Obesity, defined as abnormal or excessive fat accumulation, has become a global epidemic. Although some obese individuals are metabolically healthy a majority develop several co-morbidities such as type II diabetes, hypercholesterolemia, and cardiovascular disease. Diet and exercise can be effective strategies to lose weight, but many individuals have difficulty making lifestyle changes. Thus, new therapeutic strategies are needed to combat the growing prevalence of obesity. Our lab has focused on understanding the biological function of brown adipose tissue (BAT) and beige adipose tissue (BeAT), which are promising therapeutic tissues to combat obesity and obesity related diseases. Unlike white adipose tissue which serves a purpose of storing energy for later use, BAT and BeAT catabolize fat and glucose, uncoupling the electron transport chain from ATP production, instead generating heat. BAT and BeAT depots are primarily activated by cold exposure via β-adrenergic receptor signaling. Targeting β-adrenergic signaling to activate BAT and BeAT was a promising therapeutic approach, however β-adrenergic receptors are also found in the heart preventing widespread therapeutic use. A deeper biological understanding of BAT and BeAT may lead to new therapeutic targets. Our lab previously uncovered a novel role for actomyosin mediated-tension facilitating thermogenesis in BAT. This novel mechanism became the basis for three projects described within this dissertation. First, we probed the importance of type II myosin activity in BeAT development and thermogenic function; type II myosin 9 (Myh9) was found to regulate UCP1 mRNA expression, mitochondrial function, and lipid metabolism. Second, in collaboration with the company Atomwise we screened novel type II myosin activators in BAT to determine whether pharmacologically activating type II myosins could increase thermogenesis. Third, we sought to understand the function of the mechanosensitive transcriptional paralogs YAP and TAZ in regulating BeAT differentiation.

Bioluminescence imaging (BLI) is a powerful technology for studying molecular and cellular features in living animals. Impressive advances in luciferase engineering and ease of targeting by genetic encoding permit the broad use of bioluminescence for in vivo imaging for diverse purposes. First, we generated transgenic mice with liver-specific expression of luciferase and used BLI with a FFA probe to enable non-invasive real-time imaging of FFA flux in the liver. Our approach enabled us to observe the changes in FFA hepatic uptake under different physiological conditions in live animals. We found that our novel imaging system is a useful and reliable tool to study the dynamic changes in hepatic FFA flux in preclinical model systems.

We also utilize this imaging approach to study the role of fatty acid transport proteins in hepatic carcinogenesis. We generated a genetically engineered liver cancer mouse model via hydrodynamic transfection and determined the changes of fatty acid uptake during tumor formation. Our results demonstrated that the rate of FFA uptake is significantly decreased during the development of hepatocellular carcinoma (HCC), but not that of intrahepatic cholangiocarcinoma (ICC). We additionally showed that FATP5 knockdown suppresses ICC tumor formation and that the down regulation of FATP5 expression delays tumor growth even after the onset of ICC. These findings uncovered the critical role of FATP5 in ICC development and demonstrated the potential utility of these proteins as therapeutic targets for the prevention and treatment of ICC.

We expanded our bioluminescent imaging approaches to other nutrients and copper in particular. We demonstrated Copper-Caged Luciferin-1 (CCL-1), a bioluminescent copper-responsive probe can detect physiological changes in labile Cu+ levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of non-alcoholic fatty liver disease (NAFLD) reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain.

Lastly, we also utilized bioluminescent approaches to detect liver damage and apoptosis. We shown that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. We applied this "split luciferin" ligation reaction to the imaging of apoptosis associated with caspase 3/7. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. These imaging approaches for nutrient fluxes should open up our understandings of complex metabolic networks in various metabolic processes and diseases states.

Metabolic reprogramming is a common feature of cancers with many showing specific alterations in both micro and macronutrient metabolism. In this study we evaluate the utility of bioluminescent technology in evaluating the metabolism of liver cancers in vivo. We assess both nutrient uptake and nutrient sensing probes to establish the handling and/or concentrations of lipids, glucose, nicotinamide riboside, iron, copper, and ROS in both healthy mice and tumor bearing mice. Our studies showed promise for the lipid, copper, iron, and ROS probes and were able to measure metabolic differences in copper and lipid metabolism between our different cancer models.

