Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets

Abstract

Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide-exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. SecinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. SecinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS.

Figure 1. Expression of ARNO in INS 832/13 cells, rat islets and human islets

Panel A: Lysates from rat islets, human islets and INS 832/13 cells were separated by SDS-PAGE and probed for ARNO [48 kDa]. Panel B: Hydrophilic [HPL] and hydrophobic [HBP] phases of the homogenates from rat islets and INS 832/13 cells were isolated using Triton X-114 partition technique [see Methods for additional details]. Proteins were separated by SDS-PAGE and probed for ARNO. A representative blot from three independent experiments is shown here.

Figure 2. Overexpression of inactive mutants or siRNA-Arf6 or siRNA-ARNO markedly inhibits glucose-induced insulin secretion in INS 832/13 cells

Panel A: INS 832/13 cells were either mock-transfected or transfected with dominant mutants of Arf6 [T27N] or ARNO (E156K; see Methods). Relative degrees of expression of the mutants was verified by Western blotting. A representative blot from three independent studies yielding similar results is provided here. Panel B: INS 832/13 cells were either mock-transfected or transfected with siRNA-Arf6 or siRNA-ARNO as described in the Methods section. Relative degrees of knockdown of these proteins were verified by Western blotting. A representative blot from three independent studies yielding similar results is shown here. Panel C: INS 832/13 cells were transfected with dominant negative Arf6 [T27N] at a final concentration of 0.2 μg of DNA and cultured for 48 hr. Following this, cells were stimulated with either low [2.5 mM] or high [20 mM] glucose in KRB for 30 min at 37°C. Insulin released into the media was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock low glucose; **p < 0.05 vs. mock transfected cells treated with high glucose, and data points with similar symbol did not differ significantly. Panel D: INS 832/13 cells were transfected with dominant negative ARNO [E156K] at a final concentration of 0.2 μg of DNA and cultured for 48 hr following which cells were stimulated with either low [2.5 mM] or high [20 mM] glucose for 30 min at 37°C. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock transfected low glucose and **p < 0.05 vs. mock transfected high glucose, and data points with similar symbol do not differ significantly. Panel E: INS 832/13 cells were either mock-transfected or transfected with Arf6-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells was stimulated with low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock low glucose; **p < 0.05 vs. mock transfected cells treated with high glucose, and data points with similar symbol did not differ significantly. Panel F: INS 832/13 cells were either mock-transfected or transfected with ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from five independent experiments. *represents p < 0.05 vs. mock transfected low glucose; **p < 0.05 vs. mock transfected high glucose. Insulin release values between mock low glucose or siRNA transfected low glucose did not differ significantly. Panel G: INS 832/13 cells were either mock-transfected or transfected with ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with low [2.5 mM] or KCl [60 mM; osmolarity adjusted] for 60 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. *represents p < 0.05 vs. mock-transfected low glucose; **p < 0.05 vs. mock transfected K⁺. Insulin release values between mock low glucose or siRNA transfected low glucose did not differ significantly. Panels H and I: INS 832/13 cells were mock-tranfected or transfected either with Arf6-siRNA/ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with 1 mM glucose [Glu] or 1 mM glucose + 20 mM arginine [L-Arg] for 15 min. Insulin released into the medium was quantitated and expressed as percent of control. Data are mean ± SEM from replicates. * represents p < 0.05 vs. mock-transfected 1 mM glucose; **p < 0.05 vs. mock-transfected 1 mM Glu + 20 mM L-Arg. Insulin release values between mock or siRNA transfected under low glucose [1 mM] conditions did not differ significantly.

