Oxytocin receptor signaling contributes to the mitochondrial integrity and maintain gastric mucosal homeostasis in male mice

Figures

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  Figure 1. OXT/OXTR deficiency induces hyperplasia in the gastric mucosa (A) Representative confocal images of gastric mucosa from oxtr-VenusΔNeo/+ mice show nuclear DAPI, OXTR-Venus, H+, K+-ATPase, and a merged image. Similar results were observed in n = 3 mice. Scale bars, 100 μm. Upper right insets represent enlargements of the areas in white squares; scale bars, 10 μm. (B) Representative H&E staining of gastric mucosa from the fundus area of SD-fed wt, OXTR null, OXT null, and OXT-treated OXT null (OXT null + OXT) mice. Scale bars, 100 μm. (C) Gastric mucosal thickness in each group. n (mice) = 6, 6, 7, 8, respectively. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Holm’s multiple range test. (D) Representative confocal images of LGR5 staining of gastric mucosa from SD-fed wt, OXTR null, OXT null, and OXT-treated OXT null (OXT null + OXT) mice. Scale bars, 50 μm. Upper right insets represent enlargements of the areas in white squares; scale bars, 10 μm. (E) LGR5-positive (+) staining area (μm2) in 100 μm gastric mucosa. n (mice) = 3, 3, 3, 4. All data shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Holm’s multiple range test. All data shown as mean ± SEM.

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  Figure 2. OXTR activation reverses HFD-induced remodeling of the gastric mucosa (A) H&E staining of the gastric mucosa in control wt and OXTR null mice fed on an SD, HFD control, HFD with OXT administration. Scale bars, 100 μm. (B) Gastric mucosal thickness in each group. n (mice) = 6, 7, 8, 6, 8, 8 from left to right. ∗p < 0.05 and ∗∗p < 0.01, one-way ANOVA followed by Holm’s multiple range test. (C) Representative confocal images of H+, K+-ATPase in the wt and OXTR null mice: SD, HFD control, HFD with OXT administration. Scale bars, 50 μm. Lower insets represent enlargements of the areas in dashed squares. Scale bars, 10 μm. (D) Parietal cell areas expressed as a percentage of total mucosal area. n (mice) = 7, 7, 4, 6, 4, 4 in each group from left to right. ∗p < 0.05, NS, not significant. Two-way ANOVA followed by Holm’s multiple range test. (E) Parietal cell size. n (mice) = 6, 6, 4, 6, 4, 4 in each group from left to right. ∗∗p < 0.01, NS, not significant. Two-way ANOVA followed by Holm’s multiple range test. (F) H+, K+-ATPase fluorescence intensity in the parietal cells. n (mice) = 6, 6, 4, 6, 4, 4 in each group from left to right. ∗∗p < 0.01, NS, not significant. Two-way ANOVA…

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  Figure 3. OXTR expression in the gastric mucosa correlates with BW in both mice and humans (A) Representative confocal images of nuclear DAPI, OXTR, H+, K+-ATPase and a merged image of gastric mucosa from SD- (upper images) and HFD- (bottom images) fed wt mouse. Scale bars, 10 μm. (B) The intensity of OXTR fluorescence per parietal cell in each mouse. SD, n (mice) = 5, HFD, n (mice) = 6. (C) Mean OXTR fluorescence intensity in parietal cells in the SD and HFD groups. Mean OXTR fluorescence intensity for the SD group was set at “1.0”. n (mice) = 5, 6, respectively. ∗∗p < 0.01, unpaired t test. (D) Relative OXTR mRNA expression to GAPDH in the gastric mucosa from SD- and HFD-fed mice. Mean OXTR mRNA expression for the SD group was set at “1.0”. n (mice) = 6, 6, respectively. ∗p < 0.05, unpaired t test. (E) Plasma OXT levels in SD and HFD groups. n (mice) = 6, 6, respectively. Unpaired t test. (F) Relative OXTR mRNA expression to GAPDH in the hypothalamus in the SD and HFD groups. n (mice) = 4, 4, respectively. Unpaired t test. (G) Relative IRAP (OXT degradation enzyme) mRNA expression relative to GAPDH in the gastric mucosa. n (mice) = 6, 5, respectively. ∗p < 0.05, unpaired t test. All data…

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  Figure 4. OXT increases cytosolic Ca2+ and acid secretion from parietal cells (A) Representative image of isolated parietal cells (red) following Ca2+ imaging, fixing, and immunocytochemical staining for H+,K+-ATPase. Scale bars, 10 μm. (B–E) Representative Ca2+ responses to (B) KRB control, (C) 10–9 M OXT, (D) 10–8 M OXT and (E) 10–7 M OXT. (F) Amplitudes of peak [Ca2+]i transients evoked by each treatment. n (cells) = 151, 27, 63, 47 from 12 mice, respectively. ∗∗p < 0.01, one-way ANOVA followed by Holm’s multiple range test. (G) Representative Ca2+ responses to OXT (10–8 M) in the presence of thapsigargin (10–6 M). (H) Amplitudes of peak [Ca2+]i transients evoked by OXT treatment with or without thapsigargin. The data of left column (10–8 M OXT) is identical to the data of Figure 4F (10–8 M OXT). n (cells) = 63 from 5 mice, 38 from 3 mice, respectively. ∗∗p < 0.01, unpaired t test. (I–L) Representative Ca2+ transients in response to 10–8 M OXT and 10–8 M gastrin in parietal cells from (I) wt and (K) OXTR null mice. Amplitudes of [Ca2+]i transients evoked by each treatment in (J) wt and (L) OXTR null mice. n (cells) = 153 from 3 wt mice, 59 from 4 OXTR null mice. ∗∗p < 0.01, one-way…

