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Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction

We previously reported a depletion of murine regenerating islet-derived protein 2 (REG2) in pancreatic islets of glutathione peroxidase-1 (Gpx1) overexpressing (OE) mice. The present study was to explore if and how the REG2 depletion contributed to an augmented glucose stimulated insulin secretion (GSIS) in OE islets. After we verified a consistent depletion (90%, p < 0.05) of REG2 mRNA, transcript, and protein in OE islets compared with wild-type (WT) controls, we treated cultured and perifused OE islets (70 islets/sample) with REG2 (1 μg/ml or ml · min) and observed 30–40% (p < 0.05) inhibitions of GSIS by REG2. Subsequently, we obtained evidences of co-immunoprecipitation, cell surface ligand binding, and co-immunofluorescence for a ligand-receptor binding between REG2 and transmembrane, L-type voltage-dependent Ca2+ channel (CaV1.2) in beta TC3 cells. Mutating the C-type lectin binding domain of REG2 or deglycosylating CaV1.2 removed the inhibition of REG2 on GSIS and(or) the putative binding between the two proteins. Treating cultured OE and perifused WT islets with REG2 (1 μg/ml or ml · min) decreased (p < 0.05) Ca2+ influx triggered by glucose or KCl. An intraperitoneal (ip) injection of REG2 (2 μg/g) to OE mice (6-month old, n = 10) decreased their plasma insulin concentration (46%, p < 0.05) and elevated their plasma glucose concentration (25%, p < 0.05) over a 60 min period after glucose challenge (ip, 1 g/kg). In conclusion, our study identifies REG2 as a novel regulator of Ca2+ influx and insulin secretion, and reveals a new cascade of GPX1/REG2/CaV1.2 to explain how REG2 depletion in OE islets could decrease its binding to CaV1.2, resulting in uninhibited Ca2+ influx and augmented GSIS. These findings create new links to bridge redox biology, tissue regeneration, and insulin secretion.


Introduction
We previously found that overexpression of selenium-dependent glutathione peroxidase-1 (Gpx1) induced type 2 diabetes-like phenotypes in mice [1]. These mice were defined as OE mice and displayed a primary disorder of augmented glucose-stimulated insulin secretion (GSIS) [2,3]. In searching for molecular mechanisms to explain this disorder, we demonstrated a depletion of murine regenerating islet-derived 2 (REG2) in OE islets [4]. Subsequently, we elucidated that the depletion was due to an inhibition of Reg2 transcription by GPX1-mediated activation of reactive oxygen species (ROS) responsive transcriptional factors (activator protein-1 and albumin D box-binding protein) with binding domains in the proximal promoter of the gene [4]. A remaining key question is if and how the depletion of REG2 contributes to the augmented GSIS in those OE islets.
REG2 represents one of the 12 islet-derived regenerating REG family proteins [5]. The gene (3 kb) contains 6 exons and 5 introns, and the deduced protein peptide contains 173 amino acids (16 kDa) [6]. In the peptide, there are a C-type lectin binding domain [7] and acidic residues potentially involved in CaCO 3 crystal binding [8]. Expression of Reg2 was shown to occur predominantly in pancreatic acinar cells [9][10][11], but not in endocrine pancreas under normal conditions [9,12]. However, several groups actually detected mRNA and(or) protein of REG2 in islets of wild-type (WT) mice [4,[13][14][15]. Likewise, expression of the putative human orthologue hREG1β was detected in human pancreas and pancreatic cell lines [16], but not consistently in beta cells [17,18]. Nevertheless, Reg2 expression was activated or up-regulated in islet regeneration induced by partial pancreatectomy [19], pancreatitis [9], type 1 diabetes [14], and type 2 diabetes and obesity [20]. Similar to other REG proteins including REG1 and INGAP, REG2 is considered to act as a trophic factor in promoting pancreatic beta cell proliferation and regeneration [5]. However, this function was not proved experimentally by the knockout of Reg2 in mice [12].
With a canonical C-type lectin binding domain [7], REG2 could potentially bind glycoproteins on cell surface that may be involved in GSIS [21,22]. Indeed, L-type voltage-dependent Ca 2+ channel (VDCC) CaV1.2 is a transmembrane protein and possesses a dihydropyridine structure [23] that specifically binds to immobilized lectins [24]. Thus, CaV1.2 plays a major role in regulating the glucose-stimulated elevation of Ca 2+ influx upon membrane depolarization to induce insulin exocytosis in the K ATP -dependent pathway [25]. Deletion of CaV1.2 in mice [26], knockdown of the gene in INS-1 cells [27], and altering CaV1.2 function [28] were shown to affect or impair GSIS. Meanwhile, REG2 was reported to potentially function as an auto-, para-, or endocrine molecule [9]. The protein was produced and secreted into the media by cultured islets [4] and was co-localized with insulin in islet beta cells [14]. Therefore, we hypothesized that REG2, via its C-type lectin binding domain, could bind carbohydrate moiety of the transmembrane CaV1.2 in a ligand-receptor fashion to inhibit glucose-induced Ca 2+ influx and insulin secretion in pancreatic islets, whereas depletion of REG2 in OE islets result in a loss of this inhibition and contribute to the augmented GSIS.

