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. Author manuscript; available in PMC: 2025 Jan 15.
Published in final edited form as: Mol Cell Endocrinol. 2023 Oct 30;580:112104. doi: 10.1016/j.mce.2023.112104

Novel Regulatory Roles of Small G Protein GDP Dissociation Stimulator (smgGDS) in Insulin Secretion from Pancreatic β-Cells

Noah Gleason a,b, Carol L Williams c, Anjaneyulu Kowluru a,b
PMCID: PMC10842139  NIHMSID: NIHMS1942820  PMID: 38013223

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-minute 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: smgGDS, small G proteins, Insulin secretion, Islet beta cell, RAP1GDS1

1. Introduction

It is well established that GSIS from pancreatic β-cells requires the generation of soluble second messengers including cyclic nucleotides (e.g., cAMP), biologically active hydrolytic products of phospholipases (diacyl glycerol and inositol triphosphate), adenine nucleotides (e.g., ATP) and guanine nucleotides (e.g., GTP). Intracellular generation of inositol triphosphates leads to mobilization of calcium from the endoplasmic reticulum to the soluble compartment, which is essential for the transport of insulin-laden secretory granules to the plasma membrane for fusion and exocytotic secretion of insulin into circulation (MacDonald 1990, Newgard and McGarry 1995, Prentki, Matschinsky et al. 2013). Extant studies have demonstrated novel roles for small GTP-binding proteins (G proteins) belonging to the Rho, Ras, Rab and Arf subfamilies in insulin secretion, primarily via modulation of cytoskeletal remodeling and vesicle transport and fusion with the plasma membrane culminating in insulin secretion. Along these lines, previous studies have demonstrated roles for a number of GTP/GDP exchange factors (GEFs), including Tiam1, β-PIX and ARNO, in G protein-mediated insulin secretion (Kowluru 2020, Veluthakal and Thurmond 2021). Moreover, using a variety of pharmacological and molecular biological approaches, previous studies have demonstrated that post-translational modifications (e.g., prenylation) of Rho and Rab G proteins are necessary for insulin secretion to occur (Kowluru and Kowluru 2015, Kowluru 2020, Veluthakal and Thurmond 2021) .

In the context of functional regulation of small G proteins, emerging evidence implicates novel roles for small G protein GDP-dissociation stimulator (smgGDS) in the functional activation of a variety of small G proteins. Original studies by Yamamoto and coworkers have reported functionally active smgGDS (~ 61 kDa) in bovine brain cytosol (Yamamoto, Kaibuchi et al. 1990), which was later cloned by Kaibuchi et al. (Kaibuchi, Mizuno et al. 1991). Subsequent investigations have demonstrated GEF-like properties for smgGDS toward Rho subfamily of G-proteins, including Rac1, RhoA and Rap1B (Hiraoka, Kaibuchi et al. 1992, Williams 2003). Investigations by Chuang et al. have revealed that smgGDS stimulates GTP/GDP exchange via stabilization of nucleotide-bound and -free forms of Rac1 (Chuang, Xu et al. 1994). Subsequent studies by Hamel and coworkers have reported specific activation of RhoA and RhoC by smgGDS (Hamel, Monaghan-Benson et al. 2011). More recent investigations by Bergom and associates have revealed binding of DiRas1 to smgGDS, thereby antagonizing the interaction of smgGDS to other oncogenic small G proteins (Bergom, Hauser et al. 2016). Together, these studies provided compelling evidence for regulatory control of G protein activation by smgGDS.

In addition to its GEF-like properties for smgGDS, several recent investigations have implicated novel roles for smgGDS in the regulatory control of newly synthesized small G protein prenylation signaling modules (Brandt, Koehn et al. 2021). Two splice variants of smgGDS have been reported, each with specific and individual properties differentiating their intrinsic functionality. smgGDS-607 binds unprenylated small G proteins while smgGDS-558, which lacks one of the thirteen armadillo domains, binds prenylated small G proteins (Brandt, Koehn et al. 2021). These data display a prominent role in the prenylation pathway of small G proteins in both transport to the prenylation machinery (smgGDS-607) and from the site of prenylation to subsequent downstream localization (smgGDS-558). It is noteworthy that recent studies by Garcia-Torres and Fierke have defined dual roles for smgGDS-607 in the activation and inhibition of farnesylation of H-Ras and DiRas1, respectively, suggesting multiple modes of substrate recognition by smgGDS (García-Torres and Fierke 2019).

