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Vasopressin receptors in islets enhance glucose tolerance, pancreatic beta-cell secretory function, proliferation and survivalShruti Mohan, R. Charlotte Moffett, Keith G. Thomas, Nigel Irwin*, Peter R. FlattSAAD Centre for Pharmacy and Diabetes, University of Ulster, Coleraine, Northern Ireland, UK. *To whom correspondence should be addressed: Dr. Nigel Irwin, SAAD Centre for Pharmacy and Diabetes, University of Ulster, Coleraine, Northern Ireland, UK. E-mail: n.irwin@ulster.ac.uk. Tel: +44 28 7012 4574; Fax: ++44 (0) 28 7012 3939.Short title: Vasopressin in beta-cell functionKeywords: Beta-cell, islets, vasopressin, insulin secretion, diabetesAbstractArginine vasopressin (AVP), a peptide secreted from the posterior pituitary, is chiefly regarded as a hormone involved in the regulation of body fluid balance and osmolality. However, recent evidence has revealed that posterior pituitary hormones can exert important actions on endocrine pancreatic function. In the present study, the presence of AVP receptors, namely Avpr1a (V1a), Avpr1b (V1b) and Avpr2 (V2) was demonstrated in murine islets as well as rodent BRIN BD11 and human 1.1B4 beta-cells. Further to this, AVP was shown to induce significant concentration-dependent (10-12 – 10-6 M) increases of insulin release from both rodent and human beta-cells, as well as mouse islets. Insulinotropic actions of AVP were completely annulled by specific V1a or V1b receptor antagonists, and partially abolished by an oxytocin receptor antagonist. In addition, beta-cell insulin secretory actions of AVP were augmented by both IBMX (200 ?M) and KCl (30 mM) and linked to significantly increased cAMP production and [Ca2+]i. AVP substantially increased proliferation of rodent and human beta-cells. Moreover, AVP fully protected against cytokine-induced beta-cell apoptosis. AVP had no effect on glucagon secretion. Immunohistochemical examination of beta- and alpha-cells revealed co-expression of AVP with glucagon, and particularly insulin. Finally, administration of AVP in combination with glucose to mice significantly reduced blood glucose. These data indicate that AVP possesses novel and potentially important effects on pancreatic endocrine function. Understanding disturbances in islet AVP receptor signalling could reveal insight into the beta-cell defects associated with diabetes.1. IntroductionArginine vasopressin (AVP, also known as antidiuretic hormone (ADH)) is a 9 amino acid neuropeptide secreted by the posterior pituitary that is known to regulate fluid balance and cardiovascular function [1,2]. The biological effects of AVP are mediated through modulation of three separate G-protein coupled receptors (GPCRs), namely Avpr1a (V1a), Avpr1b (V1b) and Avpr2 (V2) [3]. While the V1a and V1b receptors are selectively linked to Gq/11 G-proteins, the V2 receptor is preferentially coupled to a Gs G-protein [4]. However, as well as controlling body fluid homeostasis and related actions, AVP has been suggested to be involved in the secretion of endocrine pancreatic hormones and overall metabolic control [5,6]. Indeed, it is now widely accepted that numerous hormones, beyond classical insulin, glucagon, somatostatin and pancreatic polypeptide, are involved in the physiological regulation of pancreatic islet cell function [7-10]. Further credence to this concept, and a possible endocrine role for AVP, is provided through recent observations with the related posterior pituitary peptide oxytocin, revealing local secretion and important pancreatic beta-cell actions of this hormone [11]. In addition, co-expression of the insulinotropic incretin hormone glucagon-like peptide-1 (GLP-1), oxytocin and AVP has been evidenced in the hypothalamus [12], again implying potentially important effects of AVP on centrally regulated metabolic processes.Studies using selective AVP receptor antagonists and mice genetically lacking either V1a or V1b receptors have suggested V1b receptors are important for AVP mediated islet effects [13]. In agreement, plasma insulin and glucagon levels are lower in V1b receptor knockout mice when compared to wildtype controls [5]. However, these V1b receptor knockout mice present with improved insulin sensitivity, implying possible adaptive responses in the face of lifelong decreased AVP action [14]. Nonetheless, positive effects of AVP on insulin secretion are absent in mice lacking V1b receptors [14]. However, the mechanisms underlying these islet and beta-cell effects are unclear, and the question whether local production of AVP is biologically significant, has not been assessed. As such, concentrations of AVP have been suggested to be significantly elevated in the pancreas of humans and rats, compared with the circulation [15]. Therefore, in the current study, we have explored the presence of AVP and its receptors in rodent and human beta-cells as well murine pancreatic islets. In vitro and in vivo effects of AVP on insulin release were also investigated, along with putative mechanisms of action. In addition, the impact of AVP on glucose homeostasis and insulin secretion was determined in normal lean mice. Finally, effects of AVP on beta-cell proliferation and protection against apoptosis were examined. 2. Materials and methods2.1 Gene expression Expression of AVP receptors was determined by extracting mRNA from pancreatic BRIN BD11 cells, 1.1B4 cells and isolated murine pancreatic islets (C57BL/6, 12-14 weeks old, Envigo Ltd.UK), using an RNeasy Mini Kit (Qiagen, UK), as described previously [16]. The extracted mRNA (1-3 ?g) was converted into cDNA using a SuperScript II reverse transcriptase kit (Invitrogen, Paisley, UK). Amplification conditions were set to 95°C for initial and final denaturation, primer annealing was set to 58°C and extension at 72°C for 40 cycles. This was followed by a melting curve analysis at temperatures between 60 - 90°C. Data obtained was analysed using ΔΔCt method and normalised to Actb/ACTB expression.2.2 ImmunohistochemistryAlpha-TC1.9 cells (courtesy of Professor Kevin Doherty, University of Aberdeen) and in-house derived BRIN BD11 beta-cells [17] were seeded on to chamber slides. Antigen retrieval was performed by incubating cells in citrate buffer at 95°C for 20 min, after which slides were blocked for 1 h with 2% BSA. Cells were then incubated with appropriate primary antibodies, followed by suitable secondary antibodies (Table 1). Importantly, the specificity of the AVP antibody was fully confirmed by blocking experiments using the native peptide. In addition, pancreatic tissue was excised from C57BL/6 mice (12-14 weeks old, Envigo Ltd.UK) and fixed in 4% paraformaldehyde (PFA) for 48 h at 4°C. Fixed tissues were subsequently dehydrated using a series of increasing strength ethanol solutions and processed for embedding in paraffin wax, as detailed previously [18]. Tissue blocks were sectioned (8 μm) using a Shandon Finesse 325 microtome (Thermo Scientific, Hemel Hempstead, UK) and picked for staining at intervals of 10 sections. After deparaffinising, sections were rehydrated using a series of decreasing strength ethanol solutions. Following antigen retrieval (citrate buffer, pH 6.0, 94°C for 20 min), sections were blocked using 2% BSA and incubated overnight at 4°C with appropriate primary antibody (Table 1). The slides were then incubated with appropriate secondary antibodies (Alexa Fluor??594 for red and Alexa Fluor??488 for green; Table 1) and stained with nuclear DAPI staining. Slides were mounted and photographed as previously described from our laboratory [8]. All staining procedures and image analysis were carried out in a blinded manner. Approximately 100 islets were analysed per group. All parameters were quantified using the ‘closed polygon’ tool in Olympus CellF analysis software. 