P2Y₁receptor‐dependent diacylglycerol signaling microdomains in β cells promote insulin secretion

Abstract

Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic β cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 ± 0.4 s) but high-amplitude elevations (up to 109 ± 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y₁, but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.

Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells

“…Data are from three to six donors. **p<0.01 and ***p<0.001 compared with 1 mmol/l glucose or as indicated agreement with the findings of a recent study [12], our findings vary from those of another study suggesting a dominant role for P2X 3 [11]. However, the latter study employed pyridoxalphosphate-6-azophenyl-2′,5′-disulfonic acid (iso-PPADS) and oxidised ATP at concentrations that also strongly inhibit P2Y 1 [21,22].…”

“…Although a recent study suggests that autocrine activation of P2Y 1 stimulates diacylglycerol production in rodent and human beta cells [12], we were unable to determine a role for phospholipase C as the inhibitor U-73122 (5-10 μmol/l) also suppressed KCl-evoked Ca 2+ responses (data not shown). In mice, P2Y 1 receptors depolarise beta cells via inhibition of K ATP channels [30].…”

“…PCR analysis Expression of P2Y receptors (P2RY [1][2][3][4][5][6][7][8][9][10][11][12][13][14] ) was analysed by RT-PCR in RNA purified from isolated human islets, using a previously described protocol [18]. Primer sequences are detailed in ESM Methods.…”

“…There is evidence for the expression of both P2X and P2Y receptors in rat and mouse beta cells, suggesting that ATP acts as an autocrine signal in islets, although it is debatable whether purinergic signalling stimulates or inhibits insulin secretion [8,9]. Overall, the few studies that have been conducted in human islets suggest a stimulatory role for ATP [10][11][12]. However, controversy exists regarding the receptor subtypes and signal transduction pathways involved.…”

“…However, controversy exists regarding the receptor subtypes and signal transduction pathways involved. While one study proposed that ATP acts principally via P2X 3 receptors, membrane depolarisation and increasing the intracellular Ca 2+ concentration ([Ca 2+ ] i ) [11], a more recent study suggested a prominent role for P2Y 1 receptors and activation of protein kinase C [12]. Involvement of P2X 7 has also been proposed [13].…”