Ablation of GPR56 Causes β-Cell Dysfunction by ATP Loss through Mistargeting of Mitochondrial VDAC1 to the Plasma Membrane

The activation of G Protein-Coupled Receptor 56 (GPR56), also referred to as Adhesion G-Protein-Coupled Ceceptor G1 (ADGRG1), by Collagen Type III (Coll III) prompts cell growth, proliferation, and survival, among other attributes. We investigated the signaling cascades mediating this functional effect in relation to the mitochondrial outer membrane voltage-dependent anion Channel-1 (VDAC1) expression in pancreatic β-cells. GPR56KD attenuated the Coll III-induced suppression of P70S6K, JNK, AKT, NFκB, STAT3, and STAT5 phosphorylation/activity in INS-1 cells cultured at 20 mM glucose (glucotoxicity) for 72 h. GPR56-KD also increased Chrebp, Txnip, and Vdac1 while decreasing Vdac2 mRNA expression. In GPR56-KD islet β-cells, Vdac1 was co-localized with SNAP-25, demonstrating its plasma membrane translocation. This resulted in ATP loss, reduced cAMP production and impaired glucose-stimulated insulin secretion (GSIS) in INS-1 and human EndoC βH1 cells. The latter defects were reversed by an acute inhibition of VDAC1 with an antibody or the VDAC1 inhibitor VBIT-4. We demonstrate that Coll III potentiates GSIS by increasing cAMP and preserving β-cell functionality under glucotoxic conditions in a GPR56-dependent manner by attenuating the inflammatory response. These results emphasize GPR56 and VDAC1 as drug targets in conditions with impaired β-cell function.


Introduction
The prevalence of metabolic diseases, in particular obesity and Type 2 diabetes (T2D), is high among the older population in western countries and is also increasing among the young around the globe. A resistance to insulin in its target organs, together with pancreatic β-cell dysfunction, plays a central role in the development of the metabolic syndrome and T2D [1][2][3]. Many studies have shown that genetic and environmental factors are interconnected in promoting the development of the disease through the failure of the β-cells to increase insulin secretion in compensation for resistance to the hormone [1,3]. In most of these conditions, suboptimal blood glucose control and dyslipidemia during years of prediabetes lead to β-cell dysfunction, dedifferentiation, and, eventually, apoptosis [1,4,5]. Thus, the prevention of β-cell dysfunction would have a great impact on the prevention of the metabolic syndrome and T2D development [5][6][7].
Glucotoxicity induces cellular stress in β-cells, such as the activation of inflammatory signals, oxidative stress, and endoplasmic reticulum stress, ultimately impairing the functions of vital subcellular organelles, including mitochondria [8,9]. Since β-cell mitochondria Female mice (C57/bl) (Janvier Laboratory, Saint Isle, France) weighing 25-30 g were used under standard conditions (12 h light/dark cycle, 22 • C) with access to standard pellet diet (B&K) and water ad libitum. The study protocol was approved by the Ethics Committee for Animal Research at Lund University (1057/2020). Isolation of pancreatic islets was performed by retrograde injection of a collagenase solution via the pancreatic duct and islets, which were then collected under a stereomicroscope at room temperature [27].

