Mechano-regulation of GLP-1 production by Piezo1 in intestinal L cells

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors intended to prove that gut GLP-1 expression and secretion can be regulated by Piezo1, and hence by mechanistic/stretching regulation. For this purpose, they have assessed Piezo1 expression in STC-1 cell line (a mouse GLP-1 producing cell line) and mouse gut, showing the correlation between Piezo1 level and Gcg levels (Figure S1). They then aimed to generate gut L cell-specific Piezo1 KO mice, and claimed the mice show impaired glucose tolerance and GLP-1 production, which can be mitigated by Ex-4 treatment (Figures 1-2). Pharmacological agents (Yoda1 and GsMTx4) and mechanic activation (intestinal bead implantation) were then utilized to prove the existence of ileal Piezo1-regulated GLP-1 synthesis (Figure 3). This was followed by testing such mechanism in a limited amount of primary L cells and mainly in the STC-1 cell line (Figures 4-7).

While the novelty of the study is somehow appreciable, the bio-medical significance is not well demonstrated in the manuscript. The authors stated (in lines between lines 78-83) a number of potential side effects of GLP-1 analogs, how can the mechanistic study of GLP-1 production on its own be essential for the development of new drug targets for the treatment of diabetes. Furthermore, the study does not provide a clear mechanistic insight on how the claimed CaMKKbeta/CaMKIV-mTORC1 signaling pathway upregulated both GLP-1 production and secretion. This reviewer also has concerns about the experimental design and data presented in the current manuscript, including the issue of how proglucagon expression can be assessed by Western blotting.

Strengths:

The novelty of the concept.

Weaknesses:

Experimental design and key experiment information.

Reviewer #2 (Public Review):

Summary:

The study by Huang and colleagues focuses on GLP-1 producing entero-endocrine (EEC) L-cells and their regulation of GLP-1 production by a mechano-gated ion channel Piezo1. The study describes Piezo1 expression by L-cells and uses an exciting intersectional mouse model (villin to target epithelium and Gcg to target GLP-1-producing cells and others like glucagon-producing pancreatic endocrine cells), which allows L-cell specific Piezo1 knockout. Using this model, they find an impairment of glucose tolerance, increased body weight, reduced GLP-1 content, and changes to the CaMKKbeta-CaMKIV-mTORC1 signaling pathway using a normal diet and then high-fat diet. Piezo1 chemical agonist and intestinal bead implantation reversed these changes and improved the disrupted phenotype. Using primary sorted L-cells and cell model STC-1, they found that stretch and Piezo1 activation increased GLP-1 and altered the molecular changes described above.

Strengths:

This is an interesting study testing a novel hypothesis that may have important mechanistic and translational implications. The authors generated an important intersectional genetics mouse model that allowed them to target Piezo1 L-cells specifically, and the surprising result of impaired metabolism is intriguing.

Weaknesses:

However, there are several critical limitations that require resolution before making the conclusions that the authors make.

(1) A potential explanation for the data, and one that is consistent with existing literature [see for example, PMC5334365, PMC4593481], is that epithelial Piezo1, which is broadly expressed by the GI epithelium, impacts epithelial cell density and survival, and as such, if Piezo1 is involved in L-cell physiology, it may be through regulation of cell density. Thus, it is critical to determine L-cell densities and epithelial integrity in controls and Piezo1 knockouts systematically across the length of the gut, since the authors do not make it clear which gut region contributes to the phenotype they see. Current immunohistochemistry data are not convincing.

(2) Calcium signaling in L-cells is implicated in their typical role of being gut chemo-sensors, and Piezo1 is a calcium channel, so it is not clear whether any calcium-related signaling mechanism would phenocopy these results.

(3) Intestinal bead implantation, while intriguing, does not have clear mechanisms - and is likely to provide a point of intestinal obstruction and dysmotility.

(4) Previous studies, some that are very important, but not cited, contradict the presented results (e.g., epithelial Piezo1 role in insulin secretion) and require reconciliation.