Figure 2. Overexpression of inactive mutants or siRNA-Arf6 or siRNA-ARNO markedly inhibits glucose-induced insulin secretion in INS 832/13 cells

Panel A: INS 832/13 cells were either mock-transfected or transfected with dominant mutants of Arf6 [T27N] or ARNO (E156K; see Methods). Relative degrees of expression of the mutants was verified by Western blotting. A representative blot from three independent studies yielding similar results is provided here. Panel B: INS 832/13 cells were either mock-transfected or transfected with siRNA-Arf6 or siRNA-ARNO as described in the Methods section. Relative degrees of knockdown of these proteins were verified by Western blotting. A representative blot from three independent studies yielding similar results is shown here. Panel C: INS 832/13 cells were transfected with dominant negative Arf6 [T27N] at a final concentration of 0.2 μg of DNA and cultured for 48 hr. Following this, cells were stimulated with either low [2.5 mM] or high [20 mM] glucose in KRB for 30 min at 37°C. Insulin released into the media was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock low glucose; **p < 0.05 vs. mock transfected cells treated with high glucose, and data points with similar symbol did not differ significantly. Panel D: INS 832/13 cells were transfected with dominant negative ARNO [E156K] at a final concentration of 0.2 μg of DNA and cultured for 48 hr following which cells were stimulated with either low [2.5 mM] or high [20 mM] glucose for 30 min at 37°C. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock transfected low glucose and **p < 0.05 vs. mock transfected high glucose, and data points with similar symbol do not differ significantly. Panel E: INS 832/13 cells were either mock-transfected or transfected with Arf6-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells was stimulated with low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock low glucose; **p < 0.05 vs. mock transfected cells treated with high glucose, and data points with similar symbol did not differ significantly. Panel F: INS 832/13 cells were either mock-transfected or transfected with ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from five independent experiments. *represents p < 0.05 vs. mock transfected low glucose; **p < 0.05 vs. mock transfected high glucose. Insulin release values between mock low glucose or siRNA transfected low glucose did not differ significantly. Panel G: INS 832/13 cells were either mock-transfected or transfected with ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with low [2.5 mM] or KCl [60 mM; osmolarity adjusted] for 60 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. *represents p < 0.05 vs. mock-transfected low glucose; **p < 0.05 vs. mock transfected K⁺. Insulin release values between mock low glucose or siRNA transfected low glucose did not differ significantly. Panels H and I: INS 832/13 cells were mock-tranfected or transfected either with Arf6-siRNA/ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with 1 mM glucose [Glu] or 1 mM glucose + 20 mM arginine [L-Arg] for 15 min. Insulin released into the medium was quantitated and expressed as percent of control. Data are mean ± SEM from replicates. * represents p < 0.05 vs. mock-transfected 1 mM glucose; **p < 0.05 vs. mock-transfected 1 mM Glu + 20 mM L-Arg. Insulin release values between mock or siRNA transfected under low glucose [1 mM] conditions did not differ significantly.

Figure 2. Overexpression of inactive mutants or siRNA-Arf6 or siRNA-ARNO markedly inhibits glucose-induced insulin secretion in INS 832/13 cells

