Mechanisms of Beta Cell Coordination

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Project Summary Type II diabetes affects over thirty million Americans, and its yearly economic cost is estimated to be over 300 billion dollars, ten times the annual NIH budget. Peripheral insulin resistance leads to progressive dysfunction of the insulin-secreting pancreatic islet. Since inadequate compensation for insulin resistance is a prime contributor to diabetes progression, there is an urgent need to understand how insulin secretory failure occurs. The prototypical pathway for insulin secretion within individual cells is well defined. Glucose metabolism leads to ATP generation, closure of ATP-sensitive K+ channels, membrane depolarization, calcium influx, and insulin secretion. Glucokinase, the glucose-sensing enzyme in pancreatic beta cells, is the rate-limiting enzyme in this pathway allowing changes in enzyme activity to control insulin secretion. Even so, the organization of beta-cell secretory responses across cells within an islet is poorly understood. Gap junctions facilitate intra-islet beta cells communication by coupling electrical and metabolic signals between cells. Recent studies have provided evidence that ‘hub’ beta cells within an islet disproportionately affect and even control islet electrical and calcium activity. These cells have elevated glucokinase levels, suggesting that cells with increased metabolic activity control islet activity. We wish to understand how glucokinase activity and expression affect the coordination of metabolic, electrical, and calcium dynamics. We hypothesize that cells with enhanced glucokinase activity and expression guide islet activity and that coordination is dependent on metabolite diffusion across gap junctions. To test our hypothesis, we will first determine whether increased glucokinase activity in single cells enhances their influence over islet glucose metabolism (Specific Aim 1). Next, we will test how metabolite diffusion across gap junctions affects islet activity (Specific Aim 2). To investigate these questions, our lab has generated two novel transgenic mouse models that express genetically-encoded tools specifically in pancreatic beta cells. We will use a glucokinase biosensor to quantify its activity in living cells, and an endoplasmic reticulum-localized channelrhodopsin2 (ER-ChR) to manipulate calcium levels in single cells. Optogenetic ER-ChR allows for precise activation of glucokinase via changes in intracellular calcium. Further, we will use a microfluidic pipette to influence glucokinase activity in small areas of the islet selectively, and mathematical modeling to probe islet coordination. Findings from this study will provide insight into how secretory failure may arise in type II diabetes and identify potential avenues for treatment.