Given the critical regulatory roles for post-translational prenylation and GEF-mediated activation of small G proteins in insulin secretion (as above) and based on emerging roles of smgGDS as a mediator of these two signaling pathways/steps, we undertook the current investigation to decipher roles for smgGDS in insulin secretion in INS-1 832/13 cells. Specifically, we assessed the roles of smgGDS in glucose-, KCl (calcium-induced), and forskolin/isobutyl-methylxanthine (cAMP-mediated) insulin secretion. We present the first evidence of expression of smgGDS in human islets, rodent islets, and clonal β-cells. We also report novel roles of these proteins in insulin secretion derived from glucose metabolic events, including calcium- and cAMP-dependent signaling steps.

2. Materials and Methods

2.1. Chemicals and reagents

Antibodies specific for smgGDS (RAP1GDS1; sc-390003), Rap1 (sc-398755), E-cadherin (sc-8426), FTase/GGTase-α unit (sc-23906), Vav2 (sc-20803), and β-PIX (sc-393184) were from Santa Cruz Biotechnology (Dallas, TX, USA). GAPDH (5174S), Epac2 (4156S), and HRP-conjugated secondary antisera were from Cell Signaling (Danvers, MA, USA). β-actin (A1974) antibody and normal goat serum were from Sigma Aldrich (St. Louis, MO, USA). Rap1.GTP (#26912) antibody was acquired from NewEast Bio (King of Prussia, PA, USA). smgGDS oligonucleotide siRNA duplexes and non-targeting scrambled siRNA were from Origene Technologies (Rockville, MA, USA). A second set of siRNA for smgGDS was purchased from Dharmacon (Lafayette, CO, USA). Lipofectamine RNAiMax transfection reagent and the MEM-Per Plus cytosolic and membrane extraction kit were obtained from Thermo Fischer Scientific (Carlsbad, CA, USA). Rat Insulin ELISA kit was purchased from ALPCO (Salem, NH, USA). Forskolin and IBMX were acquired from Cayman Chemical (Ann Arbor, MI, USA). Goat anti-mouse IgG FITC (ab6785) conjugated secondary antibody was obtained from Abcam (Waltham, MA, USA). ProLong Gold Antifade mountant with DAPI was from Invitrogen (Waltham, MA, USA).

2.2. INS-1 832/13 cells, rat islets, and human islets

Insulin-secreting INS-1 832/13 cells were obtained from Sigma Aldrich (St. Louis, MO, USA). Rat islets were isolated from Sprague-Dawley rats by the collagenase digestion technique (Jayaram, Syed et al. 2011, Arora, Syed et al. 2012, Thamilselvan and Kowluru 2019). All protocols were reviewed and approved by the Institutional Animal Care and Use Committees at Wayne State University and the John D. Dingell VA Medical Center. Human islets were acquired from Prodo Laboratories (Aliso Viejo, CA, USA). Studies involving human islets studies were approved by the Biosafety Committee of John D. Dingell VA Medical Center.

2.3. Cell culture and experimental conditions

INS-1 832/13 cells were cultured in RPMI-1640 media containing 10% FBS supplemented with 11.1 mM glucose, 100 IU/mL penicillin and 100 IU/mL streptomycin, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 10 mM HEPES (pH 7.4-7.6). Cultured cells were sub-cloned weekly by way of trypsinization. Cells were starved overnight in a low glucose/low serum growth media (2.5 mM glucose and 2.5% FBS) before specified treatment conditions exposure.

2.4. siRNA-mediated depletion of smgGDS

Endogenous expression of smgGDS was suppressed by siRNA transfection according to manufacturer’s instructions. Scrambled siRNA duplexes were used as control. Transfected cells were kept in growth medium containing no antibiotic for 72 hours prior to exposure to any treatment conditions. The degree of smgGDS knockdown (KD) was confirmed by western blot analysis.

2.5. Western Blotting

Following incubation under specific experimental conditions, cells were collected and lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 hour at room temperature with 3% BSA in PBS-T (0.1%). Nitrocellulose membranes were then incubated in primary antibody for desired proteins of interest overnight at 4 °C (1:2000 dilution for smgGDS, 1:5000 dilution for β-actin and GAPDH, and 1:1000 dilution for all other antibodies; dilutions were prepared in PBS-T containing 1.5% BSA). Primary antibodies were removed and washed with PBS-T (3 times for 5 minutes each) and then probed with appropriate secondary HRP-conjugated antibodies in PBS-T containing 1.5% BSA for 1 hour at room temperature. Secondary antibodies were removed, and membranes were washed in PBS-T (3 times for 10 minutes each). Proteins of interest were detected by chemiluminescence, and band intensities were quantified by densitometry.

