Novel regulation by Rac1 of glucose- and forskolin-induced insulin secretion in INS-1 β-cells

Abstract

Stimulation of insulin secretion by glucose and other secretagogues from pancreatic islet β-cells is mediated by multiple signaling pathways. Rac1 is a member of Rho family GTPases regulating cytoskeletal organization, and recent evidence also implicates Rac1 in exocytotic processes. Herein, we report that exposure of insulin-secreting (INS) cells to stimulatory glucose concentrations caused translocation of Rac1 from cytosol to the membrane fraction (including the plasmalemma), an indication of Rac1 activation. Furthermore, glucose stimulation increased Rac1 GTPase activity. Time course study indicates that such an effect is demonstrable only after 15 min stimulation with glucose. Expression of a dominant-negative Rac1 mutant (N17Rac1) abolished glucose-induced translocation of Rac1 and significantly inhibited insulin secretion stimulated by glucose and forskolin. This inhibitory effect on glucose-stimulated insulin secretion was more apparent in the late phase of secretion. However, N17Rac1 expression did not significantly affect insulin secretion induced by high K+. INS-1 cells expressing N17Rac1 also displayed significant morphological changes and disappearance of F-actin structures. Expression of wild-type Rac1 or a constitutively active Rac1 mutant (V12Rac1) did not significantly affect either the stimulated insulin secretion or basal release, suggesting that Rac1 activation is essential, but not sufficient, for evoking secretory process. These data suggest, for the first time, that Rac1 may be involved in glucose- and forskolin-stimulated insulin secretion, possibly at the level of recruitment of secretory granules through actin cytoskeletal network reorganization.

Dominant-Negative α-Subunit of Farnesyl- and Geranyltransferase Inhibits Glucose-Stimulated, but Not KCl-Stimulated, Insulin Secretion in INS 832/13 Cells

“…We previously reported a significant degree of translocation of Rac1 in isolated ␤-cells after stimulation by glucose; based on this, we hypothesized that this might represent a key signaling step in GSIS (8,23). In the next set of experiments, we determined the ability of glucose to promote membrane association of candidate G-proteins (e.g., Rac1) in INS 832/13 cells in which protein prenylation is compromised via overexpression of dn FTase/ GGTase.…”

“…Islets were isolated from normal Sprague-Dawley rats using the collagenase digestion method (7,23). INS 832/13 cells were provided by Dr. Chris Newgard (Duke University School of Medicine, Durham, NC) (8,23). Expression plasmid for the dn FTase/GGTase ␣-subunit.…”

“…Original observations from multiple laboratories, including our own, demonstrated critical involvement of small G-proteins, such as Rac1, Cdc42, Rap1, and ARF6 (ADP-ribosylation factor 6) in GSIS from normal rat islets, human islets, and clonal ␤-cell preparations (3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Such conclusions were primarily based on data from experiments using 1) inhibitors of requisite posttranslational modifications of these G-proteins; 2) clostridial toxins, which monoglucosylate and inactivate specific G-proteins; and 3) gene depletion (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Despite this compelling evidence, the precise biochemical mechanisms underlying glucose-mediated activation of these signaling proteins leading to GSIS remain only partially understood.…”

Cadherin Engagement Improves Insulin Secretion of Single Human β-Cells

“…Indeed, final access of secretory granules to the plasma membrane requires dissolution of a defined cortical actin web. Recent reports have underlined a possible role for the Rho-GTPases (44,47,48) in cortical F-actin reorganization during regulated insulin secretion.…”

Distinct pathways of cholesterol biosynthesis impact on insulin secretion

“…The mobilization of insulin granules and their fusion to the plasma membrane is markedly dependent on a family of proteins that require isoprenylation, the small GTPases (e.g., Rap1, Rac1, and Cdc42). These are involved in vesicle docking, cytoskeletal remodeling, and granule priming and fusion (Li et al 2004, Fukuda 2005, Pechlivanis & Kuhlmann 2006, Kowluru 2008, Wang & Thurmond 2009, Goalstone et al 2010.…”