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Liver injury instigates a proinflammatory response in tissue-resident macrophages, called Kupffer cells (KCs), resulting in the recruitment of monocytes and neutrophils. The high energy demand required for a rapid proinflammatory response in macrophages like KCs is achieved through metabolic reprogramming. This is supported by increased glycolysis. On the other hand, injury resolution requires hepatic macrophages to undergo an anti-inflammatory polarization, which relies on oxidative phosphorylation (OXPHOS). In addition to shifts in mechanisms of adenosine triphosphate (ATP) production, lipid metabolic reprogramming supplies metabolic intermediates and lipids for membrane remodeling and the production of inflammatory mediators. AMP-activated protein kinase (AMPK) is a master metabolic regulator that influences the metabolic reprogramming of macrophages. While AMPK activation promotes an anti-inflammatory polarization, disruption of activity exacerbates proinflammatory signaling. For this thesis work, we addressed whether macrophage AMPK is protective against liver injury by altering immunometabolism. Specifically, we investigated this question in the context of chronic (nonalcoholic steatohepatitis (NASH)) and acute (acetaminophen (APAP) overdose) liver injury. While APAP overdose is a robust and directly translational model of acute injury, models of NASH-induced hepatic fibrosis rely on nutrient-deficient diets like the choline-deficient high-fat diet (CDAHFD) or genetic manipulation. Despite the utility of these models, they seldom mirror the pathogenesis of human NASH, with diets like CDAHFD being completely dissociated from metabolic syndrome. Moreover, models are required to address the divergence between male and female mice. Recently, there has been a shift towards addressing other variables that drive inflammation and metabolism. At room temperature (RT) (22 °C), mice experience cold stress that alters various biological functions. Cold stress drives brown adipose tissue (BAT) activation and upregulates corticosterone production and immunosuppression, all processes that blunt NASH progression. Giles et al. (2016) demonstrated that housing mice at thermoneutrality (TN) (30 °C) exacerbated metabolic-dysfunction associated fatty liver disease (MAFLD) progression toward NASH in both male and female mice. Since then, we and others have implemented TN housing with different dietary interventions and mice strains. We determined that 16-week Western diet (WD) feeding of male and female mice at 29 °C was insufficient to drive hepatic fibrosis, however alterations in glucose tolerance and elevated liver injury enzymes as well as profibrotic gene expression in male mice may indicate that a longer timeline is necessary (24 weeks). Given that our TN NASH model did not produce hepatic fibrosis, we implemented the CDAHFD to investigate macrophage AMPK in chronic liver injury. Male and female AMPK Flox (Prkaa1 fl/fl/Prkaa2 fl/fl) and MacKO (Flox-LysM-Cre+) mice were fed CDAHFD for 8 weeks. In this time frame, CDAHFD produces a lean euglycemic phenotype with hepatic steatosis, inflammation, and fibrosis, to which AMPK MacKO had no influence. Moreover, intervention with a low dose of metformin had no effect, contrary to the reduction in hepatic steatosis observed in HFD-fed mice. Although macrophage AMPK is dispensable in the CDAHFD model of chronic liver injury, acute liver injury needed to be addressed. We found that priming with systemic activation of a direct AMPK activator MK-8722 did not influence hepatic injury and necrosis in our model of APAP-induced liver injury (AILI). Moreover, deletion of hepatocellular AMPK (Flox-Alb-Cre+) or AMPK MacKO did not influence injury at 24 hours post overdose. Despite the lack of effect of systemic AMPK activation, we were interested in a nanoparticle-based targeting of direct AMPK activator MK-8722 (NP-MK8722) delivery. We determined that PLGA-PEG nanoparticles (NPs) accumulated in hepatic macrophages as early as 2 hours post-injection, but NP-MK8722 did not alter hepatic necrosis, injury, or immune infiltration. Overall, my thesis work has advanced our knowledge of the effects of housing temperatures on NASH pathogenesis. Moreover, we are the first to address the effects of macrophage AMPK signaling in NASH and AILI. This is especially true for assessing how AMPK deficiency and targeted activation influences KC immunometabolism during injury.

