1732-P: ATP Dynamics in ß-Cell Primary Cilia Depend on Bidirectional Signaling from Ciliary Glycolysis to Mitochondria

Primary cilia are specialized antenna-like sensory and signaling organelles present in pancreatic β-cells that play important roles in mediating insulin secretion. However, it remains unclear how cilia are provided with ATP. Here, β-cell deletion of the ATP-generating enzyme pyruvate kinase (PK) was used to investigate autonomous ciliary signaling, while β-cell deletion of mitochondrial Pck2, which supplies PK with mitochondrial PEP, was used to assess extraciliary signaling from the mitochondria. The dynamics of plasma membrane and ciliary signaling were examined in intact islets using live-cell 3D imaging of 5HT₆ targeted biosensors. At elevated glucose, plasma membrane ATP/ADP, fructose 1,6-bisphosphate (FBP), and cAMP/PKA exhibit regular oscillatory patterns. Glucose elevation elicited a pronounced drop in plasma membrane ATP/ADP that contrasted with the sustained rise in ATP/ADP observed in cilia, and in many cases antiphasic ATP/ADP oscillations were observed between the two compartments. The cytosol represents a third “compartment” where glucose induced a monotonic rise in ATP/ADP, as shown with untargeted biosensors. In contrast with ATP/ADP, ciliary FBP did not rise in response to glucose, suggesting highly efficient glycolytic signaling within the axoneme. Experiments performed at low glucose with leucine stimulation were used to isolate retrograde PEP signaling from mitochondria to cilia. The leucine-dependent rise in ciliary ATP/ADP observed in control cells was strongly reduced by β-cell deletion of PCK2 or PKm1, with a further reduction in the PKm1/2 double knockout. Taken together, our findings indicate distinct signaling dynamics in the plasma membrane and ciliary domains, and demonstrate the importance of PK and an extraciliary PEP cycle for control of ciliary bioenergetics.