INS-1 832/13 Cell Culture
INS-1 832/13 cells (kindly donated by Dr. C. B. Newgaard, Duke University, Durham, NC, USA) were cultured in RPMI-1640 containing 11.1 mM of D-glucose and supplemented with 10% fetal bovine serum, 100 U/mL of penicillin (Gibco, BRL, Gaithersburg, MD, USA), 100 µg/mL of streptomycin (PAA Laboratories, Toronto, Ontario, Canada), 10 mM of N-2 hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2 mM of glutamine, 1 mM of sodium pyruvate, and 50 µM of β-mercaptoethanol (Sigma Aldrich, Saint Louis, MO, USA) at 37 • C in a humidified atmosphere containing 95% air and 5% CO2. For an assessment of the effects of elevated glucose (glucotoxicity), cells were treated with 20 mM of D-glucose (20G) for 72 h. Controls were maintained in 5 mM of glucose (5G) media. For GPR56 knockdown by siRNA, INS1 832/13 cells were cultured to 75% confluency and then subjected to GPR56-KD as previously described [18]. The siRNAs used for Gpr56-KD are shown in Supplementary Table S1. Each treatment was carried out in three biological replicates, along with scramble controls. Thereafter, the cells were washed and cultured for 6 h in a normal RPMI medium (recovery period) before being cultured in RPMI-1640 with 5 or 20 mM of glucose (5 G or 20 G) in the presence or absence of 20 ug/mL of Collagen-III (Col III) for 72 h (Figure 1), or they were subjected to incubation with indicated agents (see results Section 3.4).
2.5. Knockdown of GPR56 in Mouse Islets, INS-1 832/13, and EndoC βH1 Cells GPR56-KD in mouse islets was performed using a cocktail of three different Lentivirus delivered by shRNAs targeting the GPR56 gene (Santa Cruz, CA, USA), as described previously in [18]. For downregulation of GPR56 in INS-1 832/13 cells and EndoC βH1 cells, siRNA from ThermoFisher Scientific (Wilmington, DE, USA), with an appropriate scrambled control, were used according to the manufacturer's recommendations. Validation of GPR56-KD was determined by qPCR and confocal microscopy, as also described previously [18]. The siRNAs, or shRNAs used for GPR56-KD, are shown in Supplementary  Table S1.

Knockdown of GPR56 in Mouse Islets, INS-1 832/13, and EndoC βH1 Cells
GPR56-KD in mouse islets was performed using a cocktail of three different Lentivirus delivered by shRNAs targeting the GPR56 gene (Santa Cruz, CA, USA), as described previously in [18]. For downregulation of GPR56 in INS-1 832/13 cells and EndoC βH1 cells, siRNA from ThermoFisher Scientific (Wilmington, DE, USA), with an appropriate scrambled control, were used according to the manufacturer's recommendations. Validation of GPR56-KD was determined by qPCR and confocal microscopy, as also described previously [18]. The siRNAs, or shRNAs used for GPR56-KD, are shown in Supplementary Table S1.

Determination of Intracellular Pathways
The Effect of Collagen Type III (Col III) on the major signaling pathways was determined in scramble control (Scr control) and Gpr56-KD INS-1 832/13 cells cultured at 5 or 20 mM of glucose for 72 h. Detection of phosphorylated P70S6K, JNK, AKT, NFκb, STAT3, and STAT5 was assayed on the cell extracts by Luminex, according to the manufacturer's protocol.

Determination of Intracellular Pathways
The Effect of Collagen Type III (Col III) on the major signaling pathways was determined in scramble control (Scr control) and Gpr56-KD INS-1 832/13 cells cultured at 5 or 20 mM of glucose for 72 h. Detection of phosphorylated P70S6K, JNK, AKT, NFκb, STAT3, and STAT5 was assayed on the cell extracts by Luminex, according to the manufacturer's protocol.

Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was extracted from INS1 832/13 and EndoC-βH1 cells using RNAeasy (Qiagen, Hilden, Germany) before complementary DNA (cDNA) was synthesized using SuperScript (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. Concentration and purity of total RNA were measured with a NanoDrop ND-1000 spectrophotometer (A260/A280 > 1.9 and A260/A23 0 > 1.4) (NanoDrop Technologies LLC, Wilmington, DE, USA). RNA Quality Indicator (RQI) higher than 8.0 (Experion Automated Electrophoresis, Bio-Rad, USA) was considered to be high-quality total RNA preparation. TaqMan mastermix from Applied Biosystems (Foster City, CA, USA) was used for qPCR and performed following manufacturer's protocol and was run in a 7900 HT Fast Real-Time System (Applied Biosystems). The qPCR was carried out as follows: 50 • C for 2 min, 95 • C for 10 min, 40 cycles of 95 • C for 15 s, and 60 • C for 1 min. Changes in gene expression were calculated using the ∆∆Ct method with a fold-change cut-off at ≥1.5 and p < 0.05 considered significant. All samples were run in duplicate, and relevant negative controls were run on each plate. qPCR results were normalized to housekeeping genes (PPIA or HPRT). Primer sequences used in the qPCR assays are provided in Supplementary Table S2.

