Pancreatic islets contain several types of hormone-expressing cells to help regulate glucose homeostasis. Chief among these is beta cells which secrete insulin to lower blood glucose levels. Diabetes, which currently impacts over 37 million Americans, ensues when beta cells are destroyed by the body’s own immune system or when they become dysfunctional caused by a myriad of genetic and environmental factors. Consequently, the major question becomes how best to replace the lost and impaired beta cells. To this end, we discovered a novel population of immature virgin beta cells which may potentially serve as a new source to generate functionally mature beta cells as a therapeutic treatment for diabetes.

In this dissertation, we further characterized virgin beta cells to learn more about their unique properties that make them distinct from other known beta cell subpopulations. Using various transgenic mice, complex lineage-tracing techniques, and immunohistochemical analyses, we determined several novel insights about virgin beta cells. Chapter 1 reviews the discovery of virgin beta cells and the many unanswered questions remaining about them. Chapter 2 examined the proliferation capacity and maturation potential of virgin beta cells. Chapter 3 elucidated the role of pancreatic alpha and delta cells in the formation and maintenance of virgin beta cells. Chapter 4 interrogated the contribution of artemether, an anti-malarial drug, in the generation of new beta cells via beta cell transdifferentiation from alpha cells. Lastly, Chapter 5 summarizes all the original, including unpublished, work performed in this dissertation, concluding remarks, and various exciting direction for future studies.

In current cellular biology, we often assume that cells operate at a near steady state. Thisassumption implies that each individual cell performs the same processes at any particular moment. However, this assumption proves to be difficult to reconcile with cellular processes that are dynamic and asynchronous, such as the cell cycle, in which each cell has its own clock, or ey signaling processes such as the MAPK and AKT pathways, which are heterogeneous and dynamic from cell to cell. These heterogeneities play an essential role in cell fate decisions, including proliferation and differentiation. Cell signaling information also ‘spreads’ to other pathways and thus creates complex changes in cellular states, including in metabolic flux and gene expression. The connection between cell signaling and cell metabolism raises the question of whether cell metabolism is heterogeneous too. Furthermore, metabolic states could influence how cells respond to growth signaling cues. In the first part of this dissertation, I explore the question of whether cellular metabolism is heterogeneous in cell populations. I utilize a fluorescence-based FRET biosensor to probe AMPK activity when cellular oxidative phosphorylation (OXPHOS) is inhibited. I show that, in fact, at a single-cell level, cells do not utilize OXPHOS equally, and cellular adaptation after OXPHOS inhibition never reaches a steady state. In the second part, I expand the idea of single-cell metabolism and ask how cell metabolism regulates growth signals at the single-cell level. I developed transposase-based transfection systems that would allow expression of up to three fluorescence biosensors in one transfection to achieve this goal. I also developed an unsupervised clustering technique for multidimensional time-series data to analyze more than 300,000 single cell traces. I showed that the signaling activity under metabolic conditions is heterogeneous even in the same type of metabolic stress. However, the signaling landscape is not infinite since there are only about 30 modes ofviii responses. This study characterizes the complex interaction between cell metabolism and cellular growth signals.

The α-cells of the pancreatic islets are defined by their hormone, glucagon, whichthey release in response to signals indicating increased demand for glucose in the body. They exist in dynamic co-regulation with their neighboring β-cells, which release insulin to lower blood glucose, and δ-cells, which secrete somatostatin to impose local inhibitory feedback in the islet. The activity of all three cells are largely reliant on circulating glucose levels, but α-cells are also activated by nutrients like amino acids and hormones like epinephrine and arginine vasopressin. This system is disrupted in diabetes, and glucagon dysfunction ultimately contributes greatly to diabetic hyperglycemia and impaired counterregulation. Understanding what goes wrong in diabetes is complicated by that fact that while many physiological signals are known to regulate glucagon secretion, α-cells are often viewed as a single population and thus individual α-cell responses are not well characterized. In this dissertation, we have applied improved genetic tools for expressing fluorescent biosensors specifically in mouse α-cells in order to gain a population-level view of their activity in intact islets in real time. In doing so we are able to interrogate how intra-islet signaling and various glucagon secretagogues, as well as diabetes, shape α-cell behavior. Chapter 1 provides a comprehensive overview of islet biology, including the changes that occur in diabetes. Chapter 2 investigates the role of another β-cell hormone, Urocortin3, in inhibitory paracrine feedback on α-cells. Chapter 3 characterizes functional heterogeneity in α-cell activation by common physiological stimuli and how diabetes affects these responses. Chapter 4 provides a summary of the work performed, concluding remarks, and plans for future studies.