Metabolic STAMP for deciphering GPCR-regulated insulin secretion by pancreatic β cells.

Pancreatic β cells integrate glucose and metabolic cues to regulate insulin secretion, a process disrupted in T2D. GPCRs play a critical role in fine-tuning insulin release, yet the mechanisms by which ciliary (e.g., FFAR4) and non-ciliary (e.g., GLP1-R) GPCRs coordinate GSIS remains unclear. In this study, we employed Metabolic-STAMP (Synchronized Temporal-Spatial Analysis via Microscopy and Phosphoproteomics) in both mouse β cells (MIN6) and primary human islets to map the dynamic signaling networks governing GSIS and to link transient phosphorylation events to their functional outcomes. We systematically interrogated GPCR-mediated phosphorylation events through selective pharmacological inhibitors, resolving signaling hierarchies and consensus patterns across multiple pathways. Our multi-modal approach uncovered key insulin-secretion-associated PTMs, linked phosphorylation targets with phenotypic organelle dynamics, and provided mechanistic insights into how GLP1-R versus FFAR4 modulates GSIS through shared and GPCR-specific phospho-signatures. We highlighted key examples of stimulus-specific regulation by high glucose alone versus GPCR stimulation, including context-specific activation of the classic ERK signaling pathway, compartmentalized PKA signaling, pathway specificity in organelle dynamics and inter-organellar contacts, and HDAC6/ATAT-mediated regulation of microtubule acetylation. Collectively, these findings provided a blueprint for deconvolving pathway specificity of β cell GPCR signaling, illuminated regulatory nodes that program insulin release, and offered new therapeutic targets to enhance β-cell function.