Medicine

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

Yanling Huang
Haocong Mo
Jie Yang
Luyang Gao
Tian Tao
Qing Shu
Wenying Guo
Yawen Zhao
Jingya Lyu
Qimeng Wang
Jinghui Guo
Hening Zhai
Linyan Zhu
Hui Chen author has email address
Geyang Xu author has email address

Department of Physiology, School of Medicine, Jinan University, 601 Huangpu Avenue West, Tianhe District, Guangzhou 510632, Guangdong, China;
Department of Pathology, School of Basic Medicine, Guangzhou Medical University, Guangzhou 511436, Guangdong, China;
Biotherapy Center, Cell-gene Therapy Translational Medicine Research Center, the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, Guangdong, China
School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, Guangdong, China;
Endoscopy Center, The First Affiliated Hospital of Jinan University, 613 Huangpu Avenue West, Tianhe District, Guangzhou 510630, Guangdong, China;
Department of Pharmacology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong, China
Key Laboratory of Viral Pathogenesis & Infection Prevention and Control (Jinan University), Ministry of Education, Guangzhou 510632, Guangdong, China

https://doi.org/10.7554/eLife.97854.2

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Figures and data

Generation, Validation and Characterization of IntL-Piezo1^-/- mice
(A) Schematic description for the generation of Villin-Flippase (Vil-Flp) and Flippase-dependent Gcg-Cre (FGC) mice. Vil-Flp flip the inverted Cre gene in the Gcg-Cre cassette in IntL-Cre mice to restrict Cre expression in intestinal L cells. As shown, Locations of genotyping primers are also indicated.
(B) Tail DNA genotyping PCR results using genotyping primer for Vil-Flp, FGC and Flippase-activated Cre (IntL-Cre) mice.
(C) Intestine and pancreas DNA genotyping results. The “Original” band represents the original FGC cassette with inverted Cre, while the “Flipped” band represents recombined FGC cassette with Cre flipped into the correct direction.
(D) Schematic description for the validation of IntL-Cre efficacy by crossing with mT/mG reporter mice.
(E) Fluorescence was detected in the ileal and pancreatic tissues from mT/mG and IntL-Cre-mT/mG mice by frozen tissue confocal microscopy. Green fluorescence represents successful deletion of TdTomato and reactivation of EGFP in the Cre-expressing cells.
(F) Schematic description for the generation of Intestinal L cell-Piezo1^-/- mice (IntL-Piezo1^-/-) by crossing Piezo1^loxp/loxp mice with IntL-Cre mice.
(G) Body weight of 14- to 16-week-old male mice of the indicated genotypes fed NCD (n=6/group)
(H-I) IPGTT (H) and ITT (I) and associated area under the curve (AUC) values of 14- to 16-week-old male mice of the indicated genotypes fed NCD (n=6/group).
(J) Proglucagon mRNA levels in ileum of 14- to 16-week-old male mice of the indicated genotypes fed NCD. (n=6/group).
(K) The plasma GLP-1 levels in 14- to 16-week-old male mice of the indicated genotypes fed NCD (n=6/group).
(L) Representative images for Piezo1 RNA-FISH and GLP-1 immunofluorescent staining in the ileum of 14-week-old male mice of indicated genotypes fed NCD (n=6/group).
(M) Percentage of Piezo1-positive GLP-1 cells in total GLP-1 cells in the ileal mucosa of 14-week-old male mice of indicated genotypes fed NCD (n=6/group).
(N) A schematic diagram depicting the potential mechanisms linking the CaMKKβ/CaMKIV-mTOR signaling pathway and GLP-1 production.
(O) Representative western blots are shown for indicated antibodies in the ileal mucosa (n = 6/group).
Data are represented as mean ± SEM. Significance was determined by Student’s t test for comparison between two groups, and by one-way ANOVA for comparison among three groups or more, *p < 0.05, **p < 0.01, ***p < 0.001.