2.6. Insulin Secretion Assay

Control-si or smgGDS-si transfected cells were cultured overnight in media containing 2.5 mM glucose and 2.5% fetal bovine serum. Following pre-incubation in Krebs Ringer Bicarbonate Buffer (KRB; pH 7.4) for 1 hour, insulin secretion was stimulated in KRB supplemented with either glucose, KCl, forskolin, or IBMX. GSIS studies were conducted in the presence of low glucose (LG; 2.5 mM) or high glucose (HG; 20 mM) for 45 minutes at 37 °C. For KCl-induced insulin secretion, cells were incubated with LG (2.5 mM) or a stimulatory concentration of KCl (60 mM) for 60 minutes at 37 °C. For forskolin-, and IBMX-induced insulin secretion studies, cells were incubated in the presence of LG (2.5 mM) or HG (20 mM) with and without forskolin (2.5 μM) or IBMX (100 μM) for 45 minutes at 37 °C. Insulin secretion was quantified by ELISA per the manufacturer’s provided protocol. Data are presented as fold change from LG or LG supplemented with forskolin as indicated in text.

2.7. Isolation of Cytosolic and Membrane Fractions

INS-1 832/13 cells and rat islets were incubated under LG (2.5 mM) or HG (20 mM) conditions for 15- or 45-minutes as indicated in text. Cytosolic and membrane fractions were isolated using the Mem-PER Plus Extraction kit per manufacturer’s protocol as we described in (Thamilselvan, Gamage et al. 2020). Purity of cytosolic and membrane fractions was determined by the relative abundance of GAPDH and E-cadherin in these fractions, respectively.

2.8. Confocal Immunofluorescence

INS-1 832/13 cells were incubated with LG (2.5 mM) or HG (20 mM) for 45 minutes on chamber slides. Cells were fixed with 100% MeOH for 10 minutes at −20°C, washed with PBS for 5 minutes with gentle agitation, and blocked in 5% normal goat serum in PBS with 1% Triton X-100 added for 1 hour at room temperature. Blocking solution was aspirated and primary antibody was added (1:200) in PBS with 0.1% Triton X-100 for 2 hours at room temperature. Primary antibody was aspirated, and cells were washed 3 times with PBS with 0.1% Triton X-100 for 5 minutes each. Secondary FITC-conjugated antibody (1:500) was added for 1 hour with light exposure minimalized. Cells were subsequently washed 3 times in PBS with 0.1% Triton X-100 and mounted with DAPI then left overnight at 4 °C. Images were obtained with a 63x oil objective using a Zeiss Axio Examiner.Z1 upright microscope. Image analysis was completed using Volocity 7 software. All confocal microscopy studies were conducted at the Microscopy, Imaging and Cytometry Resources Core at Wayne State University School of Medicine.

2.9. Statistical Analysis

Data were analyzed using GraphPad Prism software version 9.5 (GraphPad Software, San Diego, CA). Data are presented as mean ± standard error of mean (SEM) from three or more independent experiments. Comparisons between two groups were done with a two-tailed Student t-test while studies incorporating multiple groups were analyzed with one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. A p value below 0.05 was considered significant.

