Obesity, a major risk factor in the development of Type 2 Diabetes (T2D), is commonly associated with insulin resistance and hyperinsulinemia. The long accepted view has been that insulin resistance drives hyperinsulinemia; however, there are multiple lines of evidence that hyperinsulinemia can precede and drive insulin resistance. The signals and mechanisms by which chronic excess nutrients promote pancreatic β-cell dysfunction remain poorly understood. This prompted us to define the signaling events that contribute to basal insulin hypersecretion induced by excess glucose. Of particular interest is signaling through mechanistic target of rapamycin complex 1 (mTORC1), a nutrient sensitive kinase complex whose hyperactivation has been shown to promote hyperinsulinemia. Clonal ß-cells (INS-1 cells) with and without mTORC1 inhibition were pre-exposed to physiological (5mM) or excess (11mM) glucose for 4 to 24 hrs. Basal insulin secretion, respiration and metabolites were measured. Pre-exposure to excess glucose resulted in sustained mTORC1 hyperactivation, basal insulin secretion, higher basal respiration and increased maximal respiratory capacity, due to accelerated mitochondrial pyruvate metabolism. Inhibition of mTORC1 reduced basal insulin secretion, basal respiration and maximal respiratory capacity. Moreover, cells challenged with excess glucose had increased levels of glycolysis and TCA cycle intermediates. Our results suggest that hyperactivation of mTORC1 induced by excess glucose results in increased energy demand and in the generation of metabolic factors that can lead to basal insulin hypersecretion. Therefore, targeting mitochondrial pyruvate metabolism and /or mTORC1 signaling could potentially lead to specific therapies to control hyperinsulinemia and diabetes progression.

Aluminium is an element of increasing clinical importance. It not only has uses as a medicinal substance but also in recent years it has been shown to be the cause of considerable toxicity, particularly in the setting of chronic renal failure. Diseases that have been shown to be associated with aluminium, or in which it has been implicated, include dialysis dementia, renal osteodystrophy and Alzheimer's disease. This thesis has studied aspects of the interaction between aluminium and the kidney. The work has addressed two major issues. Firstly, a study is described where Malvin's stop-flow technique was used to determine any excretory/absorptive tubular site for Al in the pig kidney. Al was found to be excreted in the distal nephron of the pig kidney. Secondly, the toxic effects of Al in vitro on the DNA of pig kidney cell line LLC-PKl were investigated, in an attempt to elucidate some of the mechanisms of toxic action. DNA synthesis was measured using ³H-TdR incorporation. Over increases of both time (9-72 h) and Al concentration (0.01-8.0 mM), ³H-TdR incorporation was diminished. Effects were evident at concentrations as low as 0.05 mM Al. The production of DNA strand breaks was assessed by the increase in size of cell nucleoids (ie DNA in supercoiled form). Nucleoid size was analyzed in a Epics 753 Fluorescence Activated Cell Sorter interfaced with an MDADSII data acquisition and analysis system. After 90 min incubation with Al (over the concentration range 0.001-32 mM), an increase in nucleoid size was noted at concentrations above 0.05 mM. The data demonstrate that Al exerts an effect on kidney cells in vitro which is expressed as diminished DNA synthesis and production of DNA strand breaks. These effects on DNA may have important long-term implications on various disease states associated with Al toxicity.

In 2011, Diabetes and prediabetes affected 9 million Canadians and 366 million people worldwide (Canadian Diabetes Website). The underlying pathophysiology of diabetes is beta cell dysfunction leading to loss of appropriate insulin secretion and resulting in hyperglycemia. I have focused on identifying critical molecular regulators of beta cell function and insulin secretion. The CRTC2-CREB pathway is required for maintaining beta cell mass and insulin secretion. I propose that identifying kinases that regulate CRTC-CREB activity will identify other important regulators of pancreatic beta cell survival and function. First, I have identified several AMP kinases as inhibitors of CRTC2-CREB that are activated by an upstream kinase, LKB1. I then went on to generate mice with a beta cell-specific deletion of LKB1 during adulthood. Loss of LKB1 increased insulin secretion and glucose clearance through enhanced beta cell mass and proliferation. The increased insulin secretion was largely the result of loss of AMPK activity and consequent constitutive mTor activity. AMPK is activated under starvation conditions and as such is thought to be a critical regulator of beta cell function. However, the decrease of AMPK activity in high glucose has been a strong argument against it being a critical effector of insulin secretion. I provide genetic evidence supporting the idea that AMPK activity attenuates insulin secretion. During periods of starvation where AMPK activity is high there is a chronic dampening effect on events that prepare beta cells for the next round of insulin secretion. Surprisingly, another downstream kinase of LKB1, SIK2, has opposing functions in the beta cell. I present evidence that the LKB1-AMPK axis attenuates beta cell functions and that targeting this pathway in beta cells may be of therapeutic benefit for T2D.