Panel A: INS 832/13 cells were either mock-transfected or transfected with dominant mutants of Arf6 [T27N] or ARNO (E156K; see Methods). Relative degrees of expression of the mutants was verified by Western blotting. A representative blot from three independent studies yielding similar results is provided here. Panel B: INS 832/13 cells were either mock-transfected or transfected with siRNA-Arf6 or siRNA-ARNO as described in the Methods section. Relative degrees of knockdown of these proteins were verified by Western blotting. A representative blot from three independent studies yielding similar results is shown here. Panel C: INS 832/13 cells were transfected with dominant negative Arf6 [T27N] at a final concentration of 0.2 μg of DNA and cultured for 48 hr. Following this, cells were stimulated with either low [2.5 mM] or high [20 mM] glucose in KRB for 30 min at 37°C. Insulin released into the media was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock low glucose; **p < 0.05 vs. mock transfected cells treated with high glucose, and data points with similar symbol did not differ significantly. Panel D: INS 832/13 cells were transfected with dominant negative ARNO [E156K] at a final concentration of 0.2 μg of DNA and cultured for 48 hr following which cells were stimulated with either low [2.5 mM] or high [20 mM] glucose for 30 min at 37°C. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock transfected low glucose and **p < 0.05 vs. mock transfected high glucose, and data points with similar symbol do not differ significantly. Panel E: INS 832/13 cells were either mock-transfected or transfected with Arf6-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells was stimulated with low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. mock low glucose; **p < 0.05 vs. mock transfected cells treated with high glucose, and data points with similar symbol did not differ significantly. Panel F: INS 832/13 cells were either mock-transfected or transfected with ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from five independent experiments. *represents p < 0.05 vs. mock transfected low glucose; **p < 0.05 vs. mock transfected high glucose. Insulin release values between mock low glucose or siRNA transfected low glucose did not differ significantly. Panel G: INS 832/13 cells were either mock-transfected or transfected with ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with low [2.5 mM] or KCl [60 mM; osmolarity adjusted] for 60 min. Insulin released into the medium was quantitated and expressed as ng/mL. Data are mean ± SEM from three independent experiments. *represents p < 0.05 vs. mock-transfected low glucose; **p < 0.05 vs. mock transfected K⁺. Insulin release values between mock low glucose or siRNA transfected low glucose did not differ significantly. Panels H and I: INS 832/13 cells were mock-tranfected or transfected either with Arf6-siRNA/ARNO-siRNA at a final concentration of 100 nM. After 48 hr culture in regular medium, cells were stimulated with 1 mM glucose [Glu] or 1 mM glucose + 20 mM arginine [L-Arg] for 15 min. Insulin released into the medium was quantitated and expressed as percent of control. Data are mean ± SEM from replicates. * represents p < 0.05 vs. mock-transfected 1 mM glucose; **p < 0.05 vs. mock-transfected 1 mM Glu + 20 mM L-Arg. Insulin release values between mock or siRNA transfected under low glucose [1 mM] conditions did not differ significantly.

Figure 3. Time-dependent activation of Arf6 by glucose in pancreatic β-cells

Panel A: INS 832/13 cells were incubated with KRB for 1 hr and either left unstimulated [diluent] or stimulated with high glucose [20 mM] for different time points as indicated. Cell lysates were used for detecting activated Arf6 [Arf6.GTP] by GST-GGA3-PBD pull down assay [see Methods]. Total Arf6 was used as the loading control and a representative blot from three independent experiments is shown. Panel B: Densitometric quantitation of Arf6 activation depicted in Panel A is shown here. * represents p < 0.05 vs. diluent. Statistical analysis was performed using One-way ANOVA, All pairwise multiple comparison method (Dunnetts').

Figure 4. Molecular biological or pharmacological inhibition of ARNO attenuates glucose-induced activation of Arf6 in INS 832/13 pancreatic β-cells

Panel A: INS 832/13 cells were either mock-transfected or transfected with siRNA-ARNO and cultured for 48 h following which cells were stimulated in the presence of either low glucose [LG, 2.5 mM] or high glucose [HG, 20 mM] for 30 min at 37°C. The relative amounts of activated Arf6 [i.e, Arf6.GTP] were determined by pull down assay. Total Arf6 from cell lysates was used as the loading control and a representative blot from three independent experiments is shown. Panel B: Data shown in the panel A were analyzed densitometrically and expressed as fold change in Arf6.GTP over basal and are mean ± SEM of three independent experiments. * represents p < 0.05 vs. mock transfected low glucose; **p < 0.05 vs. mock transfected high glucose, and data points with similar symbol do not differ statistically. Panel C: INS 832/13 cells were incubated overnight in the presence or absence of secinH3 [50 μM] and stimulated with either low glucose [LG, 2.5 mM] or high glucose [HG, 20 mM] in the continuous presence or absence of secinH3 [50 μM] for 30 min. Relative degrees of Arf6 activation was quantitated as described in Panel A. Total Arf6 from cell lysates was used as the loading control and a representative blot from three independent experiments is shown. Panel D: Data shown in the Panel C are analyzed densitometrically and expressed as fold change in Arf6.GTP over basal. Data are mean ± SEM from three independent experiments. * and ^# represents p < 0.05 vs. low glucose without secinH3; and ** p < 0.05 vs. high glucose without secinH3.

