Effects of Arginine Vasopressin on Islet Cells in Pancreatic Tissue Slices: Glucose-Dependent Modulation of IP₃ Receptor-Specific Responses

Reviewer #1 (Public review):

Summary:

The authors confirmed earlier findings that AVP influences α and β cells differently, depending on glucose concentrations. At substimulatory glucose levels, AVP combined with forskolin - an activator of cAMP -did not significantly stimulate β cells, although it did activate α cells. Once glucose was raised to stimulatory levels, β cells became active, and α cell activity declined, indicating glucose's suppressive effect on α cells and permissive effect on β cells. Under physiological glucose levels (8-9 mM), forskolin enhanced β-cell calcium oscillations, and AVP further modulated this activity. However, AVP's effect on β cells was variable across islets and did not significantly alter AUC measurements (a combined indicator of oscillation frequency and duration). In α cells, forskolin and AVP led to increased activity even at high glucose levels, suggesting that α cells remain responsive despite expected suppression by insulin and glucose.

Experiments with physiological concentrations of epinephrine suggest that AVP does not operate via Gs-coupled V2 receptors in β cells, as AVP could not counteract epinephrine's inhibitory effects. Instead, epinephrine reduced β cell activity while increasing α cell activity through different G-protein-coupled mechanisms. These results emphasize that AVP can potentiate α-cell activation and has a nuanced, context-dependent effect on β cells.

The most robust activation of both α and β cells by AVP occurred within its physiological osmo-regulatory range (~10-100 pM), confirming that AVP exerts bell-shaped concentration-dependent effects on β cells. At low concentrations, AVP increased β cell calcium oscillation frequency and reduced "halfwidths"; high concentrations eventually suppressed β cell activity, mimicking the muscarinic signaling. In α cells, higher AVP concentrations were required for peak activation, which was not blunted by receptor inactivation within physiological ranges.

Attempting to further dissect the role of specific AVP receptors, the authors designed and tested peptide ligands selective for V1b receptors. These included a selective V1b agonist; a V1b agonist with antagonist properties at V1a and oxytocin receptors; and a selective V1a antagonist. In pancreatic slices, these peptides seem to replicate AVP's effects on Ca²⁺ signaling, although responses were highly variable, with some islets showing increased activity and others no change or suppression. The variability was partly attributed to islet-specific baseline activity, and the authors conclude that AVP and V1b receptor agonists can modulate β cell activity in a state-dependent manner, stimulating insulin secretion in quiescent cells and inhibiting it in already active cells.

Strengths:

Overall, the study is technically advanced and provides useful pharmacological tools. However, the conclusions are limited by a lack of direct mechanistic and functional data. Addressing these gaps through a combination of signaling pathway interrogation, functional hormone output, genetic validation, and receptor localization would strengthen the conclusions and reduce the current (interpretive) ambiguity.

Weaknesses:

(1) The study is entirely based on pharmacological tools. Without genetic models, off-target effects or incomplete specificity of the peptides cannot be fully ruled out.

(2) Despite multiple claims about β cell activation or inhibition, the functional output - insulin secretion - is weakly assessed, and only in limited conditions. This aspect makes it very hard to correlate calcium dynamics with physiological outcomes.

(3) Insulin and glucagon secretion assays should be provided; the authors should measure hormone release in parallel with Ca2+ imaging, using perifusion assays, especially during AVP ramp and peptide ligand applications.

Additionally, there is no standardization of the metabolic state of islets. The authors should consider measuring islet NAD(P)H autofluorescence or mitochondrial potential (e.g., using TMRE) to control for metabolic variability that may affect responsiveness.

(4) There is a high degree of variability in response to AVP and V1b agonists across islets (activation, no effect, inhibition). Surprisingly, the authors do not fully explore the cause of this heterogeneity (whether it is due to receptor expression differences, metabolic state, experimental variability, or other conditions).

(5) There is no validation of V1b receptor expression at the protein or mRNA level in α or β cells using in situ hybridization, immunohistochemistry, or spatial transcriptomics.

(6) AVP effects are described in terms of permissive or antagonistic effects on cAMP (especially in relation to epinephrine), but direct measurements of cAMP in α and β cells are not shown, weakening these conclusions. The authors should use Epac-based cAMP FRET sensors in α and β cells to monitor the interaction between AVP, forskolin, and epinephrine more conclusively.

(7) Single-islet transcriptomics or proteomics (also to clarify variability) should be provided to analyze receptor expression variability across islets to correlate with response phenotypes (activation vs inhibition). Alternatively, the authors could perform calcium imaging with simultaneous insulin granule tracking or ATP levels to assess islet functional states.

(8) While the study implies AVP acts through V1b receptors on β cells, the signaling downstream (e.g., PLC activation, IP3R isoforms involved) is simply inferred but not directly shown.