Animals, gene expression, and immunoblotting
Our protocol was approved by the Institutional Animal Care and Use Committee of Cornell University. The genetic background of OE and WT mice (C57B1 × C3H), feeding, and pancreas harvesting were the same as previously described [3,4]. Mice were housed in shoeboxes (3-5/box) placed in the East Campus Core Facility of Cornell University (Ithaca, NY), had free access to food and water under a 12h:12h light and dark cycle, and checked daily. All mice used in this research were 2 month-old males unless otherwise indicated.
Because our previous research demonstrated that GSIS differences between WT and OE islets resembled those between the two genotypes of mice [2], we chose islets as the primary experimental model for determining roles and mechanisms of REG2 in the present study. Islets were isolated from WT and OE mice and recovered in RPMI 1640 media (Gibco, Grand Island, NY) (10% fetal bovine serum (FBS), 2 mmol glutamine/l, 10 mmol glucose/l, and 1% PenStrep for 12 h [3,4]. To verify a specific depletion of REG2 in OE islets, relative mRNA abundances of Reg1 and Reg2, along with those involved in insulin synthesis and secretion were quantified (Q-PCR) using total RNA extracted from islets (70 per sample) of WT and OE mice (n = 6) [2]. The primers are listed in Supplemental Table 1. Genotype differences in pancreatic islet (70 per sample) Reg2 mRNA levels were determined at ages of 6 weeks, 6 months, and 12 months (male, n = 6). To determine Reg1 and Reg 2 transcript numbers in pancreatic islets (70 per sample) of WT and OE mice (6-month old, male, n = 6), we amplified the two gene fragments and inserted them into the pPICZ vector (Supplemental Table 3). A serial dilution of the pPICZ constructs, ranging from 1 to 1 × 10 7 copies/μl was used to establish standard curves for both genes. Concentrations of the plasmids were measured using a fluorimeter. CT values of both genes in testing islets and standard plasmids were obtained by Q-PCR, and the corresponding transcript numbers were calculated based on the CT values against the standard curves. Immunoblotting of REG2 protein (75 μg protein per sample) in pancreatic islets of WT and OE mice (6-month old, male, n = 4) was performed using primary and secondary antibodies listed in Supplemental Table 2 [4].

Preparation of recombinant REG2 protein and mutants
To determine effects of REG2 on GSIS and the protein structure required for the action (see below), we prepared three constructs including the native form of REG2 and two mutants (REG2 M1 & M2) and overexpressed them in Pichia pastoris yeast. REG2 M1 was designed to mutate the C-type lectin binding domain [7] by substituting three amino acid residues in the domain with alanine (G142A, K157A, and D158A) ( Fig. 2B and Supplemental Fig. 2). REG2 M2 was designed to substitute the acidic residues potentially involved in CaCO 3 crystal binding [8] with glycine (E59G, D60G, E66G, D158G, E159G, and E162G) ( Fig. 2B and Supplemental Fig. 3). The NOD mouse Reg2cDNA (NM 009043, G22 to A173) [13] was inserted into a P. pastoris expression vector pPICZ (Invitrogen, Grand Island, NY) in a way that allowed expression of REG2 without non-native amino acids at the N-terminus. The REG2 mutants were created by site-directed mutagenesis of the Reg2/pPICZgamma C construct through touchdown PCR using mutagenic forward and reverse primers (Integrated DNA Technologies, Coralville, IA) with PrimeSTAR HS DNA polymerase (Takara, Otsu, Shiga, Japan) followed by Dpn1 digestion of parental DNA template (Supplemental Table 3). The isolated Reg2 plasmids were transformed into P. pastoris GS200 using electroporation (BTX, Holliston, MA), and cultured in BMMY media for 5 d. The secreted REG2 and mutant  hReg1β human regenerating islet derived protein 1 Beta  INGAP  islet neogenesis associated protein  INS-1  rat insulinoma cell line  Ins1  insulin  NBT  nitro-blue tetrazolium chloride  OE  overexpression  PDX1 pancreatic and duodenal homeobox 1 PNPP para-nitrophenylphosphate REG1 2, and 3, regenerating islet derived proteins 1, 2, and 3 REG1A regenerating islet derived protein 1 alpha REG2 M1 and M2 REG2 mutant 1 and 2 REG3A regenerating islet derived protein 3 alpha ROS reactive oxygen species WT wild-type proteins were isolated, purified by ammonium sulfate precipitation and ion-exchange chromatography, and verified by SDS-PAGE and immunoblotting [29].