3. Results

At the outset, we determined, by Western blotting, the expression of splice variants of smgGDS (i.e., smgGDS-607 and smgGDS-558) in insulin-secreting INS-1 832/13 cells, normal rat islets and human islets (from a non-diabetic donor). Data depicted in Figure 1 indicated that both splice variants of smgGDS are expressed in all three insulin-secreting cells studied. We next asked if smgGDS contributes to insulin secretion. To address this question, we quantified GSIS in INS-1 832/13 cells following knockdown (KD) of smgGDS expression using siRNA-smgGDS (smg-si). Data in Figure 2 (Panel A) represent the extent of KD of smgGDS in INS-1 832/13 cells following transfection of three individual duplexes of smgGDS provided by the vendor (Origene). We also confirmed the degree of smgGDS KD using another set of smg-si from a second vendor (Dharmacon; Figure 2 Panel B). These data indicated a significant reduction in the expression of smgGDS in INS-1 832/13 cells transfected with either smg-si. It should be noted that the smg-si we employed in these studies significantly depleted expression of both splice variants of interest, smgGDS-607 and smgGDS-558. We observed 70%, 85% and 45% inhibition of smgGDS expression with Origene Duplex 1, 2, and 3, respectively. Transfection of cells with Dharmacon siRNA yielded nearly 90% inhibition of the expression of smgGDS in INS-1 832/13 cells. It should also be noted that both smg-si duplexes we utilized in the current studies exerted no off-target effects as evidenced by no significant effects of smgGDS KD on the expression and subcellular distribution (i.e., membrane vs. cytosol) of multiple signaling proteins, which have been implicated in GSIS, including the common α subunit of farnesyl and geranylgeranyl transferase-1 (a G protein prenyl transferase), Vav2 (a GEF for Rac1), β-PIX (a GEF for Rac1 and Cdc42; additional data not shown). These findings further affirmed specificity of the KD method, and no off-target effects of smgGDS KD on key signaling proteins involved in physiological insulin secretion.

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

Figure 1:

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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.

Figure 2:

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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.

We next determined regulatory roles of smgGDS in GSIS. Data depicted in Figure 3A indicated significant inhibition (~52%) of GSIS in cells transfected with smg-si (bars 3 vs. 4). No significant effects of smg-si were noted on insulin secretion seen under basal (LG) conditions (bars 1 vs. 2). These data suggested that smgGDS plays critical roles in GSIS.

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

Figure 3:

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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.

In the next series of studies, we investigated roles of smgGDS in insulin secretion from INS-1 832/13 cells elicited by a membrane depolarizing concentration of KCl, which represents calcium-induced insulin secretion. Data in Figure 3B showed that, in a manner akin to GSIS, KCl-induced insulin secretion was significantly attenuated (~49%) following siRNA-mediated KD of smgGDS (bars 3 vs. 4). In line with data in Figure 3A, no significant effects on basal insulin secretion from cells transfected with control siRNA were seen in this set of experiments (Bars 1 vs. 2). Together, these data revealed novel regulatory roles of smgGDS in the cascade of events leading to calcium-induced insulin secretion from INS-1 832/13 cells.

We next examined roles of smgGDS in cAMP-mediated insulin secretion. This was accomplished by quantifying insulin secretion in INS-1 832/13 cells in the presence of IBMX, a known inhibitor of phosphodiesterase (i.e., prevention of cAMP degradation). Data depicted in Figure 4A demonstrated significant KD of smgGDS under the conditions where we performed insulin secretion studies. Under these conditions, we noted a significant increase in insulin secretion induced by HG and IBMX (bars 3 vs 7; fold increase ~3.2). siRNA-mediated depletion of smgGDS significantly decreased insulin secretion by approximately 28% (bars 7 vs 8) under these experimental conditions.

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

Figure 4:

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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.

In an alternate approach, we quantified the effects of smgGDS-KD on insulin secretion promoted by HG and forskolin, a known activator of adenylate cyclase (i.e., cAMP generation). Data provided in Figure 4C demonstrated ~ 2-fold increase in insulin secretion compared to LG supplemented with forskolin in cells transfected with control siRNA (Bars 1 vs. 3). A significant inhibition of HG plus forskolin-induced insulin secretion (~27%) was seen in INS-1 832/13 cells transfected with siRNA-smgGDS (bars 3 vs. 4). Again, basal secretion remained unaltered in these cells following transfection with control siRNA (Bars 1 vs. 2). Taken together, data represented in Figures 3-4 suggested novel roles for smgGDS in glucose-induced, and calcium- and cAMP-mediated insulin secretion from INS-1 832/13 cells.