(9) The interpretation that IP3R inactivation (mentioned in the title!) underlies the bell-shaped AVP effect is just hypothetical, without direct measurements. Assays in β (and/or α)-cell-specific V1b KO mice and IP3R KO mice must be provided to support these speculations.

Reviewer #2 (Public review):

Summary:

In this paper, Drs. Kercmar, Murko, and Bombek make a series of observations related to the role of AVP in pancreatic islets. They use the pancreatic slice preparation that their group is well known for. The observations on the slide physiology are technically impressive. However, I am not convinced by the conclusions of this manuscript for a number of reasons. At the core of my concern is perhaps that this manuscript appears to be motivated to resolve 'controversies' surrounding the actions of AVP on insulin and glucagon secretion. This manuscript adds more observations, but these do not move the field forward in improving or solidifying our mechanistic understanding of AVP actions on islets. A major claim in this manuscript is the beta cell expression of the V1b Receptor for AVP, but the evidence presented in this paper falls short of supporting this claim. Observations on the activation of calcium in alpha cells via V1b receptor align with prior observations of this effect.

I have focused my main concerns below. I hope the authors will consider these suggestions carefully - please be assured that they were made with the intent to support the authors and increase the impact of this work.

Strengths:

The main strength of this paper is the technical sophistication of the approach and the analysis and representation of the calcium traces from alpha and beta cells.

Weaknesses:

(1) The introduction is long and summarizes a substantive body of literature on AVP actions on insulin secretion in vivo. There are a number of possible explanations for these observations that do not directly target islet cells. If the goal is to resolve the mechanistic basis of AVP action on alpha and beta cells, the more limited number of papers that describe direct islet effects is more helpful. There are excellent data that indicate that the actions of AVP are mediated via V1bR on alpha cells and that V1bR is a) not expressed by beta cells and b) does not activate beta cell calcium at all at 10 nM - which is the same concentration used in this paper (Figure 4G) for peak alpha cell Ca2+ activation (see https://doi.org/10.1016/j.cmet.2017.03.017; cited as ref 30 in the current manuscript).

(2) We know from bulk RNAseq data on purified alpha, beta, and delta cells from both the Huising and Gribble groups that there is no expression of V2a. I will point you to the data from the Huising lab website published almost a decade ago (http://dx.doi.org/10.1016/j.molmet.2016.04.007) - which is publicly available and can be used to generate figures (https://huisinglab.com/data-ghrelin-ucsc/index.html). They indicate the absence of expression of not only AVP2 receptors anywhere in the islet, but also the lack of expression of V1bra, V1brb, and Oxtr in beta cells. Instead of the detailed list of expression of these 4 receptors elsewhere in the body, it would be more directly relevant to set up their pancreatic slice experiments to summarize the known expression in pancreatic islets that is publicly available. It would also have helped ground the efforts that involved the generation of the V1aR agonist and V2R antagonist, which confirm these known AVP/OXT receptor expression patterns.

(3) Importantly, the lack of V1br from beta cells does not invalidate observations that AVP affects calcium in beta cells, but it does indicate that these effects are mediated a) indirectly, downstream of alpha cell V1br or b) via an unknown off-target mechanism (less likely). The different peak efficacies in Figure 4G would also suggest that they are not mediated by the same receptor.

(4) The rationale for the use of forskolin across almost all traces is unclear. It is motivated by a desire to 'study the AVP dependence of both alpha and beta cells at the same time'. As best as I can determine, the design choice to conduct all studies under sustained forskolin stimulation is related to the permissive actions of AVP on hormone secretion in response to cAMP-generating stimuli. The permissive actions by AVP that are cited are on hormone secretion, which in many cell types requires activation of both calcium and cAMP signaling. Whether the activation of V1br and subsequent calcium response is permitted by cAMP is unclear. I believe the argument the authors are making here is that the activation of beta cell calcium by AVP is permitted by forskolin. i.e., the cAMP stimulated by it in beta cells. However, the design does not account for the elevation of cAMP in alpha cells and subsequent release of glucagon, particularly upon co-stimulation with AVP, which permits glucagon release by activating a calcium response in alpha cells. This glucagon could then activate beta cells. If resolving the mechanism of action is the goal, often less is more. The activation of Gaq-mediated calcium is not cAMP dependent (although the downstream hormone secretion clearly often is). As was shown, AVP does not activate calcium in beta cells in the absence of cAMP. The experiments in Figures 1, 2, and 4 should have been completed in the absence of cAMP first.

(5) It is unexpected that epinephrine in Figure 2 does not activate the alpha cell calcium? A recent paper from the same group (Sluga et al) shows robust calcium activation in alpha cells in a similar prep by 1 nM epinephrine, which is similar to the dose used here.