Overall, this study makes an interesting observation but the data are not currently strong enough to support the conclusions.

Author response:

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors intended to prove that gut GLP-1 expression and secretion can be regulated by Piezo1, and hence by mechanistic/stretching regulation. For this purpose, they have assessed Piezo1 expression in STC-1 cell line (a mouse GLP-1 producing cell line) and mouse gut, showing the correlation between Piezo1 level and Gcg levels (Figure S1). They then aimed to generate gut L cell-specific Piezo1 KO mice, and claimed the mice show impaired glucose tolerance and GLP-1 production, which can be mitigated by Ex-4 treatment (Figures 1-2). Pharmacological agents (Yoda1 and GsMTx4) and mechanic activation (intestinal bead implantation) were then utilized to prove the existence of ileal Piezo1-regulated GLP-1 synthesis (Figure 3). This was followed by testing such mechanism in a limited amount of primary L cells and mainly in the STC-1 cell line (Figures 4-7).

While the novelty of the study is somehow appreciable, the bio-medical significance is not well demonstrated in the manuscript. The authors stated (in lines between lines 78-83) a number of potential side effects of GLP-1 analogs, how can the mechanistic study of GLP-1 production on its own be essential for the development of new drug targets for the treatment of diabetes. Furthermore, the study does not provide a clear mechanistic insight on how the claimed CaMKKbeta/CaMKIV-mTORC1 signaling pathway upregulated both GLP-1 production and secretion. This reviewer also has concerns about the experimental design and data presented in the current manuscript, including the issue of how proglucagon expression can be assessed by Western blotting.

Strengths:

The novelty of the concept.

Weaknesses:

Experimental design and key experiment information.

Current GLP-1-based therapies for diabetes use GLP-1 agonists/analogs. Although generally safe, there are some side effect or risks of GLP-1 agonists/analogs. We agree to the reviewer that a mechanistic study on the regulation of GLP-1 production will not directly lead to development of new drug targets for the treatment of diabetes. However, understanding the mechanism of GLP-1 production may shed light onto alternative treatment strategies for diabetes that targeting the production of GLP-1. In our previous studies, we have elucidated the role of mTOR/S6K pathway in regulating GLP-1 production in L cells. Using STC-1 cell line and different mouse models, including Neurog3-Tsc1−/− mice, rapamycin or L-lucine treatment to stimulate mTOR activity, we have demonstrated that mTOR stimulates proglucagon gene expression and thus GLP-1 production (Diabetologia 2015;58(8):1887-97； Mol Cell Endocrinol. 2015 Nov 15:416:9-18.). Based on our previous studies, we found that Piezo1 regulated mTOR/S6K pathway and thus proglucagon expression and GLP-1 production through Ca2+/CaMKKbeta/CaMKIV in our present study. Although we could not exclude involvement of other signaling pathways downstream of Piezo1 in regulating the cleavage of proglucagon, granule maturation and the final release of GLP-1, our present study provided evidence to support the involvement of the Ca2+/CaMKKbeta/CaMKIV/mTOR pathway in mediating the role Piezo1 in proglucagon expression and GLP-1 production. The reviewer also expressed concerns on the use of western blot to detect proglucagon expression. In fact, western blot is often used in detection of proglucagon. Here are some examples from other researchers: Diabetes. 2013 Mar;62(3):789-800. Gastroenterology. 2011 May;140(5):1564-74. 2004 Jul 23;279(30):31068-75. The proglucagon antibody we used in our study was purchased from abcam (Cat#ab23468), which can detect proglucagon of 21 kDa.