Validation and phenotype of IntL-Piezo1^-/- mice fed with high-fat diet
(A) Body weight of 14- to 16-week-old male Piezo1^loxp/loxp and IntL-Piezo1^-/- mice fed with HFD for 10 weeks (n=6/group).
(B) IPGTT and associated area under the curve (AUC) values of 14- to 16-week-old male Piezo1^loxp/loxp and IntL-Piezo1^-/- mice fed with HFD (n=6/group).
(C) Proglucagon mRNA levels in the ileal mucosa of 14- to 16-week-old male Piezo1^loxp/loxp and IntL-Piezo1^-/- mice fed with HFD (n=6/group).
(D) The plasma GLP-1 level in 14- to 16-week-old male Piezo1^loxp/loxp and IntL-Piezo1^-/- mice fed with HFD (n=6/group).
(E) Double immunofluorescent staining of Piezo1, and GLP-1 in the ilea of 14- to 16-week-old male Piezo1^loxp/loxp and IntL-Piezo1^-/- mice fed HFD (n=6/group).
(F) Representative western blots are shown for indicated antibodies in the ileal mucosa (n=6/group).
(G) Body weight after 7 consecutive days infusion of saline or Ex-4 (100ug/kg body weight) in 14- to 16-week-old male Piezo1^loxp/loxpand IntL-Piezo1^-/- mice fed with HFD (n=6/group).
(H-I) IPGTT (H) and ITT (I) and associated area under the curve (AUC) values after consecutive infusion of saline or Ex-4.
(J) Proglucagon mRNA levels in the ileal mucosa (n=6/group) after consecutive infusion of saline or Ex-4.
(K) The plasma GLP-1 level after consecutive infusion of saline or Ex-4 (n=6/group). Data are represented as mean ± SEM. Significance was determined by Student’s t test for comparison between two groups, and by one-way ANOVA for comparison among three groups or more, *p < 0.05, **p < 0.01, ***p < 0.001.

Chemical and mechanical interventions of Piezo1 regulates GLP-1 synthesis in mice
(A-E) 14- to 16-week-old male C57BL/6J mice fed with HFD for 10 weeks were infused with vehicle, Yoda1 (2 μg per mouse) or GsMTx4 (250 μg/kg) by i.p. for 7 consecutive days. (n=6/group).
(A) Body weight after consecutive drug infusion.
(B) IPGTT and associated area under the curve (AUC) values.
(C) Proglucagon mRNA levels in the ileal mucosa.
(D) Plasma GLP-1.
(E) Representative western blots are shown for indicated antibodies in the ileal mucosa
(F-J) 14- to 16-week-old male IntL-Piezo1^-/- mice fed with HFD for 10 weeks were infused with vehicle, Yoda1 (2 μg per mouse) by i.p. for 7 consecutive days. (n=4 or 5/group)
(F) Body weight after 7 consecutive days’ drug infusion.
(G) Fasting blood glucose levels.
(H) Ileal mucosal Proglucagon mRNA levels.
(I) Plasma GLP-1 levels.
(J) Ileal mucosal Proglucagon protein levels.
(K-R) 14- to 16-week-old male C57BL/6J mice fed with HFD were subjected to sham operation, or intestinal bead implantation (n=6/group).
(K) Fasting blood glucose levels.
(L) IPGTT and associated area under the curve (AUC) values.
(M) Body weight.
(N and O) Piezo1 (N) and Proglucagon (O) mRNA levels in the ileal mucosa.
(P) The plasma GLP-1 levels.
(Q) Immunofluorescence staining of GLP-1 in ileum and quantification of GLP-1 positive cells.
(R) Representative western blots images and densitometry quantification for indicated antibodies in the ileal mucosa.
Data are represented as mean ± SEM. Significance was determined by Student’s t test for comparison between two groups, and by one-way ANOVA for comparison among three groups or more, *p < 0.05, **p < 0.01, ***p < 0.001.

Piezo1 regulates GLP-1 synthesis and secretion in primary cultured mouse L cells and isolated mouse ileum.
(A) Isolation of mouse L cells (GFP positive) from ileal tissue by FACS. The gating in flowcytometry for sorting of GFP positive cells.
(B) Immunofluorescent staining of Piezo1 in sorted GFP positive L cells.
(C) Intracellular Ca²⁺ imaging by fluo-4-AM calcium probe. The change of fluorescent intensity (ΔF/F0) was plotted against time
(D-F) L cells were treated with vehicle or Yoda1 (5μM) for 24 hours.
(D) Proglucagon mRNA expression.
(E) GLP-1 concentrations in the culture medium.
(F) Western blot images and densitometry quantification for the indicated antibodies. (G-J) Knockdown of Piezo1 in L cells by shRNA for 48 hours.
(G) Piezo1 mRNA expression.
(H) Proglucagon mRNA expression.
(I) GLP-1 levels in the culture medium.
(J) Western blot images and densitometry quantification for the indicated antibodies. (K-N) Ileal tissues from Piezo1^loxp/loxp and IntL-Piezo1^-/- mice were subjected to tension force (n=6/group).
(K) A representative photograph showing the traction of isolated ileum.
(L) Proglucagon mRNA levels.
(M) GLP-1 concentrations in the medium.
(N) Western blot images and densitometry quantification for the indicated antibodies. Data are represented as mean ± SEM and are representative of six biological replicates. Significance was determined by Student’s t test for comparison between two groups, and by one-way ANOVA for comparison among three groups or more, *p < 0.05, **p < 0.01, ***p < 0.001.

