ADP-ribosylation factor 6 regulates insulin secretion through plasma membrane phosphatidylinositol 4,5-bisphosphate

Abstract

ADP-ribosylation factor 6 (ARF6) is a small GTP-binding protein that regulates peripheral vesicular trafficking and actin cytoskeletal dynamics, and it has been implicated as critical to regulated secretion. Expression of a dominant-inhibitory ARF6 mutant, ARF6(T27N), impaired glucose-, depolarization-, and gamma-thio-GTP-stimulated insulin secretion in the pancreatic beta cell line, MIN6. In response to depolarization, MIN6 cells expressing ARF6(T27N) displayed an unaltered initial fast phase but an impaired subsequent slow phase of insulin secretion. Actin cytoskeletal disassembly with latrunculin A enhanced insulin secretion, whereas stabilization with jasplakinolide inhibited secretion, consistent with the actin cytoskeleton serving as a barrier to exocytosis in these cells. ARF6(T27N) led to a depolarization-dependent reduction in the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] with a time course that paralleled the inhibition of secretion. Moreover, blockade of PI(4,5)P2-dependent events by expression of a lipid-binding protein resulted in inhibition of depolarization-induced secretion in a manner identical to ARF6(T27N). These results indicate that ARF6 is required to sustain adequate levels of PI(4,5)P2 during periods of increased PI(4,5)P2 metabolism such as regulated secretion.

Fig. 1.

Role of ARF6 in glucose- and depolarization-stimulated insulin secretion. Pools of MIN6 cells that express the indicated proteins upon infection with AdCre were either mock-infected (-AdCre) or infected with AdCre (+AdCre). (A) Cells were stimulated with the indicated concentration of glucose, and the amount of insulin secreted after 1 h, as a percentage of the total, was determined. *, P < 0.05, statistically significant difference between the -AdCre and +AdCre samples. (B) Cells were either left unstimulated (low K⁺) or were depolarized in medium containing a high concentration of potassium (high K⁺) for 1 h, and the amount of insulin secreted was determined. Data are the means ± SEM of four independent experiments. *, P < 0.05, statistically significant difference between the -AdCre and +AdCre.

Fig. 2.

Role of ARF6 in GTPγS-induced insulin secretion. After either mock infection (-AdCre) or infection with AdCre (+AdCre), MIN6 cells were permeabilized with streptolysin O (SLO) and incubated in the absence (-GTPγS) or presence (+GTPγS) of GTPγS. The amount of insulin secreted after 1 h was determined. *, P < 0.05, statistically significant difference between the -AdCre and +AdCre samples.

Fig. 3.

Effect of ARF6 on the time course of insulin secretion after depolarization. MIN6 cell lines, either mock-infected (-AdCre) or infected with AdCre (+AdCre), were depolarized, and the total amount of insulin released over time was determined.

Fig. 4.

Effect of primary alcohols on depolarization-induced insulin secretion. (A) MIN6 cells were preincubated with the indicated concentration of the primary alcohols 1-butanol (1-BuOH) and ethanol (EtOH) or the control secondary alcohols 2-butanol (2-BuOH) and 2-propanol (2-Prop) for 1 h. The amount of insulin secreted in 1 h in response to depolarization in the continued presence of the alcohols was then determined. (B) The pattern of insulin secretion in response to depolarization after treatment, as in A, with the indicated concentrations of 1-butanol and 2-propanol, was determined.

Fig. 5.

Role of the actin cytoskeleton in mediating the dominant-negative ARF6(T27N) inhibition of secretion. (A) MIN6 cells were preincubated with vehicle (DMSO) or cytochalasin D (CytoD), latrunculin A (LatA), or jasplakinolide (Jas) for 1 h. The amount of insulin secreted after 1 h under both basal (low K⁺) and depolarizing (high K⁺) conditions, in the continued presence of the actin-modifying agents, was determined. (B) The pattern of insulin secretion in response to depolarization after treatment with jasplakinolide (Jas), as in A, compared with vehicle (DMSO) was determined.

Fig. 6.

Role of ARF6 in maintenance of plasma membrane PI(4,5)P₂ after depolarization-induced secretion. MIN6 cells that express ARF6(T27N) on infection with AdCre were either mock-infected (-AdCre) or infected with AdCre (+AdCre). All cells were then infected with AdPLCδPH-GFP at low titer. (A) The localization of the PI(4,5)P₂, as indicated by the fluorescence from the expressed PLCδPH-GFP, was assessed in live cells by confocal microscopy through the center of the cell every 30 sec. A composite photomicrograph at the indicated times (minutes) relative to depolarization at time 0 is presented. The full time-lapse recording can be found in Movie 1, which is published as supporting information on the PNAS web site. (B) From five time-lapse recordings for each condition, an average maximum plasma membrane (PM) to average cytosol ratio ± SD was determined; this ratio is presented for each frame captured over time relative to the initiation of depolarization. (C) Cells were ³²P_i-labeled and depolarized for 15 min in the continued presence of label. The extracted lipids from duplicate samples were resolved by TLC together with the indicated phosphoinositide standards and detected by autoradiogram. The locations of phosphoinositide standards are indicated.

Fig. 7.

Role of PI(4,5)P₂ in the secretion of insulin. (A) MIN6 cells were infected with either a high titer of AdPLCδPH-GFP or an equivalent titer of the control virus AdGFP. The amount of insulin secreted after 1 h under both basal (low K⁺) and depolarizing (high K⁺) conditions was determined. (B) The pattern of insulin secretion in response to depolarization after expression of PLCδPH-GFP or control GFP as in A was determined. (C) MIN6 cells that express ARF6(T27N) on infection with AdCre were either mock-infected (-AdCre) or infected with AdCre (+AdCre). High titers of either AdPLCδPH-GFP or AdGFP were then used to initiate expression of PLCδPH-GFP and GFP, respectively. The amount of insulin secreted after 10 and 60 min in response to depolarization was determined.