Western Blots
EndoC βH1 cells were homogenized in ice-cold RIPA buffer and kept shaking on ice for 30 min. Extracted total protein content from homogenates was measured by Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA USA). Homogenate samples (10 µg) from scramble control (Scr) or GPR56-KD cells were electrophoresed on 7.5% SDSpolyacrylamide gel (Bio-Rad, Hercules, CA, USA). After electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked in LSbuffer (10 mmol/L Tris, pH 7.4, 100 mmol/L NaCl, 0.1% Tween-20) containing 5% non-fat dry milk powder for 60 min at room temperature. Subsequently, the membranes were incubated overnight with the same VDAC1 antibody used for the confocal experiments (1:10,000) at 4 • C. After washing (three times) in LS-buffer, the membrane was finally incubated with a horseradish peroxidase-conjugated anti-rabbit antibody (1:5000) (Bio-Rad, Hercules, CA, USA). Detection of α-tubulin was with rabbit-anti-α-tubulin (Sigma, USA) and secondary anti-rabbit anti-body (1:1000). Immunoreactivity was detected using an enhanced chemiluminescence reaction (Pierce, Rockford, IL, USA). The blots were scanned with ChemiDocTM MP Imaging System (Bio-Rad), and bands corresponding to the~37-kDa (protein marker) were identified as VDAC1 protein. A typical Western blot image of entire gel, performed on the homogenates from two Scr controls and four GPR56-KD cells, is shown in Supplementary Figure S2E.

Insulin Secretion
For functional studies after recovery, the siRNA-treated INS-1 832/13, or EndoC βH1 cells, were washed and pre-incubated for 120 min at 37 • C in SAB buffer, pH 7.4, and supplemented with 10 mM of HEPES, 0.1 % bovine serum albumin, and 2.8 mM of glucose. After pre-incubation, the buffer was changed and INS-1 832/13, or EndoC βH1 cells, were incubated at 1 or 20 mM of glucose with indicated agents for 60 min at 37 • C. Immediately after incubation, an aliquot of the medium was removed and frozen for subsequent assay of insulin. The cells were then washed with PBS and stored in 100 mM of HCl containing IBMX (100 µM) for subsequent analysis of cyclic AMP.

cAMP Determination
The cAMP content in the cell lysate was measured using a direct cAMP ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer's instructions, and the values were related to protein content. The protein concentrations of the cell lysates were measured by a BCA kit (Thermo Fisher Scientific, Wilmington, DE, USA).

ATP Determination
ATP content (INS-1 832/13 and EndoC βH1 cells) and release (INS-1 832/13 cells) in incubated cells after GPR56-KD were determined using a luminometric assay kit according to manufacturer's recommendation (BioVision, Milpitas, CA, USA) and normalized to protein content. After incubation, the cells were washed with PBS buffer (three times) in Ripa buffer containing protease inhibitors and stored at −80 • C for subsequent measurements of cellular ATP, while the released ATP was measured in the 1-h incubation medium. The protein contents of each sample were analyzed by BCA protein kit (Thermo Scientific, IL, USA).

Statistics
The results are expressed as means ± SEM for the indicated number of observations or illustrated by an observation representative of the results obtained from different experiments (confocal microscopy). The significance of random differences was analyzed by Student's t-test or, where applicable, an analysis of variance was performed, followed by Tukey-Kramers' multiple comparisons test. p-value < 0.05 was considered significant.