The TIR/BB-loop mimetic AS-1 has been reported to prevent cardiac hypertrophy by inhibiting interleukin-1 receptor (IL-1R)-mediated myeloid differentiation primary response gene 88 (MyD88)-dependent signaling. To date, it remains unknown whether and if so how AS-1 contributes to mechanical stress (MS)-induced cardiac fibroblast activation, a key process in pressure overload-induced cardiac remodeling and heart failure. Here, we show that phosphorylation and expression of large tumor suppressor kinase 1 (LATS1), a key molecule in the Hippo-Yes associated protein (YAP) signaling pathway, were down-regulated in primary neonatal rat cardiac fibroblasts (NRCFs) in response to MS and in the hearts of mice subjected to transverse aortic constriction (TAC) procedure; AS-1 treatment was able to restore LATS1 phosphorylation and expression both in vitro and in vivo. AS-1 treatment suppressed the induction of proliferation, differentiation and collagen synthesis in response to MS in NRCFs. AS-1 also ameliorated cardiomyocyte hypertrophy and apoptosis through dampening paracrine secretion of stretched cardiac fibroblasts. In mice, AS-1 treatment could protect against TAC-induced cardiac hypertrophy, myocardial fibrosis and heart failure. Of note, LATS1 depletion using siRNA completely abrogated the inhibitory effects of AS-1 on NRCFs under MS including accelerated proliferation, differentiation, enhanced ability to produce collagen and augmented paracrine secretion of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) to induce cardiomyocyte hypertrophy. Therefore, our results delineate a previously unrecognized role for LATS1 in cardiac fibroblast to mediate the beneficial effects of AS-1 in preventing pressure overload-induced cardiac remodeling and heart failure.

Pancreatic beta cells sense fluctuations in circulating nutrients and adjust the rate of insulin secretion to maintain glucose homeostasis. Mitochondria integrate changes in nutrient flux to the generation of signals that modulate insulin secretion via oxidative phosphorylation. Type 2 Diabetes (T2D) is characterized by beta cell mitochondrial dysfunction and impairment of insulin secretion. Early stage progression of this disease in obese and pre-diabetic subjects is characterized by basal hypersecretion of insulin and increased insulin resistance in peripheral tissues including muscle, liver and adipose tissue. Whether basal hypersecretion of insulin or insulin resistance is the primary defect in T2D progression is still debated. The molecular mechanism underlying basal insulin hypersecretion and how it may lead to beta cell failure are not understood. Herein, we optimize a model of glucolipotoxicity that results in increased basal and reduced stimulated insulin secretion response. Furthermore, we show that pancreatic islets exposed to excess nutrients in vitro or isolated from high fat diet fed animals, have a decreased bioenergetic efficiency, which is characterized by increased mitochondrial proton leak. Leak represents the fraction of oxygen consumed that is not coupled to ATP production. We show that leak is sufficient to induce insulin secretion at basal glucose levels and that nutrient-induced insulin secretion at basal glucose is leak-dependent. Finally, we identify the mitochondrial permeability transition pore (PTP) as the source of the leak. Our findings suggest the PTP may be a potential therapeutic target to prevent/delay the onset of hyperinsulinemia in pre-diabetic subjects.

