Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis. To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes. Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity.

Obesity results from expansion of white adipose tissue. The inability of white adipose tissue to adequately store lipids leads to ectopic deposition of lipids in non-adipose tissue that can lead to systemic insulin resistance. It is well known that insulin resistance correlates with inflammation of adipose tissue in obese animals and humans. Decreasing inflammation in the adipose tissue has been proven as a therapeutic strategy for improvement of insulin sensitivity in vivo. Numerous factors secreted by immune cells, including macrophages, have been suggested as regulating adipose tissue insulin sensitivity. In the first part of my thesis, I describe the role of one such factor, CD40 in adipose tissue inflammation. The CD40-CD40L dyad acts as co-stimulation in the interaction of antigen-presenting cells, such as macrophages and dendritic cells, with effector cells, such as T cells, in adaptive immunity. We found that CD40 knockout mice were smaller but surprisingly more insulin resistant and glucose intolerant compared to wild-type mice when fed a high fat diet. Consistent with their metabolic phenotype, knockout mice displayed increased adipose tissue inflammation with infiltration of immune cells including macrophages and T cells. Consistent with increased inflammation, CD40 knockout adipose tissue displayed decreased lipid storage. Deficiency of CD40 also led to increased lipid deposition in liver, which may be due to increased lipid release into circulation from the adipose tissue as well as increased lipid synthesis in the liver. CD40 knockout mice had increased hepatic insulin resistance and increased gluconeogensis despite decreased hepatic inflammation. These findings suggest that CD40 is a novel regulator of adipose tissue inflammation in diet-induced obesity. In the second part of this thesis we examined perivascular adipose tissue and brown adipose tissue for the presence of inflammation. In contrast to visceral adipose tissue, macrophage infiltration was absent in perivascular and brown adipose tissue as defined by reduced F480+ cells by flow cytometry and immunohistochemistry. We also found that perivascular adipose tissue was similar to brown adipose tissue as shown by gross morphology and gene expression pattern. Inflammatory gene expression was not increased in brown or perivascular adipose tissue in obese mice as determined by microarray gene expression analysis. These findings suggest that perivascular adipose tissue is more similar to brown adipose tissue than white adipose tissue and that both perivascular and brown adipose tissue are resistant to inflammation. We conclude that, (1) CD40 protects against adipose tissue inflammation in diet-induced obesity, (2) the CD40 knockout mouse is an interesting model of hepatic steatosis with decreased inflammation and (3) perivascular adipose tissue is almost identical to brown adipose tissue in obese mice and that both are resistant to inflammation.

Mycobacterium tuberculosis stably adapts to respiratory limited environments by entering into a nongrowing but metabolically active state termed quiescence. This state is inherently tolerant to antibiotics due to a reduction in growth and activity of associated biosynthetic pathways. Understanding the physiology of the quiescent state, therefore, may be useful in developing new strategies to improve drug efficiency. Here, we used an established in vitro model of respiratory stress, hypoxia, to induce quiescence. We utilized metabolomic and genetic approaches to identify essential and active pathways associated with nongrowth. Our metabolomic profile of hypoxic M. tuberculosis revealed an increase in several free fatty acids, metabolite intermediates in the oxidative pathway of the tricarboxylic acid (TCA) cycle, as well as, the important chemical messenger, cAMP. In tandem, a high-throughput transposon mutant library screen (TnSeq) revealed that a cAMP-regulated protein acetyltransferase, MtPat, was conditionally essential for survival in the hypoxic state. Via 13C-carbon flux tracing we show an MtPat mutant is deficient in re-routing hypoxic metabolism away from the oxidative TCA cycle and that MtPat is involved in inhibiting fatty-acid catabolism in hypoxia. Additionally, we show that reductive TCA metabolism is required for survival of hypoxia by depletion of an essential TCA enzyme, malate dehydrogenase (Mdh) both in in vitro hypoxia and in vivo mouse infection. Inhibition of Mdh with a novel compound resulted in a significantly greater killing efficiency than the first-line anti-M. tuberculosis drug isoniazid (INH). In conclusion, we show that understanding the physiology of the quiescent state can lead to new drug targets for M. tuberculosis.