The Consequence of GPR56-KD on the Activation of Several Intracellular Pathways
The impact of long-term high glucose (20 mM, 72 h) culture on the key signaling molecules (pathways) involved in the physiology/pathophysiology of β-cells in scramble control INS-1 cells in the presence or absence of the naturally occurring GPR56 agonist Coll III was studied. The effect of Coll III on these signaling molecules was only investigated in Gpr56-KD cells under the two glucose culture conditions. Cell-signaling analysis revealed an increase in p-P70S6K, p-JNK, and p-NFκB induced by high glucose, while p-AKT, p-STAT3, and p-STAT5 were not significantly altered in Scr control INS-1 cells ( Figure 1A

The Effect of GPR56-KD on the Expression of Chrebp, Txnip, Vdac1 and Vdac2
Since we have reported that GPR56-KD is associated with β-cell dysfunction and decreased viability reminiscent of diabetic β-cells [18], we evaluated the impact of GPR56-KD on mitochondrial VDAC1 and VDAC2 expression, as we have demonstrated that the altered expression of VDAC1 and VDAC2 is associated with mitochondrial dysfunction, leading to impaired β-cell function [13,14]. We also assessed the consequence of GPR56-KD on Chrebp and txnip, two transcriptional factors of importance for the glucotoxicityinduced increase in VDAC1 expression in pancreatic β-cells [14]. As seen in Figure 2, GPR56-KD significantly increased mRNA expression of Chrebp (A), Txnip (B), and Vdac1 (C), while Vdac2 mRNA was reduced (D). The calculated efficiency of GPR56-KD showed a reduction of almost 75% of GPR56 mRNA compared to the scramble control group (Supplementary Figure S1A). To evaluate whether GPR56-KD would have any off-target effect, we have also analyzed the mRNA level of a highly expressed GPCR in β-cells i.e., Gprc5b [29,30]. The mRNA level of Gprc5b was not affected by Gpr56-KD, revealing no off-target effect (Supplementary Figure S1B).  Figure S1A). To evaluate whether GPR56-KD would have any off-target effect, we have also analyzed the mRNA level of a highly expressed GPCR in β-cells i.e., Gprc5b [29,30]. The mRNA level of Gprc5b was not affected by Gpr56-KD, revealing no off-target effect (Supplementary Figure S1B).

The Impact of Gpr56-KD on the Vdac1 Protein Expression and its Mistargeting to the Cell Surface in Mouse Islets
Since increased Vdac1 is associated with its mistargeting to the β-cell membrane contributing to β-cell decompensation [14], we next investigated the impact of Gpr56-KD on protein expression and sub-cellular localization of Vdac1 in isolated mouse pancreatic islets by confocal microscopy. Compared with the very low Vdac1 expression in scramble control islets ( Figure 3A), Gpr56-KD markedly increased Vdac1 protein expression in the

The Impact of Gpr56-KD on the Vdac1 Protein Expression and its Mistargeting to the Cell Surface in Mouse Islets
Since increased Vdac1 is associated with its mistargeting to the β-cell membrane contributing to β-cell decompensation [14], we next investigated the impact of Gpr56-KD on protein expression and sub-cellular localization of Vdac1 in isolated mouse pancreatic islets by confocal microscopy. Compared with the very low Vdac1 expression in scramble control islets ( Figure 3A), Gpr56-KD markedly increased Vdac1 protein expression in the islets ( Figure 3B). We studied the co-localization of Vdac1 with the plasma membraneassociated SNARE protein SNAP-25 (cell membrane marker) in islets after Gpr56-KD. Remarkably, confocal microscopy revealed Vdac1 co-localization with SNAP-25 in insulin positive cells, indicating Vdac1 surface localization in β-cells of Gpr56-KD islets ( Figure 3B).