We evaluated both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) models that were established through a hydrodynamic injection. Our bioluminescent study revealed a fatty acid uptake phenotype in our ICC model, but not our HCC models, which is a rare but increasingly documented phenotype. Classical cancer lipid metabolism focuses on the upregulation of de novo lipogenesis concomitant with a decreased dependence on exogenous fatty acids. Increased expression and activity of fatty acid synthase (FASN), the rate-limiting enzyme involved in de novo lipogenesis, is required for the survival and proliferation of many tumor cells, including hepatocellular carcinoma (HCC). Another study previously demonstrated that ICC development is insensitive to FASN deprivation. Our observation that ICC maintains robust fatty acid uptake rates suggests a role of exogenous fatty acids for the growth of ICC. Fatty acid transport proteins (FATPs), now classified as solute transporter family 27 (Slc27a1-6), are major transmembrane proteins for LCFA uptake and FATP2 and -5 are robustly expressed in the liver, FATP5 being liver specific. Using genetic manipulation and in vivo bio-imaging techniques, we measured the growth of ICC in an FATP5 knock out mouse model over time. Loss of FATP5 significantly impaired ICC growth indicating that tumor growth is dependent on exogenous fatty acid uptake. This was replicated using a knockdown system in both our ICC and HCC models, and only in ICC did we see a growth impairment. We also performed a survival experiment to see how long the growth effect in ICC was maintain. Over the course of 32 week, only three out of 7 mice grew robust tumor and when assayed for lipid uptake and metabolizing genes, two showed upregulation of FASN.

Metabolomic were also performed to determine the fate and function of these fatty acids comparing ICC, HCC, and non-tumorous liver. Our data showed a robust increase in free fatty acids, structural, signaling lipids, and acyl-carnitines suggesting a differential handling of exogenous versus endogenous fatty acids. We attempted to follow up on the increased levels of acyl-carnitines, which are implicated in beta-oxidation and energy generation. We treated mice with CPT1 inhibitor etomoxir and saw no significant growth effect. While it is obvious ICC is dependent on fatty acid uptake, and the inhibition of uptake is able to prevent tumor growth, the differential handling of the downstream fatty acids is more complex. In sum, this study has identified FATP5 as a therapeutic target for the treatment of ICC and potentially other liver cancers dependent on protein mediated exogenous fatty acid uptake.

The prevalence of obesity and diet-related diseases is widespread and increasing. Fatty acids have vital biological functions in the body, but can become pathophysiological in many contexts. It is well established that fatty acid uptake into cells is a regulated process mediated by membrane-bound fatty acid transporters. Gain- and loss-of-function studies of fatty acid transport proteins (FATPs) have demonstrated the essential role of fatty acid transporters in maintaining normal fatty acid metabolism and have also indicated that these proteins are involved in the development of diet-related diseases. We do not fully understand the physiological roles of each FATP in every metabolic context. The work presented here focuses on FATP1 and FATP6 activity in the heart and endocrine pancreas, particularly in the context of diabetes.

In Chapter 1, the creation of a FATP6 knockout mouse model that we utilized to study FATP6 activity and function is reported. We found that FATP6 mediated fatty acid uptake in vivo and was specifically responsible for fatty acid availability in the heart. Deletion of FATP6 resulted in reduced cardiac lipid levels, cardiac dilation, reduced systolic function, and elevated rates of apoptosis in cardiomyocytes. This phenotype was rescued by high-fat diet feeding. While the location and mechanism of FATP6 activity have not been fully defined, we hypothesize that lack of FATP6 expression leads to reduced cardiac fatty acid utilization, which in turn leads to reduced cardiac function.

In Chapter 2, our efforts to determine the role of FATP1 and FATP6 in the development of diabetic cardiomyopathy are described. Diabetic cardiomyopathy is a heart condition characterized by enhanced fatty acid utilization. We induced diabetes in wild-type and FATP1 and FATP6 knockout mice by feeding them a high-fat diet and injecting them with two low-doses of streptozotocin. This protocol produced hyperglycemia but did not result in a robust model of diabetic cardiomyopathy. Due to the lack of a strong phenotype in the heart, we did not detect significant differences in cardiac metabolism or function with deletion of FATP1 or FATP6.

In Chapter 3, a novel role for FATPs in the endocrine pancreas is presented. FATPs are differentially expressed in pancreatic islets, with FATP1 localizing to beta cells. We used islets from FATP1 knockout mice to analyze how FATP1 expression affected the susceptibility of beta cells to lipotoxicity. Deletion of FATP1 protected beta cells from palmitate-induced apoptosis, despite normal levels of fatty acid uptake and palmitate-induced ER stress in FATP1 knockout islets. Although the mechanism explaining this outcome is not clear, beta cells from FATP1 knockout mice may differentially store neutral lipid and this may make the cells less susceptible to palmitate-induced cell death.