Modulation of GLP-1 synthesis and secretion by pharmacological and mechanical activation of Piezo1 in STC-1 cells
(A) Whole-cell currents induced by Yoda1 (5μM) were recorded from STC-1 cells or STC-1 cells pretreated with GsMTx4 for 30 min
(B and C) Intracellular calcium imaging in STC-1 cells. (B) STC-1 cells were loaded with fluo-4 AM for 1 h. The representative time-lapse image showing the intracellular Ca²⁺ signals. (C) The change of fluorescent intensity (ΔF/F0) was plotted against time.
(D-F) STC-1 cells were treated with various concentrations of Yoda1 for 24 h. (D) Whole-cell extracts underwent western blot with indicated antibodies. (E) Proglucagon mRNA levels. (F) GLP-1 concentrations in the culture medium.
(G-I) STC-1 cells were treated with Yoda1 (5 μM) in the presence or absence of GsMTx4 (0.1 μM) for 24 h. (G) Whole-cell extracts underwent western blot with indicated antibodies. (H) Proglucagon mRNA levels. (I) GLP-1 concentrations in the culture medium
(J-N) STC-1 were subjected to mechanical stretch. (J) STC-1 cells were cultured in elastic chambers and the chambers were subjected to mechanical stretch by 120% extension of their original length. (K) The medium GLP-1 concentrations were detected at indicated time. (L) Piezo1 mRNA levels. (M) Proglucagon mRNA levels.
(N) Whole-cell extracts underwent western blot with indicated antibodies.
Data are represented as mean ± SEM and are representative of six biological replicates. Significance was determined by Student’s t test for comparison between two groups, and by one-way ANOVA for comparison among three groups or more, *p < 0.05, **p < 0.01, ***p < 0.001.

Genetic interference of Piezo1 regulates GLP-1 production in STC-1 cells
(A-D) STC-1 cells were transfected with mouse control or Piezo1 expression plasmids for 48h. Piezo1 (A) and Proglucagon (B) mRNA levels in STC-1 cells. (C) GLP-1 concentrations in culture medium. (D) Whole-cell extracts underwent western blot with indicated antibodies.
(E-H) Stable knockdown of Piezo1 in STC-1 cells. Piezo1 (E) and Proglucagon (F) mRNA levels in STC-1 cells. (G) GLP-1 concentrations in culture medium. (H) Whole-cell extracts underwent western blot with indicated antibodies.
Data are represented as mean ± SEM Data are represented as mean ± SEM and are representative of six biological replicates. Significance was determined by Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001.

Modulation of GLP-1 production by CaMKKβ/CaMKIV and mTOR signaling activity in STC-1 cells
(A-C) STC-1 cells were transfected with GFP, CaMKKβ or CaMKIV plasmids for 48h. (A) Proglucagon mRNA levels in STC-1 cells. (B) GLP-1 concentrations in culture medium. (C) Whole-cell extracts underwent western blot with indicated antibodies.
(D-F) STC-1 cells were treated with CaMKKβ inhibitor STO-609 (10 μmol/L) for 24 h. (D) Proglucagon mRNA levels in STC-1 cells. (E) GLP-1 concentrations in culture medium. (F) Whole-cell extracts underwent western blot with indicated antibodies. (G-I) STC-1 cells were pretreated with Rapamycin (50 nmol/L) for 1 h, then treated with Yoda1 (5 μmol/L) for 24 h. (G) Proglucagon mRNA levels in STC-1 cells. (H) GLP-1 concentrations in the culture medium. (I) Whole-cell extracts underwent western blot with indicated antibodies.
Data are represented as mean ± SEM Data are represented as mean ± SEM and are representative of six biological replicates. Significance was determined by Student’s t test for comparison between two groups, and by one-way ANOVA for comparison among three groups or more, *p < 0.05, **p < 0.01, ***p < 0.001.

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