The Impact of Gpr56-KD on the Vdac1 Protein Expression and its Mistargeting to the Cell Surface in Mouse Islets
Since increased Vdac1 is associated with its mistargeting to the β-cell membrane contributing to β-cell decompensation [14], we next investigated the impact of Gpr56-KD on protein expression and sub-cellular localization of Vdac1 in isolated mouse pancreatic islets by confocal microscopy. Compared with the very low Vdac1 expression in scramble control islets ( Figure 3A), Gpr56-KD markedly increased Vdac1 protein expression in the islets ( Figure 3B). We studied the co-localization of Vdac1 with the plasma membraneassociated SNARE protein SNAP-25 (cell membrane marker) in islets after Gpr56-KD. Remarkably, confocal microscopy revealed Vdac1 co-localization with SNAP-25 in insulin positive cells, indicating Vdac1 surface localization in β-cells of Gpr56-KD islets ( Figure  3B).

The Impact of Gpr56-KD on cAMP, ATP Content, and Insulin Secretion
Vdac1 is an ATP-conducting anion channel normally allowing the transport of ATP from mitochondria to the cytoplasm in cells [11,14]. Since immunohistochemical experiments showed an increased VDAC1 expression in pancreatic β-cells upon Gpr56-KD, we next evaluated the effect of two different VDAC1 blockers on the cellular cAMP and ATP content in relation to GSIS in scramble control and Gpr56-KD INS-1 832/13 cells. In the following experiments, the cAMP and ATP content, as well as the GSIS, were measured after 60 min incubation of INS-1 cells in the presence or absence of Coll III and VDAC1 inhibitors, i.e., VBIT-4 and VDAC1 antibodies (VD1ab) [14,31]. As shown in Figure 4A, the increased cellular cAMP content induced by high glucose was further augmented by Coll III, but not by the presence of VBIT-4 or VD1-ab during the short-term incubation of scramble control cells. In Gpr56-KD cells, both basal-and glucose-stimulated increases in cAMP were diminished, and the presence of Coll III did not alter cAMP generation. In contrast, the inhibition of VDAC1 with VBIT-4 and VDAC1 antibodies further increased glucose-stimulated cAMP generation in the KD cells. Our data also show that a glucosestimulated increase in cellular ATP level was not affected by Coll III or by VBIT-4 and VDAC1 antibodies in scramble control INS-1 832/13 cells. In Gpr56-KD cells, the diminished glucose-induced rise in ATP level was restored only in the presence of VBIT-4 and VDAC1 antibodies ( Figure 4B).
To evaluate the impact of altered cellular signaling, we monitored GSIS. Insulin secretion was potentiated by Coll III, while, as expected, VBIT-4 or VDAC1-ab did not alter secretion in scramble control INS-1 832/13 cells. In Gpr56-KD cells, the GSIS was markedly reduced and Coll III potentiation was abolished. The presence of VBIT-4 and VDAC1-ab during the final incubation improved GSIS ( Figure 4C). As mistargeting of VDAC1 to the plasma membrane causes loss of ATP [14], we next investigated the impact of GPR56-KD on the ATP release in INS-1 cells at 1 mM glucose to avoid high glucose-induced ATP generation [14]. After GPR56-KD, the INS-1 cells were incubated for 60 min at 1 mM of glucose in the presence or absence of Coll III, VBIT-4, and AKOS, which is another VDAC1 inhibitor [14,31]. The basal ATP release from scramble control INS-1 cells was not affected by Coll III, VBIT-4, or AKOS ( Figure 5). However, Gpr56-KD was associated with a markedly increased ATP loss from INS-1 832/13 cells ( Figure 5), which was not affected by the presence of Coll III. It is noteworthy that the ATP release was markedly prevented by VBIT-4 and AKOS ( Figure 5). As mistargeting of VDAC1 to the plasma membrane causes loss of ATP [14], we next investigated the impact of GPR56-KD on the ATP release in INS-1 cells at 1 mM glucose to avoid