In Chapter 4, our attempts to characterize small molecule inhibitors of FATPs are portrayed. We tested the ability of two classes of potential FATP inhibitors to reduce FATP activity in FATP-overexpressing cells and in cells that endogenously express FATPs. Phospholipid-based inhibitors did not reduce FATP-mediated fatty acid uptake at feasible concentrations. A dihydropyrimidone-based inhibitor effectively reduced FATP1- and FATP4-mediated fatty acid uptake in cell models overexpressing these proteins but did not affect endogenous FATP1 or FATP4 activity in other cell models.

Taken together, these results contribute to the growing body of evidence that establishes a role for FATPs in fatty acid transport in the body as well as in the development of diet-related diseases. Specifically, this work shows novel roles for FATP6 in cardiac metabolism and function and FATP1 in pancreatic beta cell metabolism and function. Clearly, FATPs are important regulators of metabolism, are implicated in metabolic disorders, and should be extensively studied as potential therapeutic targets.

Innovative approaches to shift energy balance are urgently needed to combat metabolic disorders such as obesity and diabetes. One promising approach has been the expansion or activation of thermogenic adipose tissues to improve metabolic homeostasis. My doctoral studies presented in the following text have identified novel approaches to translate adipose based metabolic therapeutics and the underlying mechanisms by which thermogenic adipocytes establish their therapeutically applicable metabolic capacity.

In chapter I, I present a novel biomaterial technology optimized to expand metabolically beneficial thermogenic adipose depots in vivo. This system enabled me to determine the degree of metabolic enhancement possible with the exogenous expansion of thermogenic adipose depots. To generate therapeutic adipose implants I modified hyaluronic acid-based hydrogels to support the differentiation of white fat derived multipotent stem cells (ADMSCs) into lipid accumulating, uncoupling protein 1 (UCP1) expressing thermogenic adipocytes. Subcutaneous implantation of the synthetic tissues successfully attracted host vasculature and persisted for several weeks and the implant recipients demonstrated elevated core body temperature during cold challenges, enhanced respiration rates, improved glucose homeostasis, and reduced weight gain demonstrating the therapeutic merit of this highly translatable approach.

In chapter II, I outline the experimentation leading to the discoveries presented in chapter III as well as thoroughly review pertinent tissue engineering strategies. Specifically, I sought to define the mechanism by which synthetic ECM components identified in chapter I could alter differentiation outcomes of preadipocytes to yield greater thermogenic capacity.

In chapter III, I demonstrate that actomyosin-based mechanical responses provide a critical differentiation cue for the development of thermogenic adipocytes. Since I had determined that the hydrogel optimization techniques described in chapter I were likely acting through cytoskeletal-mediated processes I examined the role of cytoskeletal structure and tension in thermogenic adipose development. I identified that the muscle-like gene expression patterns of UCP1+ adipocytes are critical for the acute induction of oxidative metabolism and uncoupled respiration and regulate mechanosensitive transcriptional co-activators, YAP/TAZ, that control thermogenic gene expression.

This dissertation establishes the role of physical mechanics in the development and function of thermogenic adipocytes which may engender future metabolic therapeutics.

The melanocortin 4 Receptor (MC4R) is a central component of the leptin-melanocortin pathway in the hypothalamus. MC4R plays a critical role in the long-term regulation of energy homeostasis and mutations in MC4R are the most common cause of monogenic obesity. However, the precise molecular and cellular mechanisms underlying the maintenance of energy balance within MC4R expressing neurons are unknown. The expression of MC4R in neurons from the Paraventricular Nucleus of the hypothalamus (PVN) has been shown to be both necessary and sufficient to regulate food intake and body weight, and we discovered that MC4R localizes at the primary cilium of those neurons. Primary cilia are unique microtubule-based organelles that protrude from the cell membrane to sense and relay extracellular signals. In Chapter one, we first mapped the expression of MC4R in the brain, to then determine the extent of MC4R localization at the primary cilia in some of the identified regions. We discovered that, in the PVN, MC4R localization at the primary cilium is dynamic and depends on age and physiological status relayed by MC4R ligands levels. Together, these results shed light on the complex regulation of MC4R bioavailability at the primary cilium. In Chapter two, we found that cilia were required specifically on MC4R-expressing neurons for the control of energy homeostasis. Moreover, these cilia were critical for pharmacological activators of MC4R to exert an anorexigenic effect. Using a combination of mouse genetic approaches, we demonstrated that MC4R signals via cilia of PVN neurons to control food intake and body weight. In Chapter three, we demonstrate that the Melanocortin Receptor Associated Protein 2 (MRAP2) is critical for the ciliary localization and weight-regulating function of MC4R. These findings demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggests that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.

