Novel regulatory roles of small G protein GDP dissociation stimulator (smgGDS) in insulin secretion from pancreatic β-cells

Abstract

Emerging evidence implicates novel roles for small G protein GDP dissociation stimulator (smgGDS) in G protein activation and subsequent targeting to relevant subcellular compartments for effector regulation. Given the well-established roles of small G proteins in insulin secretion, we undertook this investigation to determine the putative roles of smgGDS in insulin secretion. Immunoblotting studies revealed that both splice variants of smgGDS are expressed in human islets, rat islets and INS-1 832/13 cells. A significant inhibition (-52%) of glucose-stimulated insulin secretion (GSIS) was observed in INS-1 832/13 cells following siRNA-mediated depletion of smgGDS. In addition, insulin secretion elicited by a membrane depolarizing concentration of KCl (via increased calcium influx), forskolin (via increased cAMP generation) or IBMX (via inhibition of phosphodiesterase) was inhibited by -49%, -27%, and -28%, respectively. Subcellular distribution studies revealed no significant alterations in the abundance of smgGDS in the cytosolic and membrane fractions during the 45-min exposure of INS-1 832/13 cells to an insulinotropic concentration of glucose. Together, we present the first evidence of expression of smgGDS in human islets, rodent islets, and clonal β-cells. We also demonstrate novel regulatory roles of these proteins in insulin secretion derived from glucose metabolic events, including calcium- and cAMP-dependent signaling steps.

Keywords: Insulin secretion; Islet beta cell; RAP1GDS1; Small G proteins; smgGDS.

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

Western blot data demonstrating the expression and antibody specificity of smgGDS in lysates from INS-1 832/13 cells, rat islets, and human islets.

Figure 2:. siRNA-mediated depletion of smgGDS in INS-1 832/13 cells

Panel A: Representative western blot displaying the efficient depletion of smgGDS in cells treated with scrambled siRNA (con-si) or three individual smgGDS siRNA (smg-si1/2/3) duplexes from Origene Technologies at a final concentration of 20 nM using RNAiMax transfection reagent for 72 hours. These data show significant (p value < 0.05) depletion of smgGDS expression under indicated experimental conditions. Panel B: Representative western blot showing significant depletion of smgGDS in cells transfected with smgGDS siRNA (smg-si) acquired from Dharmacon at a working concentration of 100 nM using RNAiMax transfection reagent for 72 hours. These data indicate significant (p value < 0.05) depletion of endogenous smgGDS expression under specified experimental conditions acquired from both siRNA complexes used. β-actin was utilized as loading control.

Figure 3:. Depletion of smgGDS attenuates glucose- and calcium-stimulated insulin secretion in INS-1 832/13 cells.

Panel A: Con-si or smg-si transfected INS-1 832/13 cells were incubated under basal (LG; 2.5 mM) or high (HG; 20 mM) glucose for 45 minutes thereafter insulin content of the media was measured using ELISA. Data presented are mean ± SEM from five independent experiments, each with two replicates; *p < 0.05 and **p < 0.01. Panel B: Con-si or smg-si transfected INS-1 832/13 cells were exposed to basal (LG; 2.5 mM) or KCl (60 mM) for 60 minutes. Insulin content of media was quantified using ELISA. Data presented are mean ± SEM from five independent experiments, each with two replicates. ***p < 0.001, ****p < 0.0001.

Figure 4:. Depletion of smgGDS decreases cAMP-mediated insulin secretion in INS-1 832/13 cells.

Panel A: Representative western blot of smgGDS expression from cells incubated with basal (2.5 mM; LG) or high (20 mM; HG) glucose with and without the presence of IBMX (100 μM) displaying significant depletion of smgGDS in smg-si treated samples. Panel B: Fold change in insulin secretion from con-si or smg-si transfected cells incubated with basal or HG conditions with and without IBMX (100 μM) for 45 minutes. Insulin content of media was quantified using ELISA. Data presented are mean ± SEM from three independent experiments, each with two replicates. *p < 0.01. Panel C: Fold change in insulin secretion from con-si and smg-si transfected cells in either basal or HG conditions, all supplemented with forskolin (2.5 μM) for 45 minutes. Insulin content of media was quantified using ELISA. Data presented are mean ± SEM from six independent experiments, each with two replicates. *p < 0.05 and ***p < 0.001.

Figure 5:. Membrane association of smgGDS following glucose stimulation.

Panel A: Representative western blot (n=3) displaying smgGDS expression between the cytosolic and membrane fractions after treatment with basal (LG; 2.5 mM) or high (HG; 20 mM) glucose for 15- or 45-minutes as indicated. Panel B: Confocal imaging on the localization of smgGDS in INS-1 832/13 cells after treatment with basal (LG; 2.5 mM) or high (HG; 20 mM) for 45 minutes (n=2). These data provide evidence that smgGDS remains at or near the membrane for 45 minutes post glucose exposure.

Figure 6:. Subcellular localization of Rap1 and Epac2 is not altered in smgGDS depleted INS-1 832/13 cells under basal or glucose-stimulated conditions.

Representative western blot (n=3) presenting the expression of Rap1 and its GEF Epac2 from cells that have been depleted of smgGDS and treated with basal (LG; 2.5 mM) or high (HG; 20 mM) glucose for 45 minutes.

Figure 7:. Confocal imaging displaying the expression of total Rap1 and active Rap1 (Rap1-GTP)

Panel A: Representative images (n=2) acquired by confocal microscopy showing the expression and localization of total Rap1 after INS-1 832/13 cells were treated with basal (LG; 2.5 mM) or high (HG; 20 mM) glucose for 45 minutes. Panel B: Representative confocal images (n=2) displaying the active form Rap1-GTP in INS-1 832/13 cells after incubation with basal (LG; 2.5 mM) or high (HG; 20 mM) glucose for 45 minutes.