high glucose-induced ATP generation [14]. After GPR56-KD, the INS-1 cells were incubated for 60 min at 1 mM of glucose in the presence or absence of Coll III, VBIT-4, and AKOS, which is another VDAC1 inhibitor [14,31]. The basal ATP release from scramble control INS-1 cells was not affected by Coll III, VBIT-4, or AKOS ( Figure 5). However, Gpr56-KD was associated with a markedly increased ATP loss from INS-1 832/13 cells ( Figure 5), which was not affected by the presence of Coll III. It is noteworthy that the ATP release was markedly prevented by VBIT-4 and AKOS ( Figure 5).
Finally, we extended the observations in INS-1 cells to human EndoC βH1 cells, studying the impact of GPR56-KD on VDAC1 expression, apoptosis, and the release of inflammatory cytokines, as well as cAMP generation and insulin secretion. Among a panel of cytokines (IL-2, IL-6, IL-10, IFNγ, IL-12bp40, IL-12p70, and IL-17) that were analyzed, as seen in Figure 6A-C, GPR56-KD was associated with an increased release of MCP-1 (CCL2), IL-2, and TNF-α from EndoC βH1 cells, while the release of other measured cytokines was undetectable (not shown) in scramble control or GPR56-KD cells. Western blot and immunohistochemical analysis by confocal microscopy revealed that GPR56-KD was associated with increased VDAC1 protein expression (Supplementary Figure S2D). GPR56-KD clearly caused VDAC1 mistargeting to the cell surface, as revealed by costaining with Na + /K + ATPase in EndoC βH1 cells ( Figure 6D,E). GPR56-KD also resulted in an increased intensity of nuclear Hoechst staining, indicating an increased apoptotic rate [32] (Figure 6F). Interestingly, GPR56-KD (Supplementary Figure S2A-C) in EndoC βH1 cells attenuated both GSIS and glucose-induced increase in cAMP when the cells were incubated for 60 min at 20 mM glucose, while basal insulin release or cAMP content was not influenced (Figure 6G,H). The potentiation of GSIS concomitant with the cAMP generation by Coll III was also markedly attenuated by GPR56-KD ( Figure 6G,H). These results link β-cell stress after GPR56 loss-of-function to VDAC1-mediated abrogation of ATP formation and the subsequent impairment of stimulus-secretion coupling. Finally, we extended the observations in INS-1 cells to human EndoC βH1 cells, studying the impact of GPR56-KD on VDAC1 expression, apoptosis, and the release of inflammatory cytokines, as well as cAMP generation and insulin secretion. Among a panel of cytokines (IL-2, IL-6, IL-10, IFNγ, IL-12bp40, IL-12p70, and IL-17) that were analyzed, as seen in Figure 6A-C, GPR56-KD was associated with an increased release of MCP-1 (CCL2), IL-2 ,and TNF-α from EndoC βH1 cells, while the release of other measured cytokines was undetectable (not shown) in scramble control or GPR56-KD cells. Western blot and immunohistochemical analysis by confocal microscopy revealed that GPR56-KD was associated with increased VDAC1 protein expression (Supplementary Figure S2D). GPR56-KD clearly caused VDAC1 mistargeting to the cell surface, as revealed by co-staining with Na + /K + ATPase in EndoC βH1 cells ( Figure 6D,E). GPR56-KD also resulted in an increased intensity of nuclear Hoechst staining, indicating an increased apoptotic rate [32] ( Figure 6F). Interestingly, GPR56-KD (Supplementary Figure S2A-C) in EndoC βH1 cells attenuated both GSIS and glucose-induced increase in cAMP when the cells were incubated for 60 min at 20 mM glucose, while basal insulin release or cAMP content was not influenced ( Figure 6G,H). The potentiation of GSIS concomitant with the cAMP generation by Coll III was also markedly attenuated by GPR56-KD ( Figure 6G,H). These results link β-cell stress after GPR56 loss-of-function to VDAC1-mediated abrogation of ATP formation and the subsequent impairment of stimulus-secretion coupling.