We next investigated putative mechanisms underlying smgGDS-mediated events leading to GSIS. In the first set of experiments, we examined potential alterations, if any, in the subcellular distribution of smgGDS in INS-1 832/13 cells under conditions conducive for GSIS. To accomplish this, we determined relative abundance of smgGDS in membrane and cytosolic fractions isolated from INS-1 832/13 cells (see Methods for additional details) exposed to either basal or stimulatory glucose concentrations for 15- and 45-minutes. To verify the purity of the membrane and cytosolic fractions, we determined the abundance of E-cadherin and GAPDH, respectively. These data indicated presence of E-cadherin only in the membrane fraction further affirming the purity of these fractions (Figure 5A). In addition, we observed no significant changes in the membrane associated E-cadherin in cells exposed to basal and stimulatory glucose concentration. It is noteworthy that, in addition to its presence in the cytosolic compartment, we observed GAPDH in the membrane fraction, presumably via its binding to certain integral membrane proteins in the membrane (Thamilselvan and Kowluru 2019). In a manner akin to E-cadherin, despite low abundance of GAPDH in the membrane fraction derived from cells at 45 minutes, we observed no significant changes in its abundance under basal and stimulatory conditions. Data shown in Figure 5A indicated a modest increase in the membrane association of smgGDS in glucose-stimulated cells at the 15-minute time point. smgGDS appeared to remain associated with the membrane up to 45 minutes, even though no significant differences were seen in its abundance in the membrane fraction derived from INS-1 832/13 cells incubated with either basal or stimulatory glucose. We next verified these observations via confocal image analyses in INS-1 832/13 cells following exposure to basal or stimulatory glucose (45 min). Data depicted in Figure 5B suggested no discernable changes in the distribution of smgGDS under these conditions, as it appeared to be diffused throughout the cell with no evidence of nuclear association.

Figure 5: Membrane association of smgGDS following glucose stimulation.

Figure 5:

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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.

In the second set of investigations, we determined the identity of putative small G proteins that might be involved in smgGDS-mediated insulin secretion. Earlier studies from multiple laboratories, including our own, have demonstrated the requisite nature of activation of small G proteins in GSIS, including those belonging to the Rho (Cdc42, Rac1, RhoA), Ras (Rap1), Rab (Rab3A, Rab27) and ADP-ribosylation factor (Arf6) sub families (Kowluru 2010, Kowluru 2020, Veluthakal and Thurmond 2021). It should be noted that using a variety of pharmacological and gene KD approaches, these studies have demonstrated that activation of Rac1 is not requisite for calcium-mediated (KCl-induced) insulin secretion (Veluthakal, Kaur et al. 2007, Kowluru 2011, Asahara, Shibutani et al. 2013, Kowluru 2020, Veluthakal and Thurmond 2021). Published evidence also suggests that glucose- and KCl promote activation of the carboxyl methylation (CML) of Rap1 at its C-terminal cysteine, which is critical for its interaction with various effector proteins (Leiser, Efrat et al. 1995, Kowluru, Li et al. 1997). Lastly, studies by Shibasaki et al have demonstrated cAMP-mediated, protein kinase A-independent activation of Rap1 in pancreatic islets (Shibasaki, Takahashi et al. 2007). Several studies have demonstrated cAMP-mediated activation of Epac2-Rap1 signaling module in pancreatic β-cells (Leech, Chepurny et al. 2010, Takahashi, Shibasaki et al. 2015). Epac2 itself has been reported in multiple studies to participate in insulin secretion, including co-stimulation of incretins or glucose with forskolin or IBMX (Sugawara, Shibasaki et al. 2009, Hameed, Hafizur et al. 2019) . Therefore, based on the data accrued from aforementioned studies, and our current observations of significant inhibition of glucose-, KCl-, forskolin-, and IBMX-induced insulin secretion following depletion of smgGDS, we undertook the next set of investigations to examine putative roles of Rap1 in smgGDS-mediated effects in pancreatic β-cells leading to GSIS. First, we determined potential impact of smgGDS-KD on membrane vs. cytosolic distribution of Rap1 in INS-1 832/13 cells exposed to basal and stimulatory glucose concentrations. Data in Figure 6 showed significant KD of smgGDS in both cytosolic and membrane fractions isolated from INS-1 832/13 cells transfected with smg-si (top panel). Interestingly, Rap1 appeared to be exclusively associated with the membrane fraction in these cells, and no significant effects of smgGDS KD were demonstrated on the expression of Rap1 in the membrane fractions derived from either basal or glucose-stimulated cells (second panel). Furthermore, Epac2, a known GEF for Rap1, was distributed in both membrane and cytosolic fractions, and smgGDS KD resulted in a modest inhibition of Epac2 expression in the cytosolic fraction derived from cells treated with stimulatory glucose. A modest, but insignificant increase in the relative abundance of Epac2 was seen in membrane fraction from cells exposed to stimulatory glucose. Taken together, these findings revealed no significant effects of smgGDS depletion on the distribution (membrane vs. cytosolic) of Rap1 and Epac2 under basal or glucose-stimulated conditions.

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.

Figure 6:

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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.