(6) Figure 8 suggests a pharmacological activation of beta cell V1bR in the low pM range. How do the authors reconcile this comparison with the apparent absence of an effect of AVP stimulation at low pM to low nM doses in beta cells (Figure 4A)? I note that there are changes over time with sustained beta cell stimulation with 8 mM glucose, but these changes are relatively subtle, gradual, and quite likely represent the progression of calcium behaviors that would have occurred under sustained glucose, irrespective of these very low AVP concentrations. I will note that the Kd of the V1bR for AVP is around 1 nM, with tracer displacement starting around 100 pM according to the data in figure 5B, which is hard to reconcile with changes in beta cell calcium by AVP doses that start 10-100-fold lower than this dose at 1 and 10 pM (Figure 8).

Reviewer #3 (Public review):

Summary:

This work aims to better understand the role of arginine vasopressin (AVP) in the control of islet hormone secretion. This builds on previous literature in this area reporting on the actions of AVP to stimulate islet hormones. The gap in literature being addressed by these studies is primarily focused on the glucose-dependency of AVP on both insulin and glucagon secretion. A secondary objective is to explore the role of individual receptors with the use of newly generated peptides and existing tools. The methods include the use of Ca2+ imaging in pancreas slices from mice, with additional outcomes including insulin secretion in some areas. The conclusions presented are that AVP acts through V1b receptors in both alpha- and beta-cells, that this activity occurs in the high cAMP environment, and is glucose dependent.

Strengths:

The area of research is emerging with plenty of room for new contributions. The concept of AVP stimulating islet hormone secretion is important and deserving of further insight. The use of pancreas tissue to image primary cells makes the experiments physiologically relevant. The advancement of novel tools in this area should be helpful to other groups investigating the actions of AVP.

Weaknesses:

The conclusions are only modestly supported by the data and lack experimental depth and rigor. The rationale for only conducting studies at high cAMP conditions is not entirely clear and limits the conclusions that can be made. The use of Ca2+ is helpful, but it is a surrogate for hormone secretion. Additional measurements of hormone secretion are needed to enhance the robustness of these conclusions. Consideration of paracrine effects between alpha- and beta-cells is only superficially made and is likely essential in the context of the experimental design. For instance, there is clear literature that alpha-cells secrete several factors that work in paracrine interactions on beta-cells and autocrine actions back on alpha-cells. Conducting these studies in a high cAMP context only completely overlooks these interactions, skewing the interpretations made by the investigators. Finally, the clarity of the experiments and results could be significantly enhanced.

Author response:

We sincerely thank the reviewers and editors for their thoughtful and constructive evaluation of our manuscript and their recognition of its technical strengths, including advanced spatio-temporal Ca2+ imaging, image processing, and the rational design of selective AVP receptor ligands. We appreciate their acknowledgement that our study contributes to the understanding of glucose-dependent AVP effects in pancreatic islet physiology. Their comments will guide us to refine the scope of our work, which focuses on how α and β cells respond to AVP under varying glucose and hormonal conditions, rather than on linear correlations between the function and transcript levels in individual cells or metabolic profiles in individual cell. Most of the reviewers´ concerns and proposed remedies reflect a reductionist framework, for which we believe cannot not fully account for emergent behavior within the islet collective. As we and others have shown, islet cells do not behave in isolation; their responses often depend on the state of the entire cell population(1, 2). This means that even under identical experimental conditions, responses can differ depending on the islet’s current state. These patterns are not random, but reflect how the islet integrates signals dynamically(3, 4).

To take advantage of both the systems and molecular side, we do plan to address several of the reviewers' suggestions with new experiments and analyses:

First, we will add hormone, specifically glucagon, secretion assays to support our conclusions on α cell responses and possible paracrine effects. Second, we will include a targeted transcript analysis of V1bR using RNAscope and extend the pharmacological characterization of downstream signaling using selective agonists and inhibitors. Third, we will clarify the rationale for using forskolin, and added new experiments using GLP-1 analogues to selectively increase cAMP in β cells, allowing us to examine direct AVP effects. And fourth, we will reinforce presence of emergency and that variability in islet responses is not experimental noise, but a hallmark of the collective, non-linear behavior of the islet cell collective, which should later drive a rethinking of experimental designs and the interpretation of pharmacological responses. In conclusion, we believe that our study provides new insights into AVP modulation in pancreatic islets and highlights the importance of context-dependent responses in α and β cells. We are grateful for the opportunity to revise our manuscript and look forward to further strengthening it further through the review process.

(1) Jin E, Briggs JK, Benninger RKP, Merrins MJ. Glucokinase activity controls peripherally-located subpopulations of β-cells that lead islet Ca2+ oscillations. eLife Sciences Publications, Ltd; 2025.

(2) Korošak D, Jusup M, Podobnik B, Stožer A, Dolenšek J, Holme P, et al. Autopoietic Influence Hierarchies in Pancreatic β Cells. Phys Rev Lett. 2021;127(16):168101.

(3) Ball P. How life works : a user's guide to the new biology. Chicago: The University of Chicago Press; 2023. 541 pages p.

(4) Fancher S, Mugler A. Fundamental Limits to Collective Concentration Sensing in Cell Populations. Phys Rev Lett. 2017;118(7):078101.