Polyhydroxyalkanoates (PHAs) are a type of biologically-produced plastic known for their biocompatibility and biodegradability. They have the potential to replace petroleum-based plastics as an environmentally-friendly alternative. This is beneficial because the release of plastics into environments such as the ocean and the buildup of plastics in landfills are major concerns facing society today. Currently, however, PHAs are significantly more expensive than their petroleum-based counterparts. This is largely due to the cost of carbon sources and of extracting the bioplastics from bacteria. The goal of these studies was to examine replacing traditional carbon sources used in PHA production like sugar and oils with sustainable carbon sources and to improve extraction procedures by inducing secretion of PHAs in bacteria. A few sustainable carbon sources were examined for use in PHA production. First, studies focused on the conversion of food waste into PHAs were reviewed. It was shown that utilizing food wastes as carbon sources may be a viable approach to producing PHAs. A second carbon source examined was methanol. A novel isolate of Methylobacterium that demonstrated the ability to produce PHAs from methanol was identified. A system of secreting PHAs that was constructed using synthetic biological engineering approach was introduced to this isolate. This secretion system was not shown to improve extraction of PHAs in Methylobacterium in its current form.

Shigella are bacteria that are responsible for millions of infections and hundreds of thousands of deaths every year. The emergence of antibiotic resistant Shigella adds to the potentially devastating effect that these bacteria can have on human health. Shigella flexneri utilize specialized molecular machinery called the Type III secretion system to infect humans and cause disease. Research of this machinery promises to provide the knowledge, tools, and direction for the development of new avenues to combat shigellosis. This dissertation presents studies of two Shigella proteins, invasion plasmid antigens C and D (IpaC and IpaD). These proteins are part of a syringe and needle like protein structure that allows Shigella to secrete proteins directly into the host that hijack host cells to benefit support Shigella infections. IpaC and IpaD are part of a protein tip complex that is directly involved in these Shigella-host (e.g. human) interactions. We have advanced the biochemical tools for the in vitro study of IpaC by utilizing a new way to isolate it. This purification methodology allowed us to look at one of IpaC’s main roles, to interact with the host cell membranes. We examined IpaC’s role and tried to identify the parts of IpaC responsible for some specific interactions. We found that the parts of IpaC we believed were responsible were not but that the composition of the membrane IpaC is interacting with is more important than we previously believed. Finally, we examined a rare part of IpaD structure to determine its role. We determined that this rare feature is required for IpaD to sense Shigella’s host environment and prepare the bacteria to infect, making a promising target for anti-infective treatments against Shigella infections. Our findings advance the understanding of key molecular mechanisms that are required for Shigella virulence. We expect that our findings will aid future researchers as the pursuit for new treatments for shigellosis continues.

Indiana University-Purdue University Indianapolis (IUPUI) / The Gram-negative bacterium Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), a multi-protein molecular syringe that injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, it was found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. In this work, we demonstrate that mgtE expression acts through the GacAS two-component system to activate transcription of the small regulatory RNAs RsmY and RsmZ. This event ultimately leads to inhibition of exsA translation. Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli. In addition, a previous study has shown that the P. aeruginosa gene algR abrogates mgtE mediated regulation of cytotoxicity. AlgR has pleiotropic effects in P. aeruginosa, including regulation of synthesis of the exopolysaccharide alginate. In the second part of my thesis, I show that algR and mgtE genetically crosstalk to inhibit ExsA driven T3SS gene transcription. This genetic interaction between algR and mgtE seems to be specifically directed towards regulation of T3SS gene expression rather than having an indiscriminate effect on multiple virulence attributes in P. aeruginosa. Additionally, we have further demonstrated that AlgR inhibits mgtE transcription. These studies suggest the presence of a T3SS inhibitor that is inhibited by both AlgR and MgtE. Future work will involve transcriptomic and proteomic analysis to identify such an inhibitor. Taken together, this study provides important insight into the molecular mechanisms of mgtE expression and function in P. aeruginosa. We have established that mgtE has pleiotropic effects on cytotoxicity in P. aeruginosa. Thus, given the role that cytotoxicity regulation plays in shaping P. aeruginosa pathogenesis and associated clinical outcomes, mgtE might be an interesting drug target, though extensive future studies are required to validate this proposition. Nevertheless, this research, provides clues for identification of novel therapeutic targets in P. aeruginosa. Hence this work, in the long run, serve to ameliorate the morbidity and mortality in patients infected with P. aeruginosa.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18197号 / 人健博第14号 / 新制||人健||2(附属図書館) / 31055 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 藤井 康友, 教授 岡 昌吾, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18683号 / 医博第3955号 / 新制||医||1007(附属図書館) / 31616 / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 岩田 想, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM