The Kv2.2 channel mediates the inhibition of Prostaglandin E2 on glucose-stimulated insulin secretion in pancreatic β-cells
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Curated by eLife
eLife assessment
The study presents valuable findings on the molecular mechanisms of glucose-stimulated insulin secretion from pancreatic islets, focusing on the main regulatory elements of the signaling pathway in physiological conditions. While the evidence supporting the conclusions is solid, the study can be strengthened by the use of a beta cell line or knockout mice. The work will be of interest to cell biologists and biochemists working on diabetes.
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Abstract
Prostaglandin E2 (PGE2) is an endogenous inhibitor of glucose-stimulated insulin secretion (GSIS) and plays an important role in pancreatic β-cell dysfunction in type 2 diabetes mellitus (T2DM). This study aimed to explore the underlying mechanism by which PGE2 inhibits GSIS. Our results showed that PGE2 inhibited Kv2.2 channels via increasing PKA activity in HEK293T cells overexpressed with Kv2.2 channels. Point mutation analysis demonstrated that S448 residue was responsible for the PKA-dependent modulation of Kv2.2. Furthermore, the inhibitory effect of PGE2 on Kv2.2 was blocked by EP2/4 receptor antagonists, while mimicked by EP2/4 receptor agonists. The immune fluorescence results showed that EP1-EP4 receptors are expressed in both mouse and human β-cells. In INS-1(832/13) β-cells, PGE2 inhibited voltage-gated potassium currents and electrical activity through EP2/4 receptors and Kv2.2 channels. Knockdown of Kv2.2 reduced the action potential firing frequency and alleviated the inhibition of PGE2 on GSIS in INS-1(832/13) β-cells. PGE2 impaired glucose tolerance in wild-type mice but did not alter glucose tolerance in Kv2.2 knockout mice. Knockout of Kv2.2 reduced electrical activity, GSIS and abrogated the inhibition of PGE2 on GSIS in mouse islets. In conclusion, we have demonstrated that PGE2 inhibits GSIS in pancreatic β-cells through the EP2/4-Kv2.2 signaling pathway. The findings highlight the significant role of Kv2.2 channels in the regulation of β-cell repetitive firing and insulin secretion, and contribute to the understanding of the molecular basis of β-cell dysfunction in diabetes.
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Author response:
We thank the reviewers for their positive evaluation and constructive feedback on our study.
We acknowledge the concern regarding the use of HEK293T cells. In the revised manuscript, we will provide a more detailed explanation of the role of the PKA pathway in the regulation of GSIS by PGE2. To validate this regulation through Kv2.2, we will overexpress the Kv2.2 mutant channel in beta cells and assess its impact. Additionally, we will verify the specificity of the antibodies for EP1-EP4 receptors by knockdown. To confirm the receptors involved in PGE2 function, we will use additional EP receptor blockers or perform receptor knockdown experiments.
We will clarify that the described signaling pathway operates under normal physiological conditions and differs from pathological changes.
We once again thank the reviewers …
Author response:
We thank the reviewers for their positive evaluation and constructive feedback on our study.
We acknowledge the concern regarding the use of HEK293T cells. In the revised manuscript, we will provide a more detailed explanation of the role of the PKA pathway in the regulation of GSIS by PGE2. To validate this regulation through Kv2.2, we will overexpress the Kv2.2 mutant channel in beta cells and assess its impact. Additionally, we will verify the specificity of the antibodies for EP1-EP4 receptors by knockdown. To confirm the receptors involved in PGE2 function, we will use additional EP receptor blockers or perform receptor knockdown experiments.
We will clarify that the described signaling pathway operates under normal physiological conditions and differs from pathological changes.
We once again thank the reviewers for their positive evaluation and constructive suggestions.
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eLife assessment
The study presents valuable findings on the molecular mechanisms of glucose-stimulated insulin secretion from pancreatic islets, focusing on the main regulatory elements of the signaling pathway in physiological conditions. While the evidence supporting the conclusions is solid, the study can be strengthened by the use of a beta cell line or knockout mice. The work will be of interest to cell biologists and biochemists working on diabetes.