Effects of REG2 and(or) mutants on GSIS in OE islets and mice
To determine if augmented GSIS in OE islets could be rescued by administrations of REG2, we treated OE islets with the native form of REG2 and(or) the two mutants in static culture and(or) perifusion system. In the static culture, islets were isolated from OE mice (n = 4) and recovered in the RPMI 1640 media as described above for 5 h at 37 • C, 95% air, and 5% CO 2. Thereafter, the islets (70 per sample) were transferred to Krebs buffer containing glucose at 2.8 mmol/l with or without REG2 (1 μg/ml) (3 treatment groups) and incubated in a 37 • C water bath for 0.5 h. The REG2 dose was chosen from a preliminary experiment (0-10 μg/ml) and was in range of reported plasma concentrations of human and mouse REG3 proteins [30]. Subsequently, two groups of islets were treated with 30 mmol glucose/l with or without REG2 (1 μg/ml) for 1 h. The other group of islets was kept the same glucose concentration (2.8 mmol/l) without REG2 as control over the period of time. Supernatants were collected at the ends of the first 0.5 h and the later 1 h treatment for insulin analysis using an ELISA kit [3].
In the perifusion system [31], freshly isolated islets (70 per sample) from OE mice (n = 4 per treatment by genotype) were pre-conditioned with Krebs buffer (pH 7.4, 10 mmol glucose/l) for 1 h and placed on a nylon filter in a plastic perifusion chamber (Millipore, Bedford, MA). The perifusion apparatus consisted of a water bath (37 • C), a fraction collector, and a fast protein liquid chromatography (Bio-Rad, Hercules, CA). The perifusate was a Krebs bicarbonate buffer (pH 7.4) containing Ca 2+ (2.2 mmol/L) and 0.25% bovine serum albumin equilibrated with 95% O 2 /5% CO 2 , and the flow and collection rates were 1 ml/min. Effects of REG2 and REG2 M1 and M2 on GSIS were determined by perifusing OE islets with these proteins (at 1 μg/mL/min) or the Krebs buffer for 15 min, followed by a glucose ramp of 0-50 mmol/l (1 mmol/min) [31]. The maximal islet secretion response was tested at the end of each experiment with 30 mM KCl/l after a washout of glucose. Fractions were collected at 1 ml/min for insulin analysis.
To determine in vivo effects of REG2 on GSIS in OE mice (6 months old, male, n = 10), we gave them each an intraperitoneal (ip) injection of filter-sterilized REG2 protein (2 μg/g body weight) or an equal volume (10 μL/g) of phosphate-buffed saline (PBS) at 15 min before an ip injection of glucose (1 g/kg). The injected REG2 dose was chosen from a preliminary experiment (0-4 μg/g) and was similar to the dose of REG3δ injected to dogs [32]. Plasma samples were collected at − 15, 0, 15, 30, and 60 min after the glucose injection for insulin [3].
To determine if REG2 could still pull down deglycosylated Cav1.2, we expressed pcDNA-CaV1.2 or the vector in Min6 cells as described above in beta TC3 cells for 3 d. The transfected cells were lysed in 0.25% trypsin-EDTA, separated by ultracentrifuge (100,000 g, 90 min) to collect membrane fraction, and re-suspended in a deglycosylation buffer (pH 7.8) containing 1%NP-40, 150 mmol NaCl/l, 50 mmol Tris-HCl/l, and protease inhibitor cocktail (Thermo Scientific, 784251). The lysate suspension (1 mg protein) was incubated with or without a deglycosylase (PNGase F, 20 μl, 10,000 units, New England Biolabs, P0704S) for 7 h at 37 • C. Subsequently, the mixture was incubated with REG2 protein (4 μg) for 6 h before with REG2 antibody (4 μg) or rat IgG isotype (Supplemental Table 2) for 12 h at 4 • C on a rotation wheel. The immune-precipitated complex was collected by adding 40 μl of protein G-PLUS agarose (4 • C on a rotation wheel for 2 h, Santa Cruz Biotechnology, sc02003) and spinning down the beads. The target CaV1.2 in the precipitate was tested as described above.