Figure 5. secinH3, a selective inhibitor of ARNO attenuates GSIS in INS 832/13 cells and normal rat islets

Panel A: INS 832/13 cells were incubated in low serum-low glucose overnight in the continuous presence of 50 μM secinH3 or diluent and stimulated with either low [LG, 2.5 mM] or high glucose [HG, 20 mM] for 30 min in KRB. Insulin released into the medium was quantitated by ELISA and expressed as ng/mL. Data are mean ± SEM from four independent experiments. * represents p < 0.05 vs. low glucose without secinH3; **p < 0.05 vs. high glucose without secinH3 and data points with similar symbol do not differ statistically. Panel B: Normal rat islets were incubated in low serum-low glucose overnight in the continuous presence of either 50 μM secinH3 or diluent and stimulated with either low [LG, 2.5 mM] or high glucose [HG, 20 mM] for 30 min in KRB. Insulin released into the medium was quantitated by ELISA. Data are expressed as ng/mL insulin released and are mean ± SEM from four independent experiments. * represent p < 0.05 vs. low glucose with secinH3 and without secinH3; and **p < 0.05 vs. high glucose without secinH3. Data points with similar symbol do not differ significantly.

Figure 6. Molecular biological or pharmacological inhibition of ARNO function attenuates glucose-induced Rac1 or Cdc42 activation in INS 832/13 cells

Panel A: INS 832/13 cells were either mock-transfected or transfected with siRNA-ARNO at a final concentration of 100 nM and after 48 hr culture, cells were stimulated with either low glucose [LG, 2.5 mM] or high glucose [HG, 20 mM] for 30 min at 37°C. The relative amounts of activated Rac1 [i.e, Rac1.GTP] were quantitated by PAK-PBD pull down [see Methods for additional details]. Total Rac1 from cell lysates was used as the loading control. Data were analyzed densitometrically and expressed as fold change in Rac1.GTP over basal. Data are mean ± SEM of five independent experiments. * and ^# represent p < 0.05 vs. low glucose without siRNA-ARNO; and **p < 0.05 vs. high glucose without siRNA-ARNO. Panel B: INS 832/13 cells were cultured overnight in the presence or absence of secinH3 [50 μM] and further stimulated with low glucose [LG, 2.5 mM] and high glucose [HG, 20 mM] for 30 min in the continuous presence or absence of secinH3. The relative amounts of activated Rac1 [i.e, Rac1.GTP] were determined by PAK-PBD pull down assay as described in Panel A. Total Rac1 from cell lysates was used as the loading control. Data were analyzed densitometrically and expressed as fold change in Rac1.GTP over basal and are mean ± SEM of three independent experiments. * represents p <0.05 vs. low glucose without secinH3; ** p < 0.05 vs. high glucose without secinH3, and data points with similar symbol do not differ statistically. Panel C: INS 832/13 cells were starved overnight in the presence or absence of secinH3 [50 μM] and were stimulated with low glucose [LG, 2.5 mM] and high glucose [HG, 20 mM] for 3 min in the continuous presence or absence of secinH3. The relative amounts of activated Cdc42 [i.e, Cdc42.GTP] was determined by PAK-PBD pull down assay. Total Cdc42 from cell lysates was used as the loading control. Data were densitometrically analyzed and is expressed as fold change in Cdc42.GTP over basal and are mean ± SEM of three independent experiments yielding similar results. * and ** represents p<0.05 vs. low glucose without secinH3 and ^#p < 0.05 vs. high glucose without secinH3. Panel D: Lysates from rat islets, human islets and INS 832/13 cells were separated by SDS-PAGE and probed for Dbl by Western blotting.

