, mean BMI Z-score 1.25), cysteine and cystine were trending towards a significant relationship with higher baseline BAT lipid content, and were both related to elevated hepatic fat independent of adiposity (cysteine: quadratic β=-0.714, p<0.001 and cystine: quadratic β=0.592, p<0.001). Two hydroxy-proline isomers and L-carnitine were associated with reduced BAT activity. CONCLUSION: In adults, several metabolites were associated with reduced BAT activity and with a higher baseline BAT lipid content in the non-stimulated state – aconitate and creatine were related to both. Acylcarnitines or their metabolites related to BAT in both children and adults, which may suggest areas for subsequent investigation of BAT metabolism. Glutamic acid in adults and cysteine and cystine in children were weakly related to elevated baseline BAT and hepatic fat content. Further, amino acids such as glutamic acid and cysteine may be markers of increased ectopic fat accumulation – and are also associated with a whiter ambient BAT phenotype. Cumulatively, these findings highlight targets for further investigation into BAT physiology and the link to the liver. / Thesis / Master of Science (MSc)

Obesity and its comorbidities have emerged as serious healthcare concerns in the western world due to increased prevalence of nutritional overabundance and decreased physical activity. Due to the significant population affected and economic burden placed on national healthcare systems, there is a demonstrated need for effective weight management therapeutics. Obesity presents clinically diverse phenotypes that increase a person's susceptibility to comorbidities that commonly result in deteriorated health (cardiovascular disease, diabetes mellitus, hypertension, etc). A comorbidity of specific relevance is non-alcoholic fatty liver disease (NAFLD) and its advanced disease state known as non-alcoholic steatohepatitis (NASH), as it has had a documented rise in prevalence parallel to that observed with obesity. Currently there are no FDA approved therapeutics for NAFLD or NASH, with the majority in clinical development aiming to mitigate the effects caused by accumulation of adipose tissue in the liver known as steatosis. An alternative therapeutic approach is to use small molecules to uncouple oxidative phosphorylation in the mitochondria by passively shuttling protons from the mitochondrial inner membrane space into the mitochondrial matrix. Mitochondrial uncoupling results in the disruption of the proton motive force leading to an upregulation of metabolism (i.e., decrease in steatosis). Small molecule mitochondrial uncouplers have recently garnered great interest for their potential in treating the advanced disease state of NASH. In this study, we report the structure-activity relationship (SAR) profiling of a 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core, which utilizes the hydroxy moiety as the proton transporter across the mitochondrial inner membrane. We demonstrated that a wide array of substituents are tolerated with this novel scaffold that increased cellular metabolic rates in vitro using changes in oxygen consumption rate as a read-out. In particular, compound SHS4121705 (2.12i) displayed an EC50 of 4.3 M in L6 myoblast cells and excellent oral bioavailability and liver exposure in mice. In the preclinical stelic animal model (STAM) mouse model of NASH, administration of 2.12i at 25 mg kg-1day-1 resulted in decreased liver triglyceride levels and improved liver enzymes, NAFLD activity score, and fibrosis without affecting body temperature or food intake. Overall, our initial studies showcased the promise of mitochondrial uncouplers toward the treatment of NASH. While initial results were promising, the lead compound 2.12i had reduced potency compared to the alkyl derivatives reported in the SAR, unfortunately alkyl derivatives suffered from poor physiochemical properties, possibly due to metabolism of the alkyl chain. We hypothesized that addressing metabolic liabilities of these compounds could lead to increased potency with maintained efficacy in the STAM mouse model of NASH. Herein, we detail the SAR profiling of a 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core derivatized with 1,1'-biphenyl anilines capable of eliciting mild mitochondrial uncoupling. A wide array of substituents are tolerated, and demonstrated sustained and stable increases in ¬cellular oxygen consumption rates over a broad concentration range. In particular, compound SHS4091862 (3.9b) displayed an EC50 of 2.0 μM in L6 myoblast cells with a pharmacokinetic profile of Cmax = 46 μM and t1/2 = 4.7 h indicating excellent oral bioavailability. Administration of 3.9b at 60 mg kg-1 day-1 in the STAM mouse model of NASH decreased fibrosis, steatosis, and hepatocellular ballooning to result in a 1.9-point decrease in NAFLD activity score (NAS) compared to vehicle. No changes in food intake, body weight, alanine transaminase (ALT) or aspartate transaminase (AST) levels were observed with 3.9b. Positive control Resmetirom afforded a 1.2-point decrease in NAS score, but increased ALT levels. Cumulatively, our work demonstrates the therapeutic potential of small molecule mitochondrial uncouplers to address metabolic diseases, namely NAFLD. / Doctor of Philosophy / There has been a significant increase in the population suffering from metabolic diseases in the western world. Among the most concerning metabolic diseases are obesity and nonalcoholic fatty liver disease, which have been shown to arise from excessive consumption of calorie dense food and limited physical activity. A novel approach to combat these diseases is to use mitochondrial uncouplers that disrupt the body's natural process for ATP production, causing an increase in metabolism. This increase in the metabolic rate results in the reduction of fat mass including in organs such as the liver. This work describes the design, development, and biological study of mitochondrial uncouplers capable of producing an increase in metabolism; specifically, SHS4121705 (2.12i) and SHS4091862 (3.9b) were shown to be potent uncouplers in vitro and were active in mouse models of fatty liver disease.