Discussion
The rationale for the present investigations is the documented role of GPR56 in pancreatic β-cell survival and secretory function [18,19,21,25,33]. The expression level of GPR56 is positively correlated with the transcript level of a great number of genes with a beneficial impact on the β-cell fate in human pancreatic islets [18]. Moreover, GPR56 is downregulated in islets from T2D organ donors [18]. Herein, we define the mechanism

Discussion
The rationale for the present investigations is the documented role of GPR56 in pancreatic β-cell survival and secretory function [18,19,21,25,33]. The expression level of GPR56 is positively correlated with the transcript level of a great number of genes with a beneficial impact on the β-cell fate in human pancreatic islets [18]. Moreover, GPR56 is downregulated in islets from T2D organ donors [18]. Herein, we define the mechanism by which GPR56 loss-of-function causes pancreatic β-cell dysfunctionality.
As GPR56 is the most abundant GPR both in mouse and human β-cells [29], it is of interest that not only Coll III derived from islet endothelial cells [19], but also the insulinotropic amino acid L-phenylalanine has been identified as a GPR56 agonist [34]. The stimulation of insulin secretion by L-phenylalanine, a ligand for GPR142, is preserved in GPR142 KO islets, which could be explained by GPR56-mediated signaling [19,35]. The cumulating evidence makes GPR56 an interesting drug target in T2D. We and others have presented evidence that GPR56 is indeed capable of acting as a G-protein-coupling (Gαs type) to elicit downstream signaling cascades via the cAMP/PKA system in pancreatic β-cells [18,21,33], which was confirmed in the present work. A similar signal transduction has been shown for GpR56 activation by testosterone in prostate cells [24]. Although signaling via Gαq has also been reported in β-cells and neuronal cells [19][20][21]36], the cAMP/PKA system could act by lowering the threshold level of the exocytosis for intracellular Ca 2+ ([Ca 2+ ] i ) elevation, increasing the secretory response of β-cell to even small increases in [Ca 2+ ] i [15].
In the current study, we present mechanistic data revealing GPR56 as a mediator of the suppressive effect of Coll III on cellular stress-related signals, such as P70S6K, JNK, AKT, NFκb, STAT3, and STAT5 signaling, thereby providing the pathway(s) by which GPR56 activation prevents β-cell dysfunction. While it is well-established that stress kinases, NFκB, STAT3, and STAT5 signaling is required for an array of physiological/pathophysiological events, they are mostly involved in stress-or inflammation-induced β-cell dysfunction [6,37]. We show here that GPR56-KD causes cellular stress that also results in the release of certain inflammatory cytokines, such as MCP-1, IL-2, and TNFα by the β-cells. This observation confirms previous studies that GPR56 plays a role in inflammation and has been identified as an inhibitory receptor, suppressing the pro-inflammatory activity of cytotoxic lymphocytes [38]. Likewise, cytokine-induced β-cell apoptosis is prevented by Coll III, but not by a simple overexpression of GPR56 [25]. However, the delineation of the inflammatory mechanism in GPR56-deficient β-cells extends beyond the scope of the present work and merits further investigation. It should also be mentioned that the altered regulation of NFκB, STAT3, and STAT5 plays a critical role in inducing/maintaining the chronic, low-grade inflammation that conveys both β-cell dysfunction [37] and complications of diabetes, including atherosclerotic vascular lesions [39] by influencing diverse cellular gene expression programs.
Moreover, we link GPR56-KD to increased expression of the transcription factors Chrebp and Txnip that initiate the overexpression of VDAC1 with a consequent reduction of VDAC2 expression in INS-1 832/13 cells. Chrebp and Txnip are highly increased in pancreatic β-cells in glucotoxic condition [40] and in islets from T2D organ donors [14,41]. TXNIP is known to activate the NLRP3 inflammasome, generating interleukin-1β, thereby contributing to impaired β-cell function [37,42]. An increased cAMP formation exerts β-cell protection in part by promoting TXNIP proteosomal degradation [42], an effect that could explain coll III protection from cytokine-induced apoptosis in human islets [25]. The overexpression of VDAC1 and its mistargeting to the β-cell plasma membrane leads to a loss of ATP, the crucial metabolic coupling factor in GSIS [8]. We have shown previously that the prevention of ATP loss by the acute addition of VDAC1 antibodies and inhibitors completely restored the defective GSIS in islets from human T2D organ donors and diabetic db/db mice [14].