2.3 In vitro insulin secretion studiesClonal pancreatic rodent BRIN BD11 and human 1.1B4 beta-cells were used to assess the insulin secretory activity of AVP. General culture conditions and characteristics of the employed cell lines have been reported previously [17,19]. In addition, insulin secretory effectiveness of AVP was examined in murine islets (C57BL/6, 12-14 weeks old, Envigo Ltd.UK) isolated using standard collagenase methods [20]. For BRIN BD11 and 1.1B4 cells, the insulin secretory action of AVP was evaluated by seeding the cells (150,000/well) into 24-well plates (Nunc, Roskilde, Denmark) and allowing to attach overnight at 37°C, 5% CO2. After preincubation in assay KRB buffer at 1.1 mM glucose, cells were incubated for 20 min with test peptides (10-12 – 10-6 M) at either 5.6 or 16.7 mM glucose. After the 20 min incubation, supernatant was removed and stored at -20°C until determination of insulin concentrations by radioimmunoassay [21]. For isolated islets, insulin secretion was determined as above, but with a 60 min test incubation period. Following removal of the test solution, 200 ?l of acid–ethanol solution (1.5% (v/v) HCl, 75% (v/v) ethanol, 23.5% (v/v) H2O) was added with overnight extraction of cellular insulin. To assess potential mechanism of AVP (10-6 M) insulinotropic action, secretion studies were performed with BRIN BD11 cells in the presence of known modulators of insulin release, including IBMX (200 ?M), diazoxide (300 ?M), verapamil (50 ?M) and KCl (30 mM) [22]. In addition, effects of AVP (10-6 M) on membrane potential and intracellular [Ca2+]i were also studied in BRIN BD11 cells at 5.6 mM glucose using a Flexstation scanning fluorometer (FLIPR Calcium 5 assay kit, Molecular Devices, Sunnyvale, USA), as previously described [23]. KCl (30 mM) was employed as a positive control for membrane potential studies and alanine (10 mM) for [Ca2+]i studies. Furthermore, to confirm AVP receptor modulation, BRIN BD11 cells were incubated with AVP alone (10-6?M) alone or in combination with either the oxytocin receptor antagonist, L-371,257 (10-6?M; Tocris), V1a receptor antagonist SR-49059 (10-6?M; Sigma-Aldrich), V1b receptor antagonist Nelivaptan (10-6?M; Axon Medchem) or the V2 receptor antagonist Tolvaptan (10-6?M; Sigma-Aldrich) and insulin secretion assessed as above. Finally, intracellular cAMP production (20 min, n=4) in the presence of IBMX (200 ?M) and AVP or GLP-1 (both at 10-6 M) was examined in BRIN BD11 cells using a Parameter cAMP assay (R&D Systems, Abingdon, UK), as previously described [20]. 2.4 In vitro glucagon secretion studiesAlpha-TC1.9 cells were used to assess in vitro glucagon secretory activity of AVP. General culture conditions and characteristics of this cell line has been previously described [24]. Cells were seeded (100,000/well) into 24-well plates (Nunc, Roskilde, Denmark) and allowed to attach overnight at 37°C, 5% CO2. After preincubation in assay KRB buffer at 25 mM glucose (1 h), cells were incubated for 2 h with either positive control arginine (10 mM), or AVP and GLP-1 (each at 10-6 M), at 5.6 mM glucose. After the incubation period, supernatant was removed and stored at -20°C until determination of glucagon concentrations using a commercially available chemiluminescent ELISA kit (EZGLU-30K; Millipore, Watford, UK).2.5 In vitro beta-cell proliferation and cellular stress studiesThe effect of AVP beta-cell proliferation and protection against apoptosis was studied in clonal BRIN BD11 and 1.1B4 cells. Cells were seeded at a density of 150,000 cells per chamber slide and cultured overnight (18 h) at 37oC, in the presence of AVP (10-6 M), with GLP-1 (10-6 M) and culture media alone as positive and negative controls, respectively. Following the incubation period, cells were washed with PBS and fixed using 4% paraformaldehyde and antigen retrieval performed with as outline above. Slides were then incubated with rabbit anti-Ki67 primary antibody followed by Alexa Fluor??594 secondary antibody. The slides were washed and mounted for viewing using a fluorescent microscope (Olympus System Microscope, model BX51; Southend-on-Sea, UK) and photographed with a DP70 camera adapter system. Proliferation frequency was expressed as % total cells analysed. To assess the protective effect AVP on cytokine-induced DNA damage in BRIN BD11 and 1.1B4 cells, cells were seeded as above and then exposed to a cytokine cocktail (IL-1β 100 U/mL, IFN-γ 20 U/mL, TNF-α 200 U/mL), in the presence and or absence of test peptides (10-6 M), for 2 hours. A TUNEL assay (Roche Diagnostics Ltd, UK) was used to quantify the level of beta-cell apoptosis, as described previously [25, 26]. 