Figure 7. Glucose promotes association between Arf6 and ARNO in INS 832/13 cells

Panel A: Coimmunoprecipitation studies: Herein, INS 832/13 cells were incubated in the presence of low glucose [LG, 2.5 mM] or high glucose [HG, 20 mM] for 30 min at 37°C. ARNO was immunoprecipitated in the lysates using a specific antibody as described in Methods. The immunoprecipitates were separated by SDS-PAGE and probed for Arf6. A representative blot from three studies is shown. Panel B: Data from multiple studies shown in Panel A are analyzed densitometrically and expressed as densitometric units and are mean ± SEM. * represents p <0.05 vs. low glucose. Panel C: Immunofluorescence studies using confocal microscopy: INS 832/13 cells were cultured on coverslips and cultured overnight prior to the incubation with either 2.5 mM or 20 mM glucose for 30 min at 37°C. The cells were fixed in 4% paraformaldehyde solution in PBS for 15 min and permeabilized using 0.2% triton X-100 for 15 min. Fixed cells were examined for Arf6 [stained in green] and ARNO [stained in red] as described under Methods.

Figure 7. Glucose promotes association between Arf6 and ARNO in INS 832/13 cells

Panel A: Coimmunoprecipitation studies: Herein, INS 832/13 cells were incubated in the presence of low glucose [LG, 2.5 mM] or high glucose [HG, 20 mM] for 30 min at 37°C. ARNO was immunoprecipitated in the lysates using a specific antibody as described in Methods. The immunoprecipitates were separated by SDS-PAGE and probed for Arf6. A representative blot from three studies is shown. Panel B: Data from multiple studies shown in Panel A are analyzed densitometrically and expressed as densitometric units and are mean ± SEM. * represents p <0.05 vs. low glucose. Panel C: Immunofluorescence studies using confocal microscopy: INS 832/13 cells were cultured on coverslips and cultured overnight prior to the incubation with either 2.5 mM or 20 mM glucose for 30 min at 37°C. The cells were fixed in 4% paraformaldehyde solution in PBS for 15 min and permeabilized using 0.2% triton X-100 for 15 min. Fixed cells were examined for Arf6 [stained in green] and ARNO [stained in red] as described under Methods.

Figure 8. A proposed mechanism for ARNO signaling axis in GSIS via sequential activation of Arf6, Cdc42 and Rac1 in pancreatic β-cells

Based on the data described herein and our previously published data [22, 23], we propose a model for potential involvement of ARNO as a GEF for Arf6 in promoting GSIS in pancreatic β-cells. We propose that glucose activates islet endogenous ARNO to facilitate the conversition of inactive GDP-bound Arf6 to its GTP-bound biologically active conformation. Essential nature for ARNO in this signaling cascade was demonstrated via the use of molecular biological [E156 mutant and siRNA-ARNO] and pharmacological approaches [e.g., secinH3]. A role for Arf6 in GSIS was confirmed via molecular biological [i.e., Arf6-T27N mutant and siRNA-Arf6] approaches. These data confirm the original observations of Lawrence and Birnbaum [5]. We propose that the activation of ARNO/Arf6 signaling pathway leads to the activation of PLD [43] leading to the generation of fusigenic lipids [e.g., PA], which in turn, promote dissociation of Rac1/GDI complexes leading to the activation and membrane association of Rac1 [22, 23]. Our time course studies also suggest that ARNO-mediated activation of Cdc42 [within 1 min] is unstream to Rac1 activation [15-20 min] together suggesting that ARNO facilitates sequential activation of Arf6, Cdc42 and Rac1 to promote insulin secretion. Potential mechanisms underlying Arf6 mediated activation of Cdc42 remain to be determined. It might include dissociation of Cdc42/GDI complex by biologically-active lipids as in the case of Rac1 [21, 22] or inhibition of GAP activity specific for Cdc42 by ARNO/Arf6 signaling pathway [see Discussion]. These aspects are being investigated in our laboratory currently.