Abstract Over 375 million people suffer from type 2 diabetes (T2D), a disease defined by the failure of pancreatic β-cells. Insulin secretion and maintenance of endoplasmic reticulum (ER) Ca²+ levels are two essential β-cell functions known to fail in T2D. Therefore, uncovering the metabolic regulatory pathways critical to these processes will allow the development of new therapies for T2D. We propose that modulation of the glycolytic enzyme pyruvate kinase (PK) has strong potential to be of protective and therapeutic value. We have discovered that of the three PK isoforms in the β-cell, activation of PKM2 both potentiates insulin secretion and raises ER Ca²+ levels. The objective of the research described in this application is to use novel metabolic imaging methods to identify the specific steps in the β-cell metabolic and secretory pathways that are controlled by PKM2, and to determine the extent to which PKM2 activation can protect/restore insulin secretion and ER Ca²+ during diabetes progression. To do so, we will: 1) Determine how PKM2 regulates the triggering and metabolic amplifying pathways of insulin secretion, 2) Determine the role of PKM2 in ER Ca²+ homeostasis and the neurohormonal amplification of insulin secretion, and 3) Assess the contribution of impaired PKM2 activity to diabetes pathophysiology and the therapeutic potential of PKM2 activation. Completion of these aims will heighten our understanding of nutrient signaling in the pancreatic β-cell, and evaluate promising new targets for the prevention and therapy of T2D.