2.6 In vivo studies Studies were carried out in adult male NIH Swiss mice (12-14 weeks of age, Envigo Ltd, UK). All animals used were individually housed in an air conditioned room at 22±2°C with 12 hours light and dark cycle and ad libitum access to drinking water and standard rodent diet (10% fat, 30% protein and 60% carbohydrate; Trouw Nutrition, Northwich, UK). All experiments were carried out in accordance with the UK Animal Scientific Procedures Act 1986. For acute in vivo glucose homeostatic and insulin secretory response studies, blood glucose and plasma insulin were analysed immediately before and 15, 30 and 60 min after intraperitoneal injection of either glucose alone (18 mmol/kg bw) or in combination with GLP-1 or AVP (both at 25 nmol/kg bw) in 18 h fasted mice. This dose was chosen based on our previous studies with posterior pituitary hormones [11]. Blood glucose was measured directly using the Ascencia Contour blood glucose meter (Bayer Healthcare, Newbury, Berkshire, UK). Plasma insulin was evaluated by collecting blood in heparin/fluoride coated microcentrifuge tubes (Sarstedt, Numbrecht, Germany), from the cut tip on the tail vein of conscious mice. Collected blood was immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) for 5 min at 13,000 rpm and plasma stored at -20°C, prior to determination of insulin by radioimmunoassay. 2.7 Statistical analysisStatistical analyses were performed using GraphPad PRISM software (Version 5.0). Values are expressed as mean ± S.E.M. Comparative analyses between groups were carried out using a One-way ANOVA with Bonferroni post hoc test or student’s unpaired t-test, as appropriate. Difference between groups was considered significant if P < 0.05.3. Results3.1 Gene expression of AVP, GLP-1 and GIP receptorsIn BRIN-BD11 cells, expression of all AVP receptors, namely V1a, V1b and V2, was apparent but at significantly (P < 0.01 to P < 0.001) lower levels than Glp1r expression (Fig. 1A). However, Gipr mRNA levels were similar to Avpr1a and Avpr2 in BRIN BD11 cells, with Avpr1a expression being significantly higher (P < 0.01) than Gipr (Fig. 1A). In 1.1B4 cells, expression levels of Avpr1b were elevated (P < 0.01), Avpr1a reduced (P < 0.01) and Avpr2 unchanged, when comparted to Glp1r (Fig. 1B). All AVP receptors were expressed at lower intensity (P < 0.05 to P < 0.001) than Gipr in 1.1B4 beta-cells (Fig. 1B). In isolated mouse islets, mRNA expression of Avpr1a and Avpr2 was higher (P < 0.05 to P < 0.001) when compared to both Glp1r and Gipr expression, with Avpr2 expressed at similar levels as both the incretin receptors (Fig. 1C). 3.2 ImmunohistochemistryAVP was clearly evident in BRIN-BD11 cells and co-localised with approximately 50% of insulin staining in these cells (Fig. 2A,C). AVP was also observable in alpha-TC1.9 cells, but less than 10% of the alpha-cells exhibited co-localisation of AVP and glucagon (Fig. 2B,C). In murine islets, immunohistochemical AVP staining with either insulin or glucagon revealed no evidence of AVP within islet cells, and more AVP present in perivascular locations (Fig. 2D).3.3 In vitro insulin and glucagon release as well as beta-cell mechanistic studiesMurine islets demonstrated clear glucose-dependent insulinotropic properties (Fig. 3A). Notably, neither AVP nor GLP-1 induced insulin secretion from islets at glucose concentrations of 1.4 mM (Fig. 3A). However, both AVP and GLP-1 (10-6 M) stimulated significant (P < 0.01 to P < 0.001) insulin secretion from murine islets at 5.6 and 16.7 mM glucose when compared to respective glucose alone controls (Fig. 3A). In addition to this, AVP stimulated (P < 0.05 to P < 0.001) insulin release in a