Arginine-vasopressin mediates counter-regulatory glucagon release and is diminished in type 1 diabetes
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Evaluation Summary:
Although the cellular and molecular mechanisms for insulin secretion regulation are relatively well defined, the precise control of glucagon secretion remains poorly understood. This paper is an elegant and thorough investigation into the role of Arginine-vasopressin (AVP) in glucagon secretion. It is known that AVP is a robust activator of calcium response in pancreatic alpha cells leading to glucagon release. The physiological relevance and regulation of this AVP-induced glucagon secretion is unclear. This manuscript goes a long way in closing this gap.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Insulin-induced hypoglycemia is a major treatment barrier in type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (8 to 4 mM) has no significant effect on glucagon secretion in the perfused mouse pancreas or in isolated mouse islets (in vitro), and yet associates with dramatic increases in plasma glucagon. The identity of the systemic factor(s) that elevates circulating glucagon remains unknown. Here, we show that arginine-vasopressin (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Alpha-cells express high levels of the vasopressin 1b receptor (V1bR) gene ( Avpr1b ). Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of the AVP precursor peptide) and increased blood glucose; effects blocked by pharmacological antagonism of either the glucagon receptor or V1bR. AVP also mediates the stimulatory effects of hypoglycemia produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to insulin-induced hypoglycemia. AVP injection increased cytoplasmic Ca 2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. Hypoglycemia also increases circulating levels of AVP/copeptin in humans and this hormone stimulates glucagon secretion from human islets. In patients with T1D, hypoglycemia failed to increase both copeptin and glucagon. These findings suggest that AVP is a physiological systemic regulator of glucagon secretion and that this mechanism becomes impaired in T1D.
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Author Response:
Reviewer #1:
In this manuscript, Angela Kim et al. use a combination of in vitro and in vivo studies to determine how glucose-control of central AVP release controls pancreatic alpha-cell calcium influx and glucagon secretion to modulate blood glucose homeostasis. The manuscript clearly shows that activation of AVP release from magnocellular AVP neurons stimulates pancreatic islet glucagon secretion. Furthermore, the manuscript finds AVP (measured by circulating Copeptin) is elevated in plasma following insulin induced hypoglycemia, which also activates AVP neuron electrical excitability and calcium entry. To confirm that AVP release stimulates glucagon secretion via islet alpha-cell Avpr1b activation, both Avpr1b antagonists and an Avpr1b-/- mouse model were utilized. Finally, the manuscript looks at plasma AVP in …
Author Response:
Reviewer #1:
In this manuscript, Angela Kim et al. use a combination of in vitro and in vivo studies to determine how glucose-control of central AVP release controls pancreatic alpha-cell calcium influx and glucagon secretion to modulate blood glucose homeostasis. The manuscript clearly shows that activation of AVP release from magnocellular AVP neurons stimulates pancreatic islet glucagon secretion. Furthermore, the manuscript finds AVP (measured by circulating Copeptin) is elevated in plasma following insulin induced hypoglycemia, which also activates AVP neuron electrical excitability and calcium entry. To confirm that AVP release stimulates glucagon secretion via islet alpha-cell Avpr1b activation, both Avpr1b antagonists and an Avpr1b-/- mouse model were utilized. Finally, the manuscript looks at plasma AVP in humans undergoing a hypoglycemic clamp; while this results in AVP release in non-diabetic controls, AVP release is blunted following hypoglycemia in type-1 diabetic patients. Based on an extensive amount of high-quality data, the authors conclude that AVP release from magnocellular AVP neurons is involved in regulating glucagon secretion in response to hypoglycemia. The manuscript is well written and easy to follow. As the exact mechanism that controls glucagon secretion is still unknown, this manuscript adds important information for the diabetes research community detailing the importance of CNS control of islet glucagon secretion through glucose regulated AVP release. Overall, this is an excellent manuscript that will be very useful to the diabetes research community.
We are grateful for the reviewers encouraging remarks and constructive feedback on our study.
Reviewer #2:
The authors cover a lot of ground, physiologically, by expanding from the islet up through multiple regions of the brain, but they do so in a manner that is stepwise and logical. And in the end, their efforts in probing further and further up the pathway results in a clean model of hypoglycemia sensing through to glucagon release. How AVP fits into counterregulation has been unclear, but Briant and colleagues are filling that gap. The paper is well written, the data are of high quality and well presented.
We are grateful for the reviewers encouraging remarks and helpful feedback on our study.
Specific comments:
• Lines 137-139 state that reducing glucose from 8 to 4 mM does not stimulate glucagon from ex vivo islets, but the experiment does not appear to show glucose being reduced. Rather, islets were incubated in separate glucose concentrations and the glucagon from the separate wells was then measured. Methods indicate that islets were incubated at 3 mM prior to each treatment, so glucose was actually raised from 3 to 4 mM and separately from 3 to 8 mM. Suggest either changing the wording or show a perfusion secretion experiment demonstrating the drop from 8 to 4 mM.
The reviewer is correct in that the isolated islet experiments in Figure 1 are static secretion experiments. We have now reworded this section to make this clear (Line 140-145).
• Lines 195-196 & Figure 3f: If YM254890 blocks AVP-induced calcium, please indicate with statistics comparing frequency in AVP and AVP + YM
Thank you for pointing out this. The p-value for this comparison (p=0.99) has now been added to the figure legend (Lines 552-553), with statistics indicated in the figure legend. However, the important point we tried to make here is that AVP has no effect in the presence of the inhibitor (YM vs. AVP + YM).