Co-localization of REG2 and CaV1.2 in beta TC-3 cells
To test co-localization of REG2 with CaV1.2 in beta TC-3 cells, we modified the AP-Reg2 and Reg2 M1 vectors by replacing AP with EGFP to form Gfp/Gfp-Reg2/Reg2 M1 constructs (Addgene, Supplemental Tables 1 and 3). The plasmids were transfected into HEK293T cells to produce CM as described above (for the AP-REG2 binding assay). Expression of GFP-REG2 protein was measured using an Axiovert 200 M fluorescent microscope (Zeiss, Carl-Zeiss-Strasse, Oberkochen, Germany). Meanwhile, the CaV1.2 gene was inserted into pmCherry-C3 vector (Addgene, Supplemental Table 3) to generate pmCherry-CaV1.2-C3 plasmid and transfected into betaTC-3 cells as described above. The co-localization assay was run with six different combinations of two sources of CaV1.2 (cells transfected with pmCherry and pmCherry-CaV1.2) and three types of CM proteins (GFP, GFP-REG2, and GFP-REG2 M1). After 48 h of transfection, cells (seeded in μ-Dish, 35 mm high, Ibidi, Germany, at 3 × 10 5 /dish, 90% confluence at transfection) were washed twice with HBH buffer and incubated with CM containing GFP, GFP-REG2, or GFP-REG2 M1 proteins for 2 h at room temperature. Thereafter, cells were washed 3 times with ice cold HBH and viewed using a Confocal-MP Inverted microscope (Zeiss 880, Carl-Zeiss-Strasse, Oberkochen, Germany). The relative percentage of yellow fluorescence area (% of GFP positive cells in mCherry positive cells) was scored using MetaMorph® image analysis software (Molecular Devices) and was used as the indicator of co-localization.
In the perifusion system, isolated WT islets were recovered in RPMI 1640 media as described above for 12 h at 37 • C and 5% CO 2 . After a 40min pretreatment at 37 • C in 2 ml of Krebs buffer supplemented with Fura 2-AM (1 μmol/l), the loaded islet was transferred to a perifusion chamber and placed on the homeothermic platform of an inverted Zeiss AxioVision microscope. Islets were perfused with Krebs buffer at 37 • C at a flow rate of 2 ml/min, while REG2 (1 μg/ml, PBS as the control) was added into the perifusate continuously or for a given period of time. In the former (continuous) case (n = 4), the islet was perifused with 3 mmol glucose/l for 10 min and then REG2 (1 μg/ml) was added in the perifusate with glucose at 3 mmol/l for 15 min, at 16.7 mmol/l for 15 min, and at 3 mmol/l for 7 min and with KCl at 30 mmol/l for 8 min. In the latter (on/off) case (n = 9), the islet was perifused with glucose at 3 mmol/l for 15 min and at 16.7 mmol glucose/l for 10 min. Subsequently, REG2 (1 μg/ml) was added into the perifusate in which glucose concentration was maintained at 16.7 mmol/l for 10 min. Thereafter. REG2 was removed from the perifusate and the perifusion was continued to run with glucose at 16.7 mmol/l for 10 min and at 3 mmol/l for 10 min and with KCl at 30 mmol/l for 8 min. Intracellular Ca 2+ concentrations were determined by ratios of fluorescence (Fura 2-AM) detected at excitation wavelength of 340 (Ca 2+ complex) to 380 (free anion) nm with the emission wavelength at 520 nm using an Attofluor chargecoupled device camera, and calibrated by the Attofluor Ratio Vision Software [31].

Statistical analysis
Genotype differences in islet gene expression and protein production (n = 6) and various treatment effects of REG2 verse PBS/buffer controls (n = 4 to 10) were analyzed using Student t-test and one-way ANOVA (SAS 6.11 software, SAS Institute, Cary, NC). Values were expressed as mean ± SEM. Significance was defined as p < 0.05.

REG2 was depleted by GPX1 overproduction in OE islets
Compared with WT mice, OE mice had moderate elevations (32-50%, p < 0.05) of Ins1, Pdx1, and Foxa2 mRNA levels in pancreatic islets (Fig. 1A). There were no significant genotype differences in islet mRNA levels of Reg1 or other 14 insulin-related genes (Supplemental Fig. 1). In contrast, Reg2 mRNA in OE islets was decreased to only 0.7-7% (p < 0.05) of that in WT islets across the three tested ages of mice (Fig. 1B). OE islets showed a similar decrease (p < 0.05) in transcript numbers of Reg2, but not Reg1 compared with WT islets (Fig. 1C). Likewise, REG2 protein in OE islets was decreased (p < 0.05) to < 10% of that in WT islets (Fig. 1D). These results depicted a consistent diminishing of REG2 gene expression and(or) protein production in OE islets.

Inhibition of GSIS by REG2 required its C-type lectin domain
The verified REG2 depletion in OE islets led us to test if augmented GSIS in these islets could be inhibited or rescued by treating them with exogenous REG2. Compared with PBS-treated controls, the REG2 (1 μg/ mL) treatment (for 0.5 h) did not affect insulin secretion of OE islets cultured at 2.8 mmol glucose/l, but the treatment (for 1 h) decreased insulin secretion (30%, p < 0.05) of OE islets cultured at 30 mmol glucose/l ( Fig. 2A). In perifused OE islets, the REG2 treatment (1 μg/ml) suppressed the overall GSIS by 41% (p < 0.05) compared with the buffer-treated control (Fig. 2C). The inhibition became significant when the glucose ramp reached approximately 15 mmol glucose/l and was maintained up to 50 mmol glucose/l. The REG2 perifusion also caused substantial decreases (p < 0.05) in insulin secretion stimulated by KCl (30 mmol/l). In contrast, REG2 M1 (with mutations in the C-lectin binding domain) showed a major loss of the inhibition on GSIS (Fig. 2D). Meanwhile, REG2 M2 (with mutations in the acid residue domain) retained a moderate inhibition on the high side of glucose ramp (Fig. 2E). Although both mutants decreased KCl-induced insulin secretions, their effects were not statistically significant. Apparently, REG2 exerted a potent inhibition on GSIS in OE islets, and the C-type lectin domain was required for the inhibition because mutating the domain in REG2 M1 abolished the inhibition. In comparison, the retained moderated inhibition by REG2 M2 on GSIS implied that the acidic residues could modulate (decrease) the inhibition, but were not essential for the action. The loss of a significant inhibition on the KCl-stimulated insulin secretion by the two mutants in comparison with REG2 implied the importance of both C-type lectin domain and acidic residues in the regulation.

Co-immunoprecipitation of REG2 and CaV1.2 required glycosylation of CaV1.2
The inhibition by REG2, but not REG2 M1 on GSIS and the recognized role of CaV1.2 in regulating glucose-stimulated elevation of Ca 2+ influx and insulin secretion [25] gave us a strong rationale to determine if REG2, through its C-type lectin binding domain, bound the carbohydrate moiety of CaV1.2 to exert the inhibition. After lysates of beta TC3 cells overproducing CaV1.2 (Fig. 3A) were incubated with REG2 protein, REG2 antibody was able to pull down CaV1.2 from the mixture (Fig. 3B). This pull down was seen only from the mixture added with both REG2 protein and antibody, indicating a specific co-immunoprecipitation of CaV1.2 via binding to REG2. In contrast, the same REG2 antibody failed to pull down CaV1.2 from the mixture when lysates of Min6 cells overproducing CaV1.2 were deglycosylated (Fig. 3C). The CaV1.2 α1c subunit was detected in the immuno-precipitates with only glycosylated but not deglycosylated CaV1.2 in the cell lysates, verifying the essential role of the carbohydrate moiety of CaV1.2 in this putative binding.

Binding of REG2 to CaV1.2 required the C-type lectin binding domain of REG2
Because only REG2 M1 (with mutations in the C-type lectin domain), but not REG2 M2 (with mutations in the acidic residues), lost most of the inhibition on GSIS, we focused subsequent CaV1.2 binding assays with REG2 and REG2 M1. Incubating beta TC3 cells overexpressing CaV1.2 with AP-REG2 produced a 1.4-fold increase (p < 0.05) in the color staining (shading/dark pixels), compared with that in cells transfected with the vector only (Fig. 3D). The increased intensity was resulted from the enhanced AP reactions with NBT and BCIP, indicating an elevated ligand-receptor binding between AP-REG2 and CaV1.2 on the cell surface. However, neither AP-REG2 M1-treated nor AP-treated cells showed such an increase. Furthermore, that increase produced by the AP-REG2 treatment was attenuated (30-45%) by incubations of CaV1.2 antibody or excessive REG2 protein (unconjugated with AP) via competitive binding to CaV1.2. In the control cells transfected with the vector only, the AP-REG2 treatment also produced ~ 3-fold AP activity (staining, p < 0.05) of that treated by AP-5, suggesting a binding of AP-REG2 to the endogenous CaV1.2. These results revealed that the overexpressed or endogenous, transmembrane CaV1.2 in beta TC3 cells indeed bound extracellular REG2. The binding was displayed as a specific, competitive ligand-receptor interaction, and required the presence of C-type lectin binding domain in REG2.

Only REG2, but not REG2 M1, was co-localized with CaV1.2
After the pull down and AP-tag binding assays, we performed a coimmunofluorescence analysis to generate another evidence for the putative binding between REG2 and CaV1.2. Incubating CaV1.2-mCherry-overexpressing beta TC3 cells with GFP-REG2 yielded yellow fluorescence (Fig. 3E). The overlap of the green (GFP-REG2) and the red (CaV1.2-mCherry) fluorescence was verified from 4 different views (Supplemental Fig. 4). The relative percentage of yellow fluorescence area was nearly tripled (p < 0.05) by the GFP-REG2 incubation over the control. In contrast, little change in the fluorescence (yellow) was produced by either REG2 (without being conjugated with GFP) or GFP-REG2 M1 (mutations in the C-type lectin binding domain). Once again, only GFP-REG2, but not GFP-REG2 M1, when added extracellularly, was found to bind or co-localize with CaV1.2 overexpressed in beta TC3 cells.