concentration-dependent (10-12 – 10-6 M) manner in both BRIN BD11 and 1.1B4 beta-cell lines, with similar or enhanced efficacy as GLP-1 at 10-6 M (Fig. 3B,C). The V1a and V1b receptor antagonists, SR-40959 and nelivaptan (both at 10-6 M) respectively, completely annulled (P < 0.001) the insulin secretory actions of AVP in BRIN BD11 cells (Fig. 3D). Furthermore, L-371,257, the oxytocin receptor antagonist, also partially abolished AVP-induced insulin secretion, whereas V2 receptor blockade had no impact on AVP mediated insulinotropic actions (Fig. 3D). Interestingly, AVP-induced (10-6 M) insulin secretion was significantly (P < 0.05 and P < 0.01, respectively) augmented in the presence of IBMX and KCl, but inhibited (P < 0.05) in the presence of verapamil and diazoxide (Fig. 3E). Notably, an almost identical insulin release pattern was observed following incubation of GLP-1 with these beta-cell secretory modulators (Fig. 3E). Furthermore, both AVP and GLP-1 significantly (P < 0.001) augmented cAMP production in BRIN BD11 cells, albeit GLP-1 had increased (P < 0.01) efficacy than AVP (Fig. 3F). In addition to this, AVP and GLP-1 (10-6 M) had no effect on glucagon secretion from alpha-TC1.9 cells at 5.6 mM glucose, whereas 10 mM arginine evoked significant (P < 0.001) glucagon release from these cells (Fig. 3G). In terms of beta-cell membrane potential and [Ca2+]i, AVP (10-6 M) had no impact on membrane potential, but it did transiently increase (P < 0.01) [Ca2+]i when compared to 5.6 mM glucose control (Fig. 4A-D). 3.4 Beta-cell proliferation and protection against apoptosisAVP (10-6 M) significantly (P < 0.001) augmented BRIN BD11 and 1.1B4 cell proliferation, to a similar, or even elevated manner, when compared to the same concentration of GLP-1 (Fig. 5A,B). In terms of protecting against cytokine-induced apoptosis in BRIN BD11 and 1.1B4 cells, AVP was similar, or indeed more effective, than GLP-1 (Fig. 5C,D). As such, TUNEL positive staining was actually significantly (P < 0.05) reduced by AVP, but not GLP-1, when compared to media control (Fig. 5C,D). Figure 5E & F depict representative images of BRIN BD11 and 1.1B4 beta-cells stained for Ki-67 and TUNEL under each treatment condition. 3.5 Acute in vivo glucose homeostatic and insulin secretory effectsBoth AVP and GLP-1 significantly reduced (P < 0.001) individual and 0-60 min overall AUC glucose values when injected conjointly with glucose in mice (Fig. 6A,C). Corresponding individual and overall plasma insulin concentrations were significantly elevated (P < 0.05) by GLP-1 (Fig. 6B,D). Interestingly, AVP did not induce any obvious elevation of overall insulin secretory activity, despite reduced glucose levels, when compared to glucose control (Fig. 6D). 4. Discussion The relatively abundant expression of all AVP receptor subtypes in murine islets, as well as a rodent and human beta-cell lines, clearly implies a modulatory role for AVP in pancreatic islet function. Indeed, quantification of V1b receptor expression in islets revealed significantly elevated levels compared to both the classic islet incretin receptors Glp-1r and Gipr [27, 28]. These observations correspond well with the earlier suggestion that the V1b receptor is expressed in pancreatic islets [29]. Although there were slight differences in the AVP receptor expression profile in rodent BRIN BD11 and human 1.1B4 beta-cells, when compared to mouse islets, the clear presence each receptor subtype highlights applicability of these cellular models to further investigate the beta-cell effects of AVP. Previous work has raised the possibility of a role for V1b receptors in AVP-induced insulin release [29]. Our studies with pharmacological receptor antagonists, would agree with this, but also suggest an important impact of both oxytocin and V1a receptors in AVP-induced insulin secretion [1]. However, we acknowledge that generation of specific V1a, V1b and V2 receptor knockdown beta-cell lines would be useful to confirm these findings. Thus, AVP stimulated insulin release from rodent and