Autophagy is a cellular process that delivers cytoplasmic materials for degradation by the lysosomes. Autophagy-related (Atg) genes were identified in yeast genetic screens for vehicle formation under stress conditions, and Atg genes are conserved from yeast to human. When cells or animals are under stress, autophagy is induced and Atg8 (LC3 in mammal) is activated by E1 activating enzyme Atg7. Atg8-containing membranes form and surround cargos, close and mature to become the autophagosomes. Autophagosomes fuse with lysosomes, and cargos are degraded by lysosomal enzymes to sustain cell viability. Therefore, autophagy is most frequently considered to function in cell survival. Whether the Atg gene regulatory pathway that was defined in yeast is utilized for all autophagy in animals, as well as if autophagy could function in a cell death scenario, are less understood. The Drosophila larval digestive tissues, such as the midgut of the intestine and the salivary gland, are no longer required for the adult animal and are degraded during the pupal stage of development. Cells stop growing at the end of larval development, and proper cell growth arrest is required for midgut degradation. Ectopic activation of the PI3K/Akt signaling induces cell growth and inhibits autophagy and midgut degradation. Down regulating PI3K/Akt pathway by Pten mis-expression activates autophagy. In addition, mis-expression of autophagy initiator Atg1 inhibits cell growth and knocking down autophagy restore PI3K/Akt activity. Together, these results indicate that autophagy and growth signaling mutually inhibit each other. Midgut destruction relies on the autophagy gene Atg18, but not caspase activation. The intestine length shortens and the cells undergo programmed cell size reduction, a phenomenon that also requires Atg18, before cell death occurs during midgut destruction. To further investigate whether cell size reduction is cell autonomous and requires other Atg genes, we reduced the function of Atg genes in cell clones using either gene mutations or RNAi knockdowns. Indeed, many Atg genes, including Atg8, are required for autophagy and cell size reduction in a cell autonomous manner. Surprisingly, Atg7 is not required for midgut cell size reduction and autophagy even though this gene is essential for stress-induced autophagy. Therefore, we screened for known E1 enzymes that may function in the midgut, and discovered that Uba1 is required for autophagy, size reduction and clearance of mitochondria. Uba1 does not enzymatically substitute for Atg7, and Ubiquitin phenocopies Uba1, suggesting Uba1 functions through ubiquitination of unidentified molecule(s) to regulate autophagy. In conclusion, this thesis describes: First, autophagy participates in midgut degradation and cell death. Second it reveals a previously un-defined role of Uba1 in autophagy regulation. Third it shows that the Atg genes are not functionally conserved and the requirement of some Atg genes can be context dependent.

Obesity is the top risk factor for the development of type 2 diabetes mellitus in humans. Obese adipose tissue, particularly visceral depots, exhibits an increase in macrophage accumulation and is described as being in a state of chronic low-grade inflammation. It is characterized by the increased expression and secretion of inflammatory cytokines produced by both macrophages and adipocytes, and is associated with the development of insulin resistance. Based on these observations, we investigated the potential role of macrophage infiltration on whole body metabolism, using genetic and diet-induced mouse models of obesity. Using flow cytometry and immunofluorescence imaging we found that a significant percentage of macrophages proliferate locally in adipose tissue of obese mice. Importantly, we identified monocyte chemoattractant protein 1 (MCP-1) as the stimulating factor. We also found that ATMs can be targeted for specific gene silencing using glucan encapsulated siRNA particles (GeRPs). Knockdown of the cytokine osteopontin improved regulation of systemic glucose levels as well as insulin signaling in adipocytes. Conversely, targeting lipoprotein lipase (LPL) abrogated the buffering of lipid spillover from adipose tissue, resulting in increased hepatic glucose output. Finally, silencing of the master regulator of inflammation NF-κB in resident liver macrophages called Kupffer cells significantly improved hepatic insulin signaling. Thus this work demonstrates that macrophages can regulate whole body metabolism.