We next assessed the degree of activation of Rap1 in glucose-stimulated cells under conditions conducive for GSIS. This was accomplished via confocal imaging using an antibody directed against Rap1.GTP (active conformation). Data depicted in Figure 7A indicated no significant effects on stimulatory glucose on distribution of total Rap1. Interestingly, however, we noted a modest increase in the Rap1.GTP in cells exposed to stimulatory glucose concentrations. It should be noted that, compatible with Western blot data (figure 6) the relative abundance of Rap1.GTP appears to be more in the vicinity of membrane in INS-1 832/13 cells exposed to stimulatory glucose (Figure 7B; indicated by arrows). Based on these data we conclude that stimulatory glucose concentrations might promote activation of membrane associated Rap1 (see below).

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

Figure 7:

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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.

4. Discussion

The main objective of our studies was to assess the regulatory roles of smgGDS in pancreatic islet β cell function, specifically insulin secretion. To address this question, we employed siRNA methodology to selectively deplete endogenous expression of smgGDS. As stated above, we were able to significantly KD the expression of two splice variants of smgGDS. Our findings suggested novel roles for smgGDS in glucose-, calcium-, and cAMP-induced insulin secretion. As stated, smgGDS has been shown to regulate G protein function, including serving as a chaperone for pre-and prenylated G proteins. In the context of regulatory roles of G protein prenylation, several earlier studies have provided compelling evidence in support of the formulation that prenylation of small G proteins (e.g., H-Ras, Rac1 and Rab) is critical for GSIS to occur (Kowluru 2010, Kowluru 2020, Veluthakal and Thurmond 2021). In addition, extant investigations have provided compelling evidence to implicate post-translational CML of small G proteins (e.g., Rap1) in insulin secretion (Kowluru, Li et al. 1997, Kowluru 2020). For example, studies by Leiser and coworkers have reported GTP-dependent CML and membrane association of Rap1 in insulin-secreting β-TC cells (Leiser, Efrat et al. 1995). In addition, they reported an increase in the CML of Rap1 by glucose and a depolarizing concentration of KCl, which was inhibited by D600, a known calcium channel inhibitor. Using acetyl farnesyl cysteine, a known inhibitor of G protein CML, these researchers have implicated activation of Rap1 as requisite step in glucose and calcium-induced insulin secretion (Leiser, Efrat et al. 1995). Shortly thereafter, we provided additional evidence for regulatory roles of Rap1 in glucose and KCl-induced insulin secretion in intact rat islets and insulin-secreting HIT-T15 cells (Kowluru, Li et al. 1997). It may be germane to point out that, compatible with data shown in the current studies, we have shown that Rap1 was predominantly associated with the membrane fraction, and that exposure to GTPγS or inhibitors of CML did not result in alterations in the membrane association of Rap1 as late as 45 min incubation (Kowluru, Li et al. 1997). Based on these data we concluded that the CML of Rap1 may be required for calcium-dependent events, a conclusion compatible with observations of Leiser et al. (Leiser, Efrat et al. 1995). Interestingly, studies in human neutrophils have suggested critical roles for calcium and diacylglycerol in Rap1 activation, further affirming roles for calcium in Rap1 activation (M'Rabet, Coffer et al. 1998). Additional studies are needed, however, to assess the role of smgGDS in targeting Rap1 to the relevant subcellular compartment (e.g., membrane) for effector regulation leading to insulin secretion. In this context of regulatory roles of Rap1 in islet function, it is likely that it could contribute to the functional activation of phagocyte-like NADPH oxidase (Nox2) to generate superoxide anion transiently during glucose stimulation to facilitate cytoskeletal rearrangement and secretory granule transport to the membrane for fusion and secretion of insulin (Morgan, Rebelato et al. 2009, Newsholme, Morgan et al. 2009, Syed, Kyathanahalli et al. 2011). Indeed, several earlier investigations in other cell types have provided evidence in support of critical roles for Rap1 in the regulation of Nox2 (Vignais 2002, Li, Kim et al. 2012). In addition, Rap1 has been implicated in a variety of islet β-cell functions, including cell proliferation via the mammalian target of rapamycin complex 1 signaling pathway (Kelly, Bailey et al. 2010). Published evidence also implicates cAMP/Epac2 module in fusion pore expansion during exocytotic secretion of insulin (Guček, Gandasi et al. 2019). Future investigations will define roles for smgGDS-Rap1 signaling module, which we reported herein, in the cascade of events leading to insulin secretion.