human beta-cells, as well as murine islets, with the effect completely annulled by co-administration V1a or V1b receptor antagonists, and partially inhibited by oxytocin receptor blockade. These findings also further corroborate the suitability of the selected in vitro models to study AVP beta-cell effects, although diminished glucose-responsiveness of cellular models needs to be acknowledged [30]. Interestingly, insulinoptropic effects of AVP were evident at both basal and elevated glucose concentrations [6]. Thus, it has been proposed that AVP induces insulin secretion only in the presence of high glucose concentrations, whereas glucagon release is augmented at lower glucose levels [31]. However, in the current study we were unable to evidence significant inhibitory effects of AVP on glucagon secretion from alpha-TC1.9 cells, albeit at physiological glucose levels. Still, a similar dual insulin and glucagon secreting scenario, dependent solely on ambient glucose levels, has been reported for the well characterised insulinotropic incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) [32]. Interestingly, it is notable that higher concentrations of AVP are reported to be required to stimulate insulin, rather than glucagon, secretion [33] and this could be one explanation as to why we did not observe increased insulin levels following AVP administration in mice. However, AVP did improve glucose homeostasis in these mice suggesting potential insulin independent glucose lowering actions, again similar to the biological actions of GIP [34].Furthermore, although we have reported clear augmentation of insulin secretion by AVP in rodent and human beta cells, elevations of blood glucose have been reported following chronic AVP infusion in rats [35]. Whilst it is possible that this could be linked to putative elevated glucagon secretion by AVP [5], our observations in alpha-TC1.9 cells along with the proposed glucose-dependent nature of AVP-induced glucagon secretion would advocate that this is unlikely [31]. Since AVP is thought to play a role in somatostatin secretion [36], as well as potentiating corticotropin-releasing hormone induced insulin release [37], a complex mode of action for AVP on pancreatic islets is very apparent. Given this, we considered it prudent to investigate the molecular mechanism of action of AVP-induced insulin release. Interestingly, AVP provoked a remarkably similar insulin secretory profile as GLP-1 in the presence of known modulators of beta-cell function, including IBMX, KCl, diazoxide and verapamil [38-40]. In addition, similar to GLP-1, AVP increased cAMP concentrations in BRIN BD11 cells. AVP had no impact on beta-cell membrane potential, but did induce a transient increase effect of [Ca2+]i, which has also been observed with oxytocin [11]. Together, this would imply a more distal, GLP-1 like, effect of AVP on insulin secretion, and further investigation into this is warranted.Epidemiological studies reveal that altered plasma AVP concentrations represent a risk factor for type 2 diabetes [41-43]. In view of these observations, we next assessed the impact of AVP on beta-cell growth and survival, with a view to deciphering a possible link to development of diabetes, given that the human disease is associated with beta-cell loss [44]. Strikingly, AVP induced proliferation and prevented cytokine-induced apoptosis of both rodent and human beta-cells, in a similar fashion to GLP-1 [27]. In agreement with this, a proliferative effect of AVP on rat intestinal epithelial cells as well as glomerular mesangial cells and vascular smooth muscle has previously been demonstrated [45,46]. A similar beta-cell protective phenomenon has also recently been reported for oxytocin [11], implying important pancreatic effects of both posterior pituitary hormones. These positive AVP beta-cell effects are consistent with a high percentage of BRIN BD11 beta-cells displaying co-localisation of AVP and insulin. However, further immunohistochemical examination of murine islets revealed that