• Adrenergic signaling as a method of physiological glucagon stimulation is dismissed multiple times, yet is not tested/compared with AVP. The known and robust activation of calcium by AVP in alpha cells notwithstanding, epinephrine is a strong activator of alpha cell calcium responses and glucagon secretion. In multiple panels of the paper, blockade or deletion of Avp1rb reduces, but does not prevent hypoglycemia-induced glucagon secretion, which demonstrates that AVP is not the only signal stimulating alpha cells under these conditions.
We were certainly not suggesting that adrenaline is not important. The point we tried to make was that circulating levels of adrenaline are too low to stimulate glucagon secretion. In light of this comment, we have softened this section and we now acknowledge that adrenaline may contribute (Lines 371-377).
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Evaluation Summary:
Although the cellular and molecular mechanisms for insulin secretion regulation are relatively well defined, the precise control of glucagon secretion remains poorly understood. This paper is an elegant and thorough investigation into the role of Arginine-vasopressin (AVP) in glucagon secretion. It is known that AVP is a robust activator of calcium response in pancreatic alpha cells leading to glucagon release. The physiological relevance and regulation of this AVP-induced glucagon secretion is unclear. This manuscript goes a long way in closing this gap.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Angela Kim et al. use a combination of in vitro and in vivo studies to determine how glucose-control of central AVP release controls pancreatic alpha-cell calcium influx and glucagon secretion to modulate blood glucose homeostasis. The manuscript clearly shows that activation of AVP release from magnocellular AVP neurons stimulates pancreatic islet glucagon secretion. Furthermore, the manuscript finds AVP (measured by circulating Copeptin) is elevated in plasma following insulin induced hypoglycemia, which also activates AVP neuron electrical excitability and calcium entry. To confirm that AVP release stimulates glucagon secretion via islet alpha-cell Avpr1b activation, both Avpr1b antagonists and an Avpr1b-/- mouse model were utilized. Finally, the manuscript looks at plasma AVP in …
Reviewer #1 (Public Review):
In this manuscript, Angela Kim et al. use a combination of in vitro and in vivo studies to determine how glucose-control of central AVP release controls pancreatic alpha-cell calcium influx and glucagon secretion to modulate blood glucose homeostasis. The manuscript clearly shows that activation of AVP release from magnocellular AVP neurons stimulates pancreatic islet glucagon secretion. Furthermore, the manuscript finds AVP (measured by circulating Copeptin) is elevated in plasma following insulin induced hypoglycemia, which also activates AVP neuron electrical excitability and calcium entry. To confirm that AVP release stimulates glucagon secretion via islet alpha-cell Avpr1b activation, both Avpr1b antagonists and an Avpr1b-/- mouse model were utilized. Finally, the manuscript looks at plasma AVP in humans undergoing a hypoglycemic clamp; while this results in AVP release in non-diabetic controls, AVP release is blunted following hypoglycemia in type-1 diabetic patients. Based on an extensive amount of high-quality data, the authors conclude that AVP release from magnocellular AVP neurons is involved in regulating glucagon secretion in response to hypoglycemia. The manuscript is well written and easy to follow. As the exact mechanism that controls glucagon secretion is still unknown, this manuscript adds important information for the diabetes research community detailing the importance of CNS control of islet glucagon secretion through glucose regulated AVP release. Overall, this is an excellent manuscript that will be very useful to the diabetes research community.
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Reviewer #2 (Public Review):
The authors cover a lot of ground, physiologically, by expanding from the islet up through multiple regions of the brain, but they do so in a manner that is stepwise and logical. And in the end, their efforts in probing further and further up the pathway results in a clean model of hypoglycemia sensing through to glucagon release. How AVP fits into counterregulation has been unclear, but Briant and colleagues are filling that gap. The paper is well written, the data are of high quality and well presented.
Specific comments:
• Lines 137-139 state that reducing glucose from 8 to 4 mM does not stimulate glucagon from ex vivo islets, but the experiment does not appear to show glucose being reduced. Rather, islets were incubated in separate glucose concentrations and the glucagon from the separate wells was then …
Reviewer #2 (Public Review):
The authors cover a lot of ground, physiologically, by expanding from the islet up through multiple regions of the brain, but they do so in a manner that is stepwise and logical. And in the end, their efforts in probing further and further up the pathway results in a clean model of hypoglycemia sensing through to glucagon release. How AVP fits into counterregulation has been unclear, but Briant and colleagues are filling that gap. The paper is well written, the data are of high quality and well presented.
Specific comments:
• Lines 137-139 state that reducing glucose from 8 to 4 mM does not stimulate glucagon from ex vivo islets, but the experiment does not appear to show glucose being reduced. Rather, islets were incubated in separate glucose concentrations and the glucagon from the separate wells was then measured. Methods indicate that islets were incubated at 3 mM prior to each treatment, so glucose was actually raised from 3 to 4 mM and separately from 3 to 8 mM. Suggest either changing the wording or show a perfusion secretion experiment demonstrating the drop from 8 to 4 mM.
• Lines 195-196 & Figure 3f: If YM254890 blocks AVP-induced calcium, please indicate with statistics comparing frequency in AVP and AVP + YM
• Adrenergic signaling as a method of physiological glucagon stimulation is dismissed multiple times, yet is not tested/compared with AVP. The known and robust activation of calcium by AVP in alpha cells notwithstanding, epinephrine is a strong activator of alpha cell calcium responses and glucagon secretion. In multiple panels of the paper, blockade or deletion of Avp1rb reduces, but does not prevent hypoglycemia-induced glucagon secretion, which demonstrates that AVP is not the only signal stimulating alpha cells under these conditions.
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