Lastly, extant studies have suggested that smgGDS plays a role as a GEF for a variety of small G proteins, including Rap1A, Rap1B, K-Ras4B, RhoA, RhoC, Rac1, Rac2 and Cdc42, even though such a postulation remains under debate since more recent studies from Hamel et al. suggest that smgGDS acts in a GEF manner for only RhoA and RhoC (Hamel, Monaghan-Benson et al. 2011). In addition, studies of Shin and coworkers have reported have demonstrated critical requirement for smgGDS-mediated β-PIX-induced activation of Rac1 in the cascade of events leading to fibroblast growth factor-induced neurite outgrowth (Shin, Lee et al. 2006). Potential cross talk between smgGDS and β-PIX to promote insulin secretion might represent a fruitful area of investigation, especially based on studies of Kepner et al. demonstrating novel roles of β-PIX as a GEF to promote activation of Cdc42 leading to GSIS (Kepner, Yoder et al. 2011).

5. Conclusion

Based on these data we conclude that smgGDS plays key regulatory roles in glucose-induced, calcium- and cAMP-mediated signaling events leading to insulin secretion. We postulate that Rap1 might represent one of the target proteins that is under the regulatory control of smgGDS. However, several questions remain to be addressed including potential roles of smgGDS in facilitating functional regulation of G proteins leading to insulin secretion. Lastly, we realize that it is necessary to further validate roles of smgGDS in insulin secretion elicited by glucose, calcium and cAMP in rodent islets following knockdown of each of the two splice variants using adenoviral transfection protocol. If successful, it would be ideal to further evaluate this formulation the mouse model in which smgGDS is conditionally deleted in β-cells.

Highlights.

  • Both splice variants of Small G protein GDP-dissociation stimulator (smgGDS-558 and smgGDS-607) are expressed in human islets, rat islets and INS-1 832/13 cells.

  • siRNA-mediated depletion of these splice variants of smgGDS attenuates glucose, KCl, and forskolin/IBMX-induced insulin secretion in INS-1 832/13 cells.

  • We postulate that Rap1 might represent one of the target proteins that is under the regulatory control of smgGDS.

Acknowledgments

This research is supported by a Merit Review Award (BX004663) from the US Department of Veterans Affairs and the National Institutes of Health; EY022230 to AK and CA188871 to CLW. AK is the recipient of a Senior Research Career Scientist Award (K6 BX005383) from the US Department of Veterans Affairs. AK would like to thank Wayne State University for Distinguished Professorship award. NG is supported by a T32 predoctoral fellowship from the Detroit Cardiovascular Research Training Program (NIH-2T32HL120822). The authors thank the Microscopy, Imaging and Cytometry Core of Wayne State University, which is supported, in part, by NIH center grants P30 CA22453 to the Karmanos Cancer Institute and R50 CA251068-01 to Dr. Moin, Wayne State University, and the perinatology Research Branch of the National Institutes of Child Health and Development.

Abbreviation Used

Arf6

ADP-ribosylation factor 6

CARD9

Caspase recruiting domain containing protein 9

Cdc42

Cell division control protein 42

CML

Carboxylmethylation

Epac2

Exchange protein activated by cyclic-AMP

GEF

Guanine nucleotide exchange factor

GSIS

Glucose-stimulated insulin secretion

GDP

Guanosine diphosphate

GTP

Guanosine triphosphate

HG

High glucose

IBMX

Isobutylmethylxanthine

LG

Low glucose

Rac1

Ras related C3 botulinum toxin substrate 1

Rap1

Ras-related protein 1 or Ras proximate protein 1

smgGDS

Small G protein GDP dissociation stimulator

Footnotes

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CrediT authorship contribution statement

Noah Gleason: participated in Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing.

Carol L. Williams: participated in Conceptualization and Writing – review and editing.

Anjaneyulu Kowluru: participated in Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, writing – review and editing. All authors have read and agreed to the published version of the manuscript.

Portions of these findings were presented in the Annual Meetings of the American Society of Biochemistry and Molecular Biology held in Seattle in March 2023 (J Biol Chem 299 (3S) S717, Abstract 1465).

Statement of Ethics

All protocols involving animal care and use were reviewed and approved by Wayne State University and John D. Dingell VA Medical Center Institutional Animal Care and Use Committees. Studies involving human islets were approved by the Biosafety Committee at the John D. Dingell VA Medical Center.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.

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