Reviewer #2 (Public Review):

Summary:

The study by Huang and colleagues focuses on GLP-1 producing entero-endocrine (EEC) L-cells and their regulation of GLP-1 production by a mechano-gated ion channel Piezo1. The study describes Piezo1 expression by L-cells and uses an exciting intersectional mouse model (villin to target epithelium and Gcg to target GLP-1-producing cells and others like glucagon-producing pancreatic endocrine cells), which allows L-cell specific Piezo1 knockout. Using this model, they find an impairment of glucose tolerance, increased body weight, reduced GLP-1 content, and changes to the CaMKKbeta-CaMKIV-mTORC1 signaling pathway using a normal diet and then high-fat diet. Piezo1 chemical agonist and intestinal bead implantation reversed these changes and improved the disrupted phenotype. Using primary sorted L-cells and cell model STC-1, they found that stretch and Piezo1 activation increased GLP-1 and altered the molecular changes described above.

Strengths:

This is an interesting study testing a novel hypothesis that may have important mechanistic and translational implications. The authors generated an important intersectional genetics mouse model that allowed them to target Piezo1 L-cells specifically, and the surprising result of impaired metabolism is intriguing.

Weaknesses:

However, there are several critical limitations that require resolution before making the conclusions that the authors make.

(1) A potential explanation for the data, and one that is consistent with existing literature [see for example, PMC5334365, PMC4593481], is that epithelial Piezo1, which is broadly expressed by the GI epithelium, impacts epithelial cell density and survival, and as such, if Piezo1 is involved in L-cell physiology, it may be through regulation of cell density. Thus, it is critical to determine L-cell densities and epithelial integrity in controls and Piezo1 knockouts systematically across the length of the gut, since the authors do not make it clear which gut region contributes to the phenotype they see. Current immunohistochemistry data are not convincing.

We appreciate the reviewer’s comment. We agree that Piezo1 may affect L-cell density and epithelial integrity. We will do quantification of L-cell density and test the epithelial integrity by examining the expression of tight junction proteins (ZO-1 and Occludin) and determine the transepithelial resistance in different regions of the gut

(2) Calcium signaling in L-cells is implicated in their typical role of being gut chemo-sensors, and Piezo1 is a calcium channel, so it is not clear whether any calcium-related signaling mechanism would phenocopy these results.

We will examine whether other calcium-related signaling mechanism also contribute the phenotype seen in the IntL-Piezo1-/- mice.

(3) Intestinal bead implantation, while intriguing, does not have clear mechanisms - and is likely to provide a point of intestinal obstruction and dysmotility.

To ascertain if intestinal bead implantation led to intestinal obstruction and dysmotility, we conducted a bowel transit time test. The results revealed no difference in bowel transit time between the sham-operated mice and those implanted with beads.

(4) Previous studies, some that are very important, but not cited, contradict the presented results (e.g., epithelial Piezo1 role in insulin secretion) and require reconciliation.

Overall, this study makes an interesting observation but the data are not currently strong enough to support the conclusions.

We will cite more previous studies on GLP-1 production and discuss the discrepancy between our study and others’ studies. The lack of changes in blood glucose seen in Villin-Piezo1-/- mice reported by Sugisawa et. al. is not surprising (Cell. 2020 Aug 6;182(3):609-624.e21.). Actually, in another recent study from our group, we found similar results when the Villin-Piezo1-/- mice Piezo1fl/fl control mice were fed with normal chow diet. Since Villin-1 is expressed in all the epithelial cells of the gut, including enterocytes and various types of endocrine cells, the effect of L-cell Piezo1 loss may be masked by other cell types under normal condition. However, impair glucose tolerance was seen in Villin-Piezo1-/- mice compared to the Piezo1fl/fl control mice after high fat diet for 8 weeks. We further found that Piezo1 in enterocytes exerted a negative effect on the glucose and lipid absorption. Loss of Piezo1 in enterocytes led to over-absorption of nutrients under high-fat diet （Tian Tao, Qing Shu, Yawen Zhao, Wenying Guo, Jinting Wang, Yuhao Shi, Shiqi Jia, Hening Zhai, Hui Chen, Cunchuan Wang*, Geyang Xu*, Mechanical regulation of lipid and sugar absorption by Piezo1 in enterocytes, Acta Pharmaceutica Sinica B , Accepted, 2024，https://doi.org/10.1016/j.apsb.2024.04.016）.