Translocase of outer mitochondrial membrane 40 (TOMM40, aka TOM40) is the major channel-forming subunit of the translocase of outer mitochondrial membrane (TOM) complex that regulate mitochondrial function by importing nuclear-encoded proteins. TOMM40 is located at mitochondria-endoplasmic reticulum contact sites (MERCs), which play a key role in cellular signaling, with known effects on mitochondria bioenergetics and lipid synthesis. Furthermore, we have used RNA-seq to assess global transcriptional response to statin exposure in immortalized lymphoblastoid cell lines (LCLs) derived from participants in a clinical trial who were treated with 40mg/day of simvastatin for 6 weeks. Our previous studies using this model system identified a number of novel genes affecting cholesterol metabolism and in vivo statin LDL (low-density lipoprotein) cholesterol response, including TOMM40.

Statins are the drugs most commonly used for lowering plasma LDL cholesterol and cardiovascular disease risk. Although generally well tolerated, statins can induce myopathy, a major cause of non-adherence to treatment. Cellular studies have identified statin-induced myopathy to be associated with impaired mitochondrial function and morphology. In Chapter 2, we show that TOMM40 and TOMM22 play a key role in mediating statin-induced mitochondrial dysfunction as well as disruption of mitochondrial dynamics in skeletal myotubes. These findings suggest a novel mechanism by which adverse effects of statin treatment on mitochondrial dynamics that may contribute to the development of statin-induced myopathy are mediated by reduced TOMM40 and TOMM22 gene expression.

Beyond the mitochondria, we explore the role of TOMM40 on regulation of lipid metabolism via MERCs. Fundamentally, the gene encoding TOMM40 is adjacent to that for APOE, a protein with central functions in lipid and lipoprotein transport. Recent GWAS studies have shown that a number of SNPs in linkage disequilibrium between these two genes are significantly associated with plasma lipid levels. While these associations have generally been attributed to APOE, no previous studies have addressed the possibility that TOMM40 has an independent effect on lipid metabolism. In Chapter 3, our investigation of molecular mechanisms underlying the effects of TOMM40 knockdown on lipid metabolism has revealed a number of novel findings, including a key role for the LXR transcription factor due both to transcriptional upregulation and increased activation by oxysterols that are generated by mitochondria-induced reactive oxygen species. Interestingly, the increased LXR activity also results in reduced intracellular cholesterol via both upregulation of the membrane cholesterol transporter ABCA1 and increased production and secretion of oxysterol-derived cholic acid. Finally, and importantly, we have shown using a number of experimental approaches that the LXR-mediated effects described above result from disruption of mitochondria-ER contact sites (MERCs), where TOMM40 plays a key role, and that lipid droplet contact sites are consequently shifted from the ER to mitochondria.

Overall, these findings demonstrate TOMM40 plays a key role in connecting mitochondrial function and lipid metabolism.

Obesity and the metabolic diseases that result from it continue to increase across the developing and developed world little slow down in sight. As such, novel mechanisms for the study and treatment of these metabolic outcomes are needed to combat the ever-increasing epidemic. Our lab has focused on the development of two mechanisms geared toward the understanding of adipose tissue’s role in these abnormal metabolic processes through the development of an in situ forming implant whose goal is to burn excess circulating glucose and lipids, and through the characterization of a microphysiological device (MPS) to model insulin resistance formation in adipocytes. We have found that our hyaluronic acid (HA)-based implants are ideal candidates to perform the three main goals of the therapeutic implant showing robust capabilities to perform the three main goals: recruit stem cells, differentiate those stem cells to a beige adipocyte fate, and then maintain activation of these cells over a long period of time. Using two well characterized chemistries, thiol-acrylate Michael addition and Strain Promoted Azide Alkyne click (SPAAC), we can tailor our hydrogels to the targeted niche using short adhesion peptides, growth factors, degradable crosslinkers, and compounds that bind receptors on a cell’s surface. To this end we have shown that molecular weight plays no role in adipocyte function in vitro, but altering the hydrogel in increase the loss, or viscous, modulus of a hydrogel can. In addition, peptides and growth factors seem to enhance the differentiation of the cells plated on these substrates in ways that may provide further tailoring opportunities down the road for this therapeutic. I also created a bifunctional HA version of our implant with the goal of pushing forward aim three of the hydrogel project. Modifying the HA backbone with a DBCO group would allow us to conjugate in a tri-azole modified compound like Mirabegron, a β3-adrenergic agonist responsible for the differentiation of beige adipocytes. I was able to illustrate the efficacy of this bi-chemical modification and determine the final ability of the gels to perform in both capacities, which will be crucial for the beige adipocyte therapeutic in the future. Finally, we created a MPS that reproduces obese metabolic disease states in adipose tissue using induced pluripotent stem cells (iPSC) to differentiate them toward adipocytes and macrophages, modelling the interactions and development of these two cell types to validate this method as a testable platform for insulin resistance.