pancreatic AVP expression is largely confined to perivascular tissue, as previously observed [47]. This could suggest an additional role for locally produced pancreatic AVP on the vasculature of the gland, known to be important for overall pancreatic function [48], and in keeping with the recognised effects of AVP on vascular tone [1,2]. Indeed, investigation of blood pressure changes following AVP injection in mice may have been interesting in this regard. Thus, further investigation into the impact of local islet cell secretion and action of AVP is required, especially since pancreatic AVP concentrations are known to be elevated [15]. Such paracrine actions of AVP are likely to greatly augment the effects of AVP released from the posterior pituitary and delivered to the beta-cells via islet vasculature. In conclusion, this study reveals a novel and potentially important role of AVP in the regulation of pancreatic islet function. Similar to another posterior pituitary hormone oxytocin [11], AVP receptors are highly abundant in islets where they mediate important positive actions on insulin secretion as well as beta-cell growth and survival. This raises the possibility that disturbances in the modulation of islet AVP receptor signalling might contribute to beta-cell defects in diabetes.AcknowledgmentsThe study was supported by an EFSD/Boehringer Ingelheim grant, donation from the SAAD Trading and Contracting Company and award of an Ulster University Vice Chancellor’s research scholarship to SM. The authors declare that no conflicting interests exist.Conflict of interests The authors declare that no conflicting interests existReferencesT.A. Koshimizu, K. Nakamura, N. Egashira, M. Hiroyama, H. Nonoguchi, A. Tanoue, Vasopressin V1a and V1b Receptors: From Molecules to Physiological Systems, Physiol. Rev. 92 (2012) 1813-1864L.P. Renaud, Vasopressin, Encyclopedia of Stress (Second Edition) (2007) 824-829.M. 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Haeften, T. van Haeften W., Type 2 diabetes: principles of pathogenesis and therapy, Lancet. 365 (2010) 1333-46.P.M. Ghosh, M. Mikhailova, R. Bedolla, J.I. Kreisberg, Arginine vasopressin stimulates mesangial cell proliferation by activating the epidermal growth factor receptor, Am. J. Physiol. Renal Physiol. 280 (2001) F972-F979.G.P. Mavani, M.V. DeVita, M.F. Michelis, A review of the nonpressor and nonantidiuretic actions of the hormone vasopressin, Front. Med. (Lausanne). 2 (2015) 19.I. Méchaly, F. Macari, M. F. Laliberté , C. Lautier, J.J. Serrano, G. Cros, F. Grigorescu, Identification by RT-PCR and immunolocalisation of arginine vasopressin in rat pancreas, Diabetes Metab. 25 (1999) 498-501.B.M. Henry, B. Skinningsrud, K. Saganiak, P.A. P?kala, J.A. Walocha, K.A, Tomaszewski, Development of the human pancreas and its vasculature - An integrated review covering anatomical, embryological, histological, and molecular aspects, Ann Anat. 221 (2019) 115-124.Table 1. Target, host, dilution and source of primary and secondary antibodiesTargetHostDilutionSourceInsulin (primary)Mouse1:500Abcam, ab6995Glucagon (primary)Guinea pig1:200Raised in-house, PCA2/4AVP (primary)Rabbit1:200Abcam, ab68669Ki-67 (primary)Rabbit1:200Abcam, ab15580IgG (secondary)Goat1:400Alexa Flour 594, Invitrogen, UKIgG (secondary)Goat1:400Alexa Flour 488, Invitrogen, UKFigure legendsFig. 1. Expression of AVP receptors, Avpr1a, Avpr1b and Avpr2, in rodent and human beta-cells and in isolated mouse islets. AVP receptors mRNA expression in (A) rodent BRIN-BD11 cells, (B) human 1.1B4 cells and (C) C57BL/6 mouse islets. mRNA expression was compared against the mRNA expression of Glp1r and Gipr. Values are mean ± SEM (n=5). *P < 0.05, **p <0.01 and ***P < 0.001 compared to Glp1r. ΔP < 0.05, ΔΔP < 0.01 and ΔΔΔP < 0.001 compared to Gipr. All mRNA expression was normalized to Actb/ACTB expression. Fig. 2. Co-localisation of AVP with either insulin or glucagon in rodent BRIN BD11 and alpha-TC1.9 cells as well as mouse islets. Representation images of (A) BRIN-BD11 cells stained for