Binding of REG2 to CaV1.2 decreased glucose-stimulated Ca 2+ influx in islets
To explore metabolic impacts of the above-tested binding of REG2 to CaV1.2, we determined if that binding affected Ca 2+ influx of OE and WT islets stimulated by glucose or KCl. Compared with PBS, REG2 substantially attenuated or blocked the fluorescence enhancements of Fluo 4-AM (increases in Ca 2+ influx) caused by glucose (16.7 and 30 mmol/l) and KCl (30 mmol/l) (Fig. 4A). The fluorescence increases in the PBS-treated OE islets after these stimulations were in an ascending order of 16.7 and 30 mmol glucose/l and 30 mmol KCl/l, with little change in those islets continuously incubated with 2.8 mmmol glucose/ l. In addition, only REG2 and nifedipine (a CaV1.2 inhibitor), but not REG2 M1 or PBS, decreased the 30 mM glucose-induced increase of Ca 2+ influx in the cultured OE islets (Supplemental Fig. 5).
When REG2 was added continuously into the perifusate starting at 3 mmol glucose/l, the inhibition of REG2 on Ca 2+ influx appeared only when the glucose concentration in the ramp reached 16.7 mmol/l (Fig. 4B). The overall inhibition was 25% (p < 0.05, REG2 vs. buffer control). Meanwhile, adding REG2 in the perifusate for 10 min after the glucose concentration reached 16.7 mmol/l quickly suppressed the Ca 2+ influx by 17% (p < 0.05 vs. without REG2) (Fig. 4C). The inhibition Fig. 1. Depletion of REG2 mRNA, transcript, and protein in pancreatic islets of mice by GPX1 overproduction Relative mRNA levels of Ins1, Pdx1, Foxa2, Reg1, and Reg2 in pancreatic islets (70 per sample) of OE mice to those of WT (as 1) mice (2-month old, male, n = 6) after normalization with the respective beta-actin mRNA (A), depletion of Reg2 mRNA in pancreatic islets (70 per sample) of OE mice across three ages compared with the WT (as 1) mice (male, n = 6) after normalization with the respective beta-actin mRNA levels (B), comparisons of Reg1 and Reg2 transcript numbers/ng cDNA in pancreatic islets (70 per sample) between OE and WT mice (6-month old, male, n = 6) (C), depletion of REG2 protein in pancreatic islets (75 μg protein per sample) of OE mice compared with WT mice (6-month old, male, n = 4) shown by immunoblotting (upper panel, a representative of four independent experiments), and relative densities (lower panel) of bands were quantified with Image J (the WT as 100) after normalization with beta-actin protein density. Data are mean ± SE, *, p < 0.05 for differences between genotypes. disappeared or the Ca 2+ influx was restored (p < 0.05 vs. with REG2) after REG2 was removed from the perifusate (Fig. 4C). In both settings, REG2 was able to inhibit the Ca 2+ influx induced by 16.7 and(or) 30 mmol glucose/l and 30 mmol KCl/l in OE and WT islets. The inhibition was acute and reversible, with minimal effects at the baseline or low glucose concentrations (2.8 or 3 mmol/l).

Exogenous REG2 administration inhibited GSIS in OE mice
To explore the physiological relevance and clinical potential of the inhibition on GSIS by REG2 in islets, we gave OE mice an ip injection of the purified recombinant REG2 protein. Compared with PBS, the REG2 injection (2 μg/g of body weight) to OE mice decreased their overall plasma insulin concentration by 46% (p < 0.05, Fig. 5A) and elevated their overall plasma glucose concentration by 25% (p < 0.05, Fig. 5B) during a 60 min period after glucose challenge. This depicted an in vivo effectiveness of the exogenous REG2 administration.