<p>In obesity, nonalcoholic fatty liver disease (NAFLD) has been associated with the development of hepatic insulin resistance. Acetyl coA carboxylase (ACC), which exists as two separate isoforms (ACC1 and ACC2), is an important metabolic enzyme which controls the production of the metabolic intermediate malonyl coA, and hence, fat metabolism. AMP-activated protein kinase (AMPK) has been shown to inhibit ACC activity by phosphoryating ACC1 at Ser79 and ACC2 at Ser221. The objectives of this were to determine the physiological importance of AMPK phosphorylation of ACC as it relates to the development of NAFLD and insulin resistance.</p> <p>We examined the metabolic phenotype of C57Bl6 mice with a targeted ACC1 Ser79 to Ala and ACC2 Ser221 to Ala double knock-in mutation (ACC DKI), which would inhibit AMPK phosphorylation of ACC and compared them to wild-type (WT) mice. Basic body characteristics, assessment of insulin sensitivity, and assessment of liver steatosis were used.</p> <p>ACC DKI body mass and energy expenditure were not different compared to WT. Liver ACC activity and malonyl coA were higher in ACC DKI mice. The livers of ACC DKI mice displayed greater triacylglycerol accumulation and aggregation of neutrophils. ACC DKI mice were insulin resistant as shown by: higher fasting blood glucose and insulin, glucose and insulin intolerance, liver insulin resistance, and impaired insulin-stimulated glucose disposal rate.</p> <p>In summary, we have shown that the phosphorylation of ACC1 Ser79 and ACC2 Ser221 is critical for maintaining ACC activity and malonyl coA levels in the liver. The dysregulation of this pathway results in liver fat accumulation and the development of insulin resistance. These studies demonstrate that AMPK phosphorylation of ACC is essential for maintaining metabolic homeostasis.</p> / Master of Science (MSc)

<p>Current evidence suggests that both prediabetes and diabetes can reverse to normoglycemia; however, predictors of remission of these conditions are poorly understood. We performed analyses on 1,209 people with impaired fasting glucose and/or impaired glucose tolerance treated with placebo rosiglitazone and placebo ramipril in the DREAM trial. Normoglycemia was defined as a fasting plasma glucose <5.6 mmol/L and 2-hour plasma glucose <7.8 mmol/L on a 75 g oral glucose tolerance test (OGTT).</p> <p>The effects of baseline ALT and waist to hip ratio (WHR) on regression of prediabetes to normoglycemia 2 years later were found to be interdependent (p-value for interaction 0.01). Adjusted odds ratios ORs (95% CI) of regression to normoglycemia per 10 U/L increase in ALT were 0.79 (0.66-0.94) when WHR was at the mean minus 1 standard deviation (SD), 0.90 (0.80-1.02) when WHR was at the mean of 0.91, and 1.03 (0.90-1.18) when WHR was at the mean plus 1 SD. Adjusted ORs of regression to normoglycemia per 0.1 unit increase in WHR were 0.75 (0.60-0.95) when ALT was at the mean minus 1 SD, 0.91 (0.76-1.08) when ALT was at the mean of 25 U/L, and 1.09 (0.89-1.35) when ALT was at the mean plus 1 SD.</p> <p>Similarly, the effects of baseline ALT and WHR on AUC_{glucose0-120 min} obtained from the OGTT were found to be interdependent (p-value for interaction 0.056). A 10 U/L increase in ALT was associated with an adjusted AUC_{glucose0-120 min} increase of 19.5 (95% CI 5.3 to 33.7) min*mmol/L when WHR was at the mean minus 1 SD, 11.0 (1.4 to 20.6) min*mmol/L when WHR was at the mean of 0.91, and 2.5 (-9.2 to 14.1) min*mmol/L when WHR was at the mean plus 1 SD. A 0.1 unit increase in WHR was associated with an adjusted AUC_{glucose0-120 min} increase of 30.3 (10.2 to 50.3) min*mmol/L when ALT was at the mean minus 1 SD, 18.3 (3.8-32.9) min*mmol/L when ALT was at the mean of 25 U/L, and 6.4 (-11.5 to 24.3) min*mmol/L when ALT was at the mean plus 1 SD.</p> <p>In conclusion, high baseline ALT and WHR predict a lower likelihood of regression of prediabetes to normoglycemia and an increase in AUC_{glucose0-120 min} 2 years later; however, the effects of ALT and WHR on these outcomes are interdependent.</p> / Master of Science (MSc)

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.