Novel Roles for Geranylgeranyl Transferase-III (GGTase-III) in Insulin Secretion

Abstract

Background/aims: Post-translational prenylation of G proteins is implicated in physiological insulin secretion. It has been reported recently that GGTase-III participates in the functional regulation of Ykt6, a synaptobrevin homolog, via geranylgeranylation. However, potential localization and putative regulatory roles of GGTase-III in insulin secretion remains unknown. The current study is aimed at determining the expression and contributory roles of GGTase-III in glucose- and KCl-induced insulin secretion from pancreatic β-cells.

Methods: Mouse islets were isolated by the collagenase digestion method. Human islets were from Prodo Laboratories. INS-1 832/13 cells were transfected with either control (scrambled) or siRNA-PTAR1 (the α-subunit of GGTase-III) using lipofectamine RNAiMax. Insulin released into the medium was quantified using a commercially available Insulin ELISA kit. Expression of GGTase-III subunits and ykt6 was determined by Western blotting and quantified by densitometry.

Results: Western blotting revealed that both subunits of GGTase-III (PTAR1 and RabGGTB) are expressed in human islets, mouse islets and INS-1 832/13 cells. Transfection of INS-1 832/13 cells with siRNA-PTAR1 resulted in significant reduction (~50%) in the expression of PTAR1. siRNA-mediated knockdown of PTAR1 significantly attenuated (~60%) glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 cells. Furthermore, insulin secretion elicited via KCl-induced membrane depolarization was markedly reduced (~69%) in INS-1 832/13 cells following PTAR1 depletion. Lastly, immunoblotting data suggested expression of Ykt6, a known substrate for GGTase-III, in human islets, rodent islets, and INS-1 832/13 cells.

Conclusion: GGTase-III-dependent signaling step is necessary for glucose- and KCl-induced insulin secretion.

Keywords: Protein prenylation ; GGTase-III ; Islet β-cell ; Insulin secretion ; G proteins ; Diabetes.

Fig. 1.

Schematic depiction of subunit composition of prenyltransferases and their substrates: At least four types of prenyltransferases, namely FTase-I, GGTase-I, GGTase-II and GGTase-III have been identified in mammalian cells. They exist as heterodimers, comprising of α- and β-subunits. As highlighted in the Fig., FTase and GGTase-I share a common α-subunit, but distinct β-subunits. GGTase-II and GGTase-III (focus of this studies; highlighted in green) share common β-subunit, but distinct α-subunits. PTAR1 represents the α-subunit of GGTase-III. FTase mediates farnesylation of a variety of proteins, including Ras, nuclear lamins and ykt6 (focus of these studies; highlighted in green). GGTase-I promotes geranylgeranylation of a smgs belonging to Rho subfamily, including Rac1, Cdc42 and RhoA. GGTase-II mediates geranylgeranylation of smgs belonging to Rab subfamily. GGTase-III is involved in geranylgeranylation of ykt6 and FBXL2.

Fig. 2.

Immunological evidence for the expression of α- (PTAR1) and β-(RabGGTB) subunits of GGTase III and ykt6 in INS-1 832/13 cells, mouse islets and human islets. Lysates derived from INS-1 832/13 cells, mouse islets and human islets were employed for immunodetection of the α-(PTAR1) and β-(RabGGTB) and ykt6 by Western blotting. β-actin was used as loading control.

Fig. 3.

Depletion of endogenous expression of PTAR1 attenuates glucose- and KCl-induced insulin secretion from INS-1 832/13 cells. Panel A: siRNA-mediated knockdown of PTAR1 in INS-1 832/13 cells: Cells were transfected with either scrambled siRNA (Con-si) and siRNA-PTAR1 (100 nM each) using lipofectamine RNAiMax transfection reagent. The cells were incubated for 72 hours in media containing no antibiotic and Opti-MEM mix to achieve optimal protein depletion. Representative blot showing transfections from duplicate studies is shown here. Panel B: siRNA-mediated knockdown of PTAR1 markedly attenuates GSIS in INS-1 832/13 cells: Con-si or PTAR1-si transfected INS-1 832/13 cells were incubated under low (LG; 2.5 mM) or high (HG; 20 mM) glucose for 45 minutes. Insulin released into the medium was quantified using a commercially available insulin ELISA detection kit. Data are presented as mean ± SEM from four independent studies, each sample having 2 replicates. Lane a: cells transfected with Con-si and incubated with LG; lane b: cells transfected with siRNA-PTAR1 and incubated with LG; lane c: cells transfected with Con-si and incubated with HG; and lane d: cells transfected with siRNA-PTAR1 and incubated with HG. *p<0.05. Panel C: siRNA-mediated depletion of PTAR1 markedly inhibits KCl-induced insulin secretion in INS-1 832/13 cells: Con-si or PTAR1-si transfected INS-1 832/13 cells were exposed to low glucose (LG; 2.5 mM) or KCl (60 mM) for 60 minutes. Insulin released into the medium was quantified using a commercially available insulin ELISA detection kit. Data are presented as mean ± SEM from four independent studies, each sample having 2 replicates. Lane a: cells transfected with Con-si and incubated with LG; lane b: cells transfected with siRNA-PTAR1 and incubated with LG; lane c: cells transfected with Con-si and exposed to KCl; and lane d: cells transfected with siRNA-PTAR1 and exposed to KCl. *p<0.05.

Fig. 4.

Schematic representation of potential mechanisms underlying FTase-GTTase-III mediated double prenylation of ykt6 leading to glucose-stimulated insulin secretion. Published evidence demonstrated an increase in the catalytic activities of prenyltransferases (FTase and GGTase-I) in INS-1 832/13 cells and rat islets under conditions of glucose-stimulated insulin secretion. Pharmacological and molecular biological evidence also implicates protein farnesylation is requisite for GSIS to occur. Data accrued from the current investigations suggest that siRNA-mediated knockdown of PTAR1 culminates in loss of GSIS. Based on these observations we propose that GSIS might involve activation of FTase and GGTase-III leading to double prenylation of ykt at C194 (geranylgeranylation) and C195 (farnesylation) thus favoring translocation and docking of insulin containing secretory granules at the plasma membrane for exocytotic secretion of insulin. Note that this remains to be validated experimentally (see text for additional details).