Discussion
The present study reveals a novel role and mechanism of REG2 depletion in the augmented GSIS of OE islets [2]. Specifically, REG2 transcript and protein in OE islets were depleted by GPX1 overproduction. Administrations of exogenous REG2 inhibited GSIS in OE islets and mice, and the inhibition required the C-type lectin binding domain in the protein. Exogenous or extracellular REG2 could pull down, bind, and co-localize with VDCC protein CaV1.2 overexpressed in beta TC3 cells, indicating a ligand-receptor binding between REG2 and CaV1.2. This putative binding required the C-type lectin binding domain of REG2 and glycosylation of CaV1.2, and suppressed glucose-and KCl-stimulated Ca 2+ influx in cultured OE islets and perifused WT islets. Along with our previous findings [2][3][4], the present study identifies a novel GPX1/REG2/CaV1.2 cascade to help explain how the REG2 depletion in OE islets could decrease its binding to CaV1.2, resulting in uninhibited Ca 2+ influx and insulin secretion after glucose stimulation. Because GPX1 overproduction initiates or drives this cascade [4] and dietary Se deficiency [3], but not diet restriction [2], precludes GPX1 overproduction in OE mice, it is now conceivable that only the former but not the latter prevented the augmented GSIS in those mice [2,3]. Linking GPX1 overproduction to GSIS and Ca 2+ influx through regulating REG2 production and its interactions with CaV1.2 provides a new paradigm to describe long-term metabolic mechanisms for GPX1 and other redox enzymes [40] as modulator of intracellular ROS [41]. While physiological levels of intracellular peroxides are required to trigger GSIS [42,43], there are various impacts of transient or acute elevation of intracellular ROS on oxidative modifications of protein kinases or phosphatases related to insulin signaling [43][44][45], excessive ROS on expression and function of beta cell-related transcriptional factors [46][47][48], and chronic exposure of ROS on blunting signaling of GSIS [49,50]. Clearly, our findings add a new mode of action or pathway for redox enzymes and ROS to regulate GSIS and perhaps other metabolic events.
The inhibition of GSIS by REG2 was demonstrated in both OE islets In all panels (except for B), data are mean ± SE, *, p < 0.05 for differences between REG2 or REG2 M2 verse buffer or PBS controls. In panel A, **, p < 0.05 for difference between 2.8 G and 30 G in insulin secretion. In panels C, D, and E, values on the top are areas under curve (AUC) of insulin secretion stimulated by glucose or KCl for the perifusion with buffer or REG2 and mutants, *p < 0.05 for difference between the buffer and treatments (REG2 or REG2 M2); G: glucose; mM, mmol/l; and the gradient bar above the trace: glucose ramp from 0 to 50 mmol/l. and mice. When the mutations of the C-type lectin binding domain in REG2 M1 led to a loss of inhibition on GSIS and Ca 2+ influx, the variant was no longer able to bind to or co-localize with CaV1.2 in beta TC3 cells. Clearly, the C-type lectin binding domain in REG2 was essential for the inhibition. In contrast, mutating the acidic residues of REG2 (REG2 M2) retained a moderate inhibition of GSIS on the high side of glucose ramp. The acidic residues in hREG1α were shown to be involved in CaCO 3 binding [8] and were highly conserved between the mouse REG2 and hREG1α (Supplemental Fig. 3). Seemingly, these residues might be involved in, but not essential, for the inhibition. Neither mutant (REG2 M1 or M2) exhibited significant inhibition on the KCl-induced insulin release. We do not have a clear structure explanation to this. Although we used relatively high glucose concentrations (30 or 50 mM) to stimulate the islets, our glucose ramp covered a wide range of GSIS from minimal to above maximal physiological concentrations. The perifused REG2 maintained similar inhibitions on GSIS from 15 mmol glucose/l to the end of glucose ramp, indicating a broad effectiveness of REG2 at both physiological and superphysiological glucose conditions. Such a broad spectrum of REG2 action was shown by its effects on GSIS and glucose tolerance in the OE mice that were hyperglycemia and hyperinsulinemia [1]. Consistently, REG2 exerted comparable inhibitions on Ca 2+ influx induced by 16.7 and 30 mmol glucose/l and 30 mmol KCl/l in OE islets, and reversible inhibition on the 16.7 mmol glucose-induced Ca 2+ influx in the WT islets. Notably, REG2 displayed little or insignificant effect on insulin secretion or Ca 2+ influx induced by low glucose concentrations including 2.8 and 3 mM in OE and(or) WT islets. Demonstrating effectiveness of exogenous REG2 in inhibiting GSIS of OE islets and mice has biomedical implications. Firstly, it confers a new role for REG2 other than beta cell proliferation [5] and regeneration [19,51]. Although trophic functions have been an exclusive focus of REG2 research since its discovery [6], a lack of impact by the Reg2 deletion in young mice [12] on beta cell proliferation failed to support that premise. Secondly, illustrating the inhibition on GSIS by exogenous REG2 in OE islets and mice provides a direct evidence for the proposed endo-, para- and autocrine functions of the protein [9]. It may underscore metabolic potentials for REG2 secreted by cultured islets into media [4] and by exocrine pancreas under various stress conditions [9,10,12]. Intriguingly, the old Reg2− /− mice showed an impaired GSIS [12], whereas our REG2-depleted OE mice displayed an augmented GSIS [2]. This GSIS-phenotype discrepancy may be partially explained by the decreased beta cell proliferation and mass in the former in contrast to the hypertrophy of beta cells, upregulation of pancreatic and duodenal homeobox 1 (PDX1) and insulin synthesis, and down-regulation of uncoupling protein 2 in the latter. Nevertheless, it will be interesting to find out if repeated injections of REG2 to both OE and WT mice for an extended time affect their pancreatic beta cell mass and insulin synthesis, in addition to GSIS. Conversely, it will be relevant to determine if glucose exposure and uptake affect REG2 expression and function in islets.
It is exciting to reveal the ligand-receptor binding of REG2 to CaV1.2 in beta TC3 cells and the subsequent effect on the glucose-stimulated Ca 2+ influx in OE and WT islets. This finding represents a dual breakthrough in the field, which qualifies REG2 as a novel regulator of CaV1.2 and CaV1.2 as a new cognate receptor/binding protein of REG2. None of the REG proteins has been shown to bind CaV1.2 or affect its role in Ca 2+ influx and GSIS [25][26][27][28]52]. Although the REG family proteins are a group of secretory proteins requiring transmembrane receptors to exert their functions, only exostosin like glycosyltransferase 3 (EXTL3) and epidermal growth factor receptor (EGFR) were shown as the receptor of or to bind human REG1α and human and mouse REG3A [37,53,54]. Despite the presence of the C-type lectin binding domain in all REG proteins, only the present study has succeeded in finding CaV1.2 as one of those transmembrane proteins with a carbohydrate moiety to bind that domain of REG2.
Clarifications, cautions, and future research may be required to interpret our novel findings. Notably, the physiological sources and routes of REG2 for regulating GSIS need a precise characterization. This is because production or secretion of REG2 by endocrine cells of pancreas remains uncertain [9,10,12]. Result discrepancies among different groups may arise from genetic background or (and) age-dependent and pancreatic-specific expressions of mouse REG2 [10] and specificity of reagents (antibodies) [4,[13][14][15]. The same controversy also exists on expression of the putative human orthologue hREG1β in beta cells [55] or islets [18]. However, the highly expressed hREG1β, as a relatively small molecule found in the blood, in exocrine pancreas [16] might reach islets to affect GSIS. Meanwhile, the effectiveness of exogenous REG2 administration in inhibiting GSIS in OE mice may suggest a distant pre-clinical potential of using hREG1β and its antagonists to treat GSIS disorders in hyperinsulinemic and diabetic patients. While GPX1 is considered to protect against various chronic diseases [56][57][58], GPX1 polymorphism [59][60][61][62][63] and (or) GPX activity alterations [64][65][66][67][68][69] are associated with insulin-related disorders. However, the exact pathological implication of the GPX1/REG2/CaV1.2 cascade in T2DM and other diseases remains a future research question, because changes of REG2 in different types of T2DM were not unilateral [4,20].
The revealed ligand-receptor interaction between REG2 and CaV1.2 may be substantiated by performing Ca 2+ current measurements in depolarized beta cells in the absence and presence of REG2 and the mutants. Because CaV1.2 and other VDCC proteins contain not only the α1c but also α2δ1 subunit [23,24], it will be useful to test if REG2 binds the α1c subunit directly or via an auxiliary subunit, and if the inhibition on GSIS is mainly due to this liagnd-receptor binding or partially related to functions of peptides of REG2 degradation [14]. It will also be important to find out how the mutations of acidic residues in REG2 retained some inhibition on GSIS, but lost most inhibition on the KCl-stimulated insulin release, if the inhibition on GSIS by REG2 involves both Ca 2+ -influx-triggered and metabolically amplified steps, and how differences between human and mouse β-cells in the expression of other Ca 2+− channels [70] affects the REG2 interaction with the L-type channels.