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Reviewer #1 (Public Review):
Summary:
This study investigated the mechanism by which PGE2 inhibits the release of insulin from pancreatic beta cells in response to glucose. The researchers used a combination of cell line experiments and studies in mice with genetic ablation of the Kv2.2 channel. Their findings suggest a novel pathway where PGE2 acts through EP2/EP4 receptors to activate PKA, which directly phosphorylates a specific site (S448) on the Kv2.2 channel, inhibiting its activity and reducing GSIS.
Strengths:
- The study elegantly demonstrates a potential pathway connecting PGE2, EP2/EP4 receptors, PKA, and Kv2.2 channel activity, using embryonic cell line.
- Additional experiments in INS1 and primary mouse beta cells with altered Kv2.2 function partially support the inhibitory role of PGE2 on GSIS through Kv2.2 inhibition.Weak…
Reviewer #1 (Public Review):
Summary:
This study investigated the mechanism by which PGE2 inhibits the release of insulin from pancreatic beta cells in response to glucose. The researchers used a combination of cell line experiments and studies in mice with genetic ablation of the Kv2.2 channel. Their findings suggest a novel pathway where PGE2 acts through EP2/EP4 receptors to activate PKA, which directly phosphorylates a specific site (S448) on the Kv2.2 channel, inhibiting its activity and reducing GSIS.
Strengths:
- The study elegantly demonstrates a potential pathway connecting PGE2, EP2/EP4 receptors, PKA, and Kv2.2 channel activity, using embryonic cell line.
- Additional experiments in INS1 and primary mouse beta cells with altered Kv2.2 function partially support the inhibitory role of PGE2 on GSIS through Kv2.2 inhibition.Weaknesses:
- A critical limitation is the use of HEK293T cells, which are not pancreatic beta cells. Functional aspects can differ significantly between these cell types.
- The study needs to address the apparent contradiction of PKA activating insulin secretion in beta cells, while also inhibiting GSIS through the proposed mechanism.
- A more thorough explanation is needed for the discrepancies observed between the effects of PGE2 versus Kv2.2 knockdown/mutation on the electrical activity of beta cells and GSIS. -
Reviewer #2 (Public Review):
The authors identified new target elements for prostaglandin E2 (PGE2) through which insulin release can be regulated in pancreatic beta cells under physiological conditions. In vitro extracellular exposure to PGE2 could directly and dose-dependently inhibit the potassium channel Kv2.2. In vitro pharmacology revealed that this inhibition occurs through the EP2/4 receptors, which activate protein kinase A (PKA). By screening specific sites of the Kv2.2 channel, the target phosphorylation site (S448) for PKA regulation was found. The physiological relevance of the described signaling cascade was investigated and confirmed in vivo, using a Kv2.2 knockdown mouse model.
The strength of this manuscript is the novelty of the (EP2/4-PKA-Kv2.2 channel) molecular pathway described and the comprehensive methodological …
Reviewer #2 (Public Review):
The authors identified new target elements for prostaglandin E2 (PGE2) through which insulin release can be regulated in pancreatic beta cells under physiological conditions. In vitro extracellular exposure to PGE2 could directly and dose-dependently inhibit the potassium channel Kv2.2. In vitro pharmacology revealed that this inhibition occurs through the EP2/4 receptors, which activate protein kinase A (PKA). By screening specific sites of the Kv2.2 channel, the target phosphorylation site (S448) for PKA regulation was found. The physiological relevance of the described signaling cascade was investigated and confirmed in vivo, using a Kv2.2 knockdown mouse model.
The strength of this manuscript is the novelty of the (EP2/4-PKA-Kv2.2 channel) molecular pathway described and the comprehensive methodological toolkit the authors have relied upon.
The introduction is detailed and contains all the information necessary to place the claims in context. Although the dataset is comprehensive and a logical lead is consistently built, there is one important point to consider: to clarify that the described signaling pathway is characteristic of normal physiological conditions and thus differs from pathological changes. It would be useful to carry out basic experiments in a diabetes model (regardless of whether this is in mice or rats).
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