Proper functioning of endocrine cells is crucial for organismal homeostasis. The underlying mechanisms that fine-tune the amount, and the timing of hormone secretion are not clear. JIP1 / MAPK8IP1 (JNK interacting protein 1) is a scaffold protein that mediates cellular stress response, and is highly expressed in endocrine cells, including insulin secreting b-cells in pancreas islets. However, the role of JIP1 in b-cells is unclear. This study demonstrates that b-cell specific Jip1 ablation results in decreased glucose-induced insulin secretion, without a change in Insulin1 and Insulin2 gene expression. Inhibition of both JIP1-kinesin interaction, and JIP1-JNK interaction by genetic mutations also resulted in decreased insulin secretion, suggesting that JIP1 may mediate insulin vesicle trafficking through interacting with kinesin and JNK. Autophagy is a cellular recycling mechanism and implicated in the b-cell function. Both JIP1 and JNK are proposed to regulate autophagy pathway. However, it is unclear whether JNK plays a role in the promotion or suppression of autophagy. The findings of this study show that JNK is not essential for autophagy induction, but can regulate autophagy in a cell and context specific manner. The results in this thesis implies a mechanism that link cellular trafficking and stress signaling pathways in the regulated hormone secretion. In addition to the known role of JIP1 in metabolism and insulin resistance, this finding may also be relevant to endocrine pathologies.

The nucleolus is a plurifunctional organelle with dynamic protein exchange involved in diverse aspects of cell biology. Additionally, the nucleolus has been shown to have a role in the replication of numerous viruses, which includes HIV-1. Several groups have reported HIV-1 vRNA localization within the nucleolus. Moreover, it has been demonstrated the HIV-1 Rev protein localizes to the nucleolus and interacts with nucleolar proteins, including NPM1. Despite evidence for a nucleolar involvement during replication, a functional link has not been demonstrated. I investigated whether introncontaining vRNAs have a Rev-mediated nucleolar localization step prior to export. Furthermore, I examined whether NPM1 mediates Rev nucleolar localization, participates in Rev function, and/or post-transcriptional events during viral replication. I used coupled RNA fluorescence in situhybridization and indirect immunofluorescence to visualize intron-containing vRNA relative to the nucleolus in the absence or presence of Rev expression. An RNAi-based approach was used to examine the role of NPM1 in Rev function and viral replication in cell lines and primary human macrophages. My research findings support a model for a Rev-independent nucleolar localization step of introncontaining vRNA prior to export. Intriguingly, my results also suggest NPM1 does not participate in Rev nucleolar localization or Rev-mediated vRNA export, as previously proposed. Rather, my findings support a novel role for NPM1, the cytoplasmic localization and utilization of a select class of Rev-dependent vRNAs. Collectively, my findings provide novel insight for a functional role of the nucleolus and NPM1 in HIV-1 replication, which enhances our current understanding of HIV-1 biology.

Early post-implantation vertebrate embryos are shaped by complex cellular and molecular mechanisms. In mice, the visceral endoderm, an extraembryonic cell lineage that appears before gastrulation, provides several important functions such as nutrition and mechanical protection. My thesis research focused on the role of the visceral endoderm in embryo patterning, a newly discovered function for this tissue. My results showed that an interplay between two subpopulations of visceral endoderm the anterior and posterior visceral endoderm, located on the opposite sides of the developing conceptus, are critical for the establishment of the anteroposterior body axis of the embryo. I also found that senescence-associated β-galactosidase activity delineates the visceral endoderm marking apical vacuole, a lysosomal-like organelle. This however indicates the nutritional function of visceral endoderm cells rather than a senescent population. My studies highlight the fundamental role of extraembryonic tissues in patterning mammalian embryos as opposed to housekeeping roles. They also reveal important difference when conducting studies at the organismal level rather than in cells in culture.