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  Figure 5. OXT increases glucose uptake by the stomach (A–D) Representative PET/CT images of (A) SD control, (B) SD with OXT administration, (C) HFD control, and (D) HFD with OXT administration. Dotted circle indicates the position of the stomach. Value of SUVmax (maximum standardized uptake value) are shown within each image. R, right side; L, left side. (E) 18F-FDG uptake (expressed as the percentage of injected dose per gram of tissue; %ID/g) from the stomach of SD control (n (mice) = 4), SD OXT (n (mice) = 4), HFD control (n (mice) = 5) and HFD OXT groups (n (mice) = 5) 60 min after intravenous injection of 18F-FDG. ∗p < 0.05, two-way ANOVA followed by Holm’s multiple range test. (F) Representative confocal images of pAMPK (upper images), H+,K+-ATPase (middle images), and merged images (bottom images) in SD control (left column), HFD control (middle column), and HFD OXT (right column) groups; scale bars, 50 μm. Lower insets represent enlargements of cells shown by dotted squares; scale bars, 10 μm. (G) pAMPK intensities in parietal cells analyzed from confocal images (n (mice) = 5 in each group). ∗p < 0.05, one-way ANOVA followed by Holm’s multiple range test. Mean pMAPK fluorescence…

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  Figure 6. OXT activates AMPK through OXTR signaling in the parietal cells (A) Isolated parietal cells were obtained from SD-fed wt and OXTR null mice. In the gastric gland isolation, two plates were made from one mouse for paired experiments. Scale bars, 10 μm. (B) Relative pAMPK fluorescence intensity. Mean pAMPK fluorescence intensity for wt control groups was set at “100%”. n (plate) = 4 from 4 wt mice and 4 OXTR null mice, respectively. ∗∗p < 0.01, NS, not significant. Two-way ANOVA followed by Holm’s multiple range test. (C) Isolated parietal cells were obtained from 8-week HFD-fed wt mice (n = 5). The glass-bottom dishes were divided into 4 groups, control, OXT (10–8 M), compound C (10 μM), Compound C + OXT. Compound C was added 30 min prior to treatment with OXT. OXT was treated for 60 min. Scale bars, 10 μm. (D) Relative pAMPK fluorescence intensity. All data shown as mean ± SEM. Mean pAMPK fluorescence intensity for control (no OXT and Compound C medium) was set at “100%”. n (plate) = 5 from 5 mice in each group. ∗p < 0.05, ∗∗p < 0.01, NS, not significant. Two-way ANOVA followed by Holm’s multiple range test. All data shown as mean ± SEM.

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  Figure 7. OXT administration restores COX4 expression in the gastric mucosa following an HFD (A) Representative confocal images of COX4, H+,K+-ATPase and merged images in SD control, SD OXT, HFD control and HFD OXT groups. Scale bars, 50 μm. Lower insets represent enlargements of cells shown by white arrows. Scale bars, 10 μm. (B) COX4 fluorescence intensities in parietal cells analyzed from confocal images. n (mice) = 9, 10, 8, 8 in each group. Mean COX4 fluorescence intensity for the SD control group was set at 100%. ∗p < 0.05, NS, not significant. Two-way ANOVA, Holm’s multiple range test. (C) Immunoblots of COX4 expression in gastric mucosa from SD (upper) and HFD (bottom)-fed mice. (D) COX4 expression (17 kDa) relative to GAPDH (35 kDa) loading control in the gastric mucosa analyzed by western blot. n (mice) = 5 in each group. ∗∗p < 0.01, Two-way ANOVA followed by Holm’s multiple range test. (E) Representative confocal images of COX4 in the wt SD control, wt HFD control, wt HFD OXT, OXTR null SD control, OXTR null HFD control and OXTR null HFD OXT groups. Scale bars, 50 μm. Lower insets represent enlargements of the areas in dashed squares. Scale bars, 10 μm. (F) COX4 fluorescence…

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  Figure 8. OXT treatment reverses HFD-induced mitochondrial damage in the gastric mucosa (A–D) Representative transmission electron micrographs of parietal cells in (A) SD control, (B) SD OXT, (C) HFD control, and (D) HFD OXT groups. Scale bars, 1 μm. (E–H) Enlarged images of areas in dashed squares in A-D, respectively. Arrows indicate abnormal mitochondrial ultrastructure. Scale bars, 1 μm. (I) Abnormal mitochondria expressed as a percentage of total mitochondria. n (mice) = 4, 5, 5, 5 in each group from left to right. ∗p < 0.05 and ∗∗p < 0.01, Two-way ANOVA followed by Holm’s multiple range test. Data shown as mean ± SEM.