Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

Abstract

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca2+ channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

“…Beta cells were identified by their large size and response to glucose. Previously docked SGs are known to be preferentially released during high-K + stimulation [18,20,24], and this occurs mostly within the first few minutes. Consistently, 50 mmol/l KCl triggered exocytosis of mainly previously docked (predocked) SGs in the first few minutes from control and Syn-4-KD cells (Fig.…”

“…Remarkably, these newcomer SGs are recruited to the PM to undergo exocytosis with minimal to no residence time on the PM [4,5]. We had identified the exocytotic machinery mediating newcomer SGs [10,[16][17][18][19]]-a second SM-SNARE complex comprised of Munc18b, Syn-3, VAMP8 and SNAP25. In that body of work, we showed that depletion of endogenous Syn-3 [10] or VAMP8 [17] reduced newcomer SGs and that when exogenously expressed they restored the deficient newcomer SGs without affecting pre-docked SGs.…”

“…Electrophysiology Capacitance measurements were performed as we previously reported [18], and described in detail in the ESM Methods. Exocytotic events were elicited by a train of ten 500 ms depolarisation pulses from −70 mV to 0 mV.…”

“…A monolayer of human beta cells was infected with adenovirus neuropeptide Y (NPY)-enhanced green fluorescent protein (EGFP) and cultured for 24-36 h before performing TIRFM. Fusion events, indicated by abrupt brightening of NPY-EGFP fluorescence, were manually selected and analysed as previously reported [10,18,19].…”

Structural and functional changes of pancreatic beta-cells during the progression of disease in the Leprdb model of type 2 diabetes

“…However, using the total internal reflection fluorescence microscope (TIRFM), the above model has been revisited recently with the introducing of a new model where the insulin-containing granules were not distinguished based on their physical location in the beta-cell but rather by their modes of exocytosis (Shibasaki, Takahashi et al 2007;Seino, Takahashi et al 2009;Seino, Shibasaki et al 2011;Xie, Zhu et al 2013;Zhu, Koo et al 2013). The first mode of exocytosis, also called old face, characterises by the immediate fusion of the pre-docked granules with the plasma membrane upon stimulation.…”