Skeletal muscle insulin resistance is a major characteristic of obesity and type 2 diabetes. Although obesity-mediated inflammation is causally associated with insulin resistance, the underlying mechanism is unclear. Our lab and others have shown that a chronic low-grade inflammation takes place in skeletal muscles during diet-induced obesity, as evidenced by increased macrophage markers and pro-inflammatory cytokine levels. Interleukin (IL)-10 is a Th2-type cytokine that inhibits the synthesis and activity of pro-inflammatory cytokines and counteracts the Toll-like receptor-mediated inflammation. Our lab has previously demonstrated the preventive role of IL-10 against insulin resistance. Here, I have analyzed the effects of IL-10 on the skeletal muscle glucose metabolism and myogenesis in three different insulin resistant states (high fat diet-induced, leptin-deficiency-induced and aging-induced). The first model involved long-term (16 weeks) high-fat diet (HFD) feeding that resulted in markedly obese and hyperglycemic mice, representative of obese type 2 diabetic subjects. In mice overexpressing IL-10 specifically in the skeletal muscle (MIL10), we observed improved whole-body and skeletal muscle insulin sensitivity as compared to wild-types after long-term high fat diet feeding. The improved insulin sensitivity in the skeletal muscle was due to increased Akt signaling and decreased muscle inflammation. Leptin is an important adipocyte-derived hormone that is elevated in obesity, and it regulates numerous physiological functions including the energy balance and inflammation. Thus, my second model examined the effects of muscle-specific overexpression of IL-10 on glucose metabolism in the hyperphagic, leptin-deficient ob/ob mice. We detected improved whole-body insulin sensitivity compared to the control mice. My third model examined the effects of increased IL-10 expression using MIL10 mice during aging-induced insulin resistance. In 18-month old MIL10 mice, we found enhanced whole-body and skeletal muscle insulin sensitivity due to improved insulin signaling and decreased muscle inflammation as compared to wild-type mice. Last, to test whether direct signaling of IL-10 on skeletal muscle is responsible for the beneficial effects of IL-10 on muscle glucose metabolism, I generated mice lacking IL-10 receptor 1 type chain selectively in skeletal muscle (M-IL10R-/-). We observed more prominent muscle inflammation and whole-body insulin resistance in HFD-fed M-IL10R-/- mice as compared to wild-type mice. Interestingly, when studying insulin resistance in the IL-10 transgenic mouse models, we identified a consistent increased lean mass phenotype, and conversely decreased lean mass in the HFD-fed M-IL10R-/- mice. Quantitative RT-PCR on HFD-fed MIL10 group muscles to measure myogenesis-related gene expression identified a correlation between lean mass and both IL-10 and MyoD mRNA expression levels. In support of this, I showed that IL-10 caused an increase in in vitro cultured myoblast proliferation rates. Together, these results highlight the potential benefits of IL-10 expression not only in muscle glucose metabolism but also in maintaining muscle mass during insulin resistant states. Overall, these results demonstrate that selective expression of IL-10 in skeletal muscle suppresses inflammation, improves glucose metabolism and muscle growth in obese and aging mice, and further establishes that these effects are at least partially mediated by direct activation of IL-10 signaling in skeletal muscle.

This thesis comprises two separate studies that focus on the consequences of cellular damage. The first investigates the effects of DNA damage on centriole behavior and the second characterizes phototoxicity during live-cell imaging. Cancer treatments such as ionizing radiation and/or chemotherapeutic DNA damaging agents are intended to kill tumor cells, but they also damage normal proliferating cells. Although centrosome amplification after DNA damage is a well-established phenomenon for transformed cells, it is not fully understood in untransformed cells. The presence of extra centrosomes in normal cell populations raises the chances of genomic instability, thus posing additional threats to patients undergoing these therapies. I characterized centriole behavior after DNA damage in synchronized untransformed (RPE1) human cells. Treatment with the radiomimetic drug, Doxorubicin, prolongs G2 phase by at least 72hrs, where 52% of cells display disengaged centrioles and 10% contain extra centrioles. This disengagement is mediated by Plk and APC/C activities both singly and in combination. Disengaged centrioles are associated with maturation markers suggesting they are capable of organizing spindle poles. Despite the high incidence of centriole disengagement, only a small percentage of centrioles reduplicate due to p53/p21 dependent inhibition of Cdk2 activity. Although all cells become prolonged in G2 phase, 14% eventually go through mitosis, of which 26% contain disengaged or extra centrioles. In addition to cancer treatments, cellular damage can be acquired from various external conditions. Short wavelengths of light are known to be toxic to living cells, but are commonly used during live-cell microscopy to excite fluorescent proteins. I characterized the phototoxic effects of blue (488nm) and green (546nm) light on cell cycle progression in RPE1. For unlabeled cells, I found that exposure to green light is far less toxic than blue light, but is not benign. However, the presence of fluorescent proteins led to increased sensitivity to both blue and green light. For 488nm irradiations, spreading the total irradiation durations out into a series of 10s pulses or conducting single longer, but lower intensity, exposures made no significant changes in phototoxicity. However, reducing oxidative stress by culturing cells at physiological (~3%) oxygen, or treatment with a water-soluble antioxidant, Trolox, greatly improved the cells tolerance to blue light. Collectively, my work offers an explanation for centrosome amplification after DNA damage and demonstrates the importance of proper centriole regulation in untransformed human cells. Further, it provides a practical assessment of photodamage during live-cell imaging.