Conclusion
Our laboratory previously demonstrated that overproduction of GPX1 led to augmented GSIS and depletion of REG2 via transcriptional inhibition in pancreatic islets of mice. Because REG2 contains a canonical C-type lectin binding domain that could potentially interact with glycoproteins on cell surface to regulate GSIS, we have explored the role and mechanism of REG2 in binding L-voltage-dependent Ca 2+ channel CaV1.2, a transmembrane protein with key roles in the glucosestimulated elevation of Ca 2+ influx and the induced insulin exocytosis. Our results have revealed a potent inhibition by REG2 on GSIS and(or) the glucose-induced Ca 2+ influx in cultured and perifused islets and(or) mice, along with a ligand-receptor binding of REG2 to CaV1.2 and their co-localization in beta cells. Altogether, our work identifies a novel pathway of GPX1/REG2/CaV1.2 in regulating GSIS and creates new links to bridge redox biology, tissue regeneration, and insulin secretion.

Data and resource availability
Data sharing: Datasets generated and/or analyzed during the current study are available from corresponding authors upon reasonable request.

Resource sharing
Resources generated during the current study are available from corresponding authors upon reasonable request.

Funding
This research was supported by a NIH grant DK53018 (to XGL).

Contributions of authors
X.Y. and Z.P.Z contributed equally to designing and performing experiments, analyzing data, and writing the manuscript. J.-W.Y. helped in determining Reg2 expression. J. W. helped in designing, producing, and testing the native and mutant REG2 proteins. CAR helped the immuneblotting. T.S. performed the co-immunoprecipitation of deglycosylated CaV1.2 and the Ca 2+ influx in the cultured islets. N.M.D. helped the Ca 2+ influx assay in the perifused islets. F. H. helped in performing the AP-Tag and mCherry assays. CCM and HS helped the data interpretation and the manuscript writing. M.Z.V. contributed to experimental design, bioinformatics of REG2, islets perfusion experiments, data analysis, and manuscript preparation. X.G.L conceived, designed, and supervised this research and was responsible for writing the final version of the manuscript. XY and CAR take responsibilities of data presented in Figs. 1 and 2; XY, ZZ, TS, and NMD take responsibilities of data presented in Figs. 3-5. XGL and MZV are the guarantor of this work and, as such, have full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Data availability
Data will be made available on request.