Remarkably, confocal microscopy revealed that VDAC1 surface mistargeting occurs in GPR56-KD mouse islet β-cells, as shown by the co-localization with the plasma membraneassociated SNARE protein SNAP-25. A similar VDAC1 mistargeting was observed after GPR56 KD in human EndoC βH1 cells. It is noteworthy that VBIT-4 and AKOS, two chemical VDAC1 inhibitors [14,31], as well as the VDAC1 antibody efficiently restored glucose-induced rises in cellular ATP levels, the generation of cAMP, and the stimulation of insulin secretion in the GPR56 KD cells. We show in INS-1 GPR56-KD cells that this is mainly due to the attenuation of the high rate of ATP leakage. The most plausible explanation for the acute restoration of cAMP generation is the increase in cellular ATP, which drives the cAMP formation during glucose stimulation [43]. In contrast, the stimulatory effects of Coll III are not restored, further validating GPR56 as the collagen receptor. Plasma membrane-resident gated VDAC1 has been documented in various mouse and human tissues with the mitochondrial surface residues facing the extracellular space [44][45][46]. Of note, oxidative stress in neurons activates the conductance of the neurolemmal VDAC1, initiating apoptosis, which is prevented by antibodies directed against the extracellular N-terminus of VDAC1 [45]. Interestingly, the association of GPR56 deletion with the mistargeting of mitochondrial VDAC1 to the cell surface further emphasizes the importance of GPR56 in the regulation of insulin secretion. Loss of GRP56 function in T2D [18] thus participates in altered gene expression and β-cell dysfunction. At first sight, this conclusion seems to be at variance with the only mild glucose intolerance of the GPR56 KO mouse [19]. We speculate that the deletion of GPR56 during fetal development may upregulate other adhesion GPRs, which would not necessarily occur during short-term KD of the receptor.
VDAC1 upregulation and oligomerization is caused by oxidative and nitrosative stress, not only in β-cells in T2D but also in neurodegenerative diseases, in particular, Alzheimer's disease [47,48]. It is, therefore, of great interest that VBIT-4, which inhibits VDAC1 conductance and oligomerization, prevents onset of diabetes in db/db mice [14] and markedly improves the phenotype in a mouse model of Alzheimer's disease [48]. VBIT-4, thus, has both acute effects on cell signaling by preventing ATP loss through VDAC1 expressed in the plasma membrane and long-term actions on gene expression and cell function in diseases linked to oxidative stress and mitochondrial dysfunction.
Taken together, the present data show that GPR56 activation by Coll Type III is associated with the suppression of P70S6K, JNK, AKT, NFκb, STAT3, and STAT5 phosphorylation/activity and increased CREB signaling. Since GPR56 positively modulates the activity of the cAMP-PKA system in the β-cell ( [18] and present work), the Coll Type III-mediated beneficial effects on the β-cell function seem to be through cAMP signaling concomitant with the suppression of the aforementioned stress kinases and transcriptional factors. A further signaling pathway implicating GPR56 is its link to integrin function [20,21]. This is relevant for β-cell senescence in which, among others, STAT3 is upregulated [49].

Conclusions
In the current work, we have linked the dysregulation and mistargeting of the diabetes executer protein VDAC1 [14] to the suppression of GPR56, thereby linking this adhesion GPCR to β-cell mitochondrial dysfunction with impaired ATP accumulation and compromised insulin secretion. GPR56 could, therefore, constitute a novel drug target preventing the loss of β-cell function in prediabetes and diabetes.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom13030557/s1. The information about siRNA, shRNA and primers used in this study is shown in Table S1 and Table S2. Figure S1: represents qPCR analysis for Gpr56-KD efficiency (A) and off-target effects (B) in INS-1 832/13 cells. Figure S2  Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and the animal study protocol was approved by the local Ethics Committee for Animal Research at Lund University (1057/2020).

Informed Consent Statement: Not applicable.
Data Availability Statement: All presented data is in the article.