AVP and insulin and (B) alpha-TC1.9 cells stained for AVP and glucagon. Nuclei are indicated by DAPI staining. (C) Quantification of cells displaying co-localisation of expression for AVP and insulin or AVP and glucagon, as appropriate. (D) Image of NIH Swiss mouse islet stained for AVP and insulin or glucagon, arrows indicate AVP staining. Values are mean ± SEM (n=3). ***P < 0.001 compared to AVP co-localisation with glucagon. Fig. 3. Effect of AVP on insulin release from (A) isolated mouse islets (B,D,E) rodent BRIN-BD11 cells and (C) human 1.1B4 cells as well as (F) cAMP generation in BRIN-BD11 cells and (G) glucagon release from alpha-TC1.9 cells. (A-C) Cells or islets were incubated (A 60 mins; B,C 20 mins) in either 1.4, 5.6 or 16.7 mM glucose, as appropriate, with AVP or related peptides (10-12 -10-6 M), and insulin secretion assessed. (D) Effects of specific V1a (SR-49059), V1b (nelivaptan), V2 (tolvaptan) and oxytocin (L-371,255) receptor antagonists (each at 10-6 M) on AVP-induced (10-7M) insulin secretion form BRIN-BD11 cells at 16.7 mM glucose. (E) Effects of known modulators of insulin secretion, namely IBMX (200 ?M), diazoxide (300 ?M), verapamil (50 ?M) and KCl (30 mM), on AVP-induced (10-6 M) insulinotropic actions in BRIN BD11 cells at 5.6 mM glucose, with GLP-1 (10-6 M) as a direct comparison. (F) BRIN-BD11 cells were incubated with IBMX (200 ?M) alone and in combination with AVP or GLP-1 (both at 10-6 M) for 20 min at 5.6 mM glucose and intracellular cAMP concentrations measured using ELISA. (G) Cells were incubated for 2 h in 5.6 mM glucose with alanine (10 mM), AVP or GLP-1 (both at 10-6 M), and glucagon secretion assessed. Values are a mean ± SEM (A,B,D,E,G n=8; C,F n=4). *P < 0.05, **P < 0.01 and ***P < 0.001 compared to respective glucose control. (A) ΔP < 0.05, ΔΔΔP < 0.001 compared to same incubation conditions at 1.4 mM glucose; +++ P < 0.05 compared to same incubation conditions at 5.6 mM glucose alone. (D,E) ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 compared to respective peptide control. (F) ΔΔP < 0.01 compared to GLP-1. (G) ΔΔP < 0.01 compared to arginine.Fig. 4. Effect of AVP on membrane potential and [Ca2+]i in rodent BRIN-BD11 cells. (A-D) Cells were incubated with 5.6 mM glucose in the presence of AVP (10-6 M) and (A) membrane potential or (B) [Ca2+]i assessed over a 5 minute period, with KCl (30 mM) or alanine (10 mM) as positive controls, respectively. (C, D) Area under curve data are also shown. Values are a mean ± SEM (n=6) *P < 0.05, ***P < 0.001 compared to respective glucose control. Fig. 5. Effect of AVP on proliferation and protection from cytokine induced stress in rodent BRIN-BD11 cells. (A,B) Proliferation in the presence of AVP and GLP-1 (both at 10-6 M) was assessed in (A) BRIN-BD11 and (B) 1.1B4 beta-cells. (C,D) TUNEL positive apoptotic cells were assessed following 2 h exposure to a cytokine cocktail (IL-1β 100 U/mL, IFN-γ 20 U/mL, TNF-α 200 U/mL) with or without co-culture in the presence of AVP or GLP-1 (both at 10-6 M) in (C) BRIN BD11 and (D) 1.1B4 beta-cells. (E,F) Representative images showing Ki-67 and TUNEL stained cells under each culture condition. The arrows point towards the proliferating or apoptotic cells, as appropriate. Values are mean ± SEM (n=4). *P < 0.05, **P < 0.01, ***P < 0.001 compared to control cultures. ΔΔΔP < 0.001 compared to cytokine cocktail.Fig. 6. Acute effects of AVP on glucose tolerance and insulin secretion in mice. (A) Blood glucose and (C) plasma insulin levels were assessed before and after intraperitoneal administration of glucose (18 mmol/kg, bw) alone or in combination with AVP or GLP-1 (both at 25 nmol/kg, bw) in overnight fasted mice. (B,D) Respective 0-60 min area under the curve data for (B) blood glucose and (D) plasma insulin. Values are mean ± SEM (n=6). *P < 0.05, **P < 0.01, ***P < 0.001 compared to glucose control. 10930890-3307742Figure 1Figure 1Figure 10141578778786087464Figure 6Figure 6Figure 612722067144 ................
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