TMEM55A-mediated PI5P signalling regulates alpha cell actin depolymerisation and glucagon secretion

Aims/hypothesis

Diabetes is associated with the dysfunction of glucagon-producing pancreatic islet alpha cells, although the underlying mechanisms regulating glucagon secretion and alpha cell dysfunction remain unclear. While insulin secretion from pancreatic beta cells has long been known to be controlled partly by intracellular phospholipid signalling, very little is known about the role of phospholipids in glucagon secretion. Using patch-clamp electrophysiology and single-cell RNA sequencing, we previously found that expression of PIP4P2 (encoding TMEM55A, a lipid phosphatase that dephosphorylates phosphatidylinositol-4,5-bisphosphate [PIP2] to phosphatidylinositol-5-phosphate [PI5P]) correlates with alpha cell function. We hypothesise that TMEM55A is involved in glucagon secretion and aim to validate the role of TMEM55A and its potential signalling molecules in alpha cell function and glucagon secretion.

Methods

Correlation analysis was generated from the data in www.humanislets.com. Human islets were isolated at the Alberta Diabetes Institute IsletCore. Electrical recordings were performed on dispersed human or mouse islets with scrambled siRNA or si-PIP4P2 (si-Pip4p2 for mouse) transfection. Glucagon secretion was measured using an islet perfusion system with intact mouse islets. TMEM55A activity was measured using an in vitro on-beads phosphatase assay and live-cell imaging. GTPase activity was measured using an active GTPase pull-down assay. Confocal microscopy was used to quantify F-actin intensity using primary alpha cells and alphaTC1–9 cell lines after chemical treatment.

Results

TMEM55A regulated alpha cell exocytosis and glucagon secretion. TMEM55A knockdown in both human and mouse alpha cells reduced exocytosis at low glucose levels and this was rescued by the direct reintroduction of PI5P. PI5P, instead of PIP2 increased the glucagon secretion using intact mouse islets. This did not occur through an effect on Ca²⁺ channel activity but through a remodelling of cortical F-actin dependent on TMEM55A lipid phosphatase activity, which occurred in response to oxidative stress. TMEM55A- and PI5P-induced F-actin remodelling depends on the inactivation of GTPase and RhoA, instead of Ras-related C3 botulinum toxin substrate 1 or CDC42.

Conclusions/interpretation

We reveal a novel pathway by which TMEM55A regulates alpha cell exocytosis by controlling intracellular PI5P and the F-actin network.

Graphical Abstract