Fig 1: Insulin-induced AVP secretion is mediated by A1/C1 neurons.(a) Upper: AAV-DIO-ChR2-mCherry was injected into the VLM of ThCre/+ mice or AvpGFP/+::ThCre/+ mice, targeting A1/C1 neurons. Lower: CRACM. Excitatory post-synaptic currents (EPSCs) were recorded in voltage-clamp mode in GFP+ (AVP) and GFP- neurons in the SON and PVH. The number (n) of neurons that responded to opto-activating A1/C1 terminals. Total of 36 neurons recorded from five mice. See Figure 5—figure supplement 1a. (b) Upper: Viral expression of ChR2-mCherry in A1/C1 neurons. Lower: A1/C1 neuron terminals co-localize with AVP-immunoreactive neurons. Representative from three mice. (c) Left: EPSCs evoked by opto-activation of A1/C1 terminals with 473 nm light pulses (arrows). Right: Light-evoked EPSCs following application of DNQX (20 µM). Representative of five recordings from three mice. (d) EPSC waveforms in a single GFP+ (AVP) neuron in response to repeated opto-activation of A1/C1 neuron terminals. Black line and shaded are=Mean ± SD of EPSCs. Light pulse=blue bar. Representative of three recordings from three mice. (e) EPSCs evoked by opto-activating A1/C1 terminals at baseline (left) and following addition of TTX (1 µM; middle) and 4-AP (1 mM; right). Representative of three recordings from three mice. (f) AAV-DIO-hM3Dq was injected into ThCre/+ mice, targeting A1/C1 neurons. CNO (1 mg/kg) was then injected (i.p.). Antagonists (or vehicle) for the V1bR (SSR149415, 30 mg/kg) or glucagon receptor (GCGR; LY2409021, 5 mg/kg) were injected 30 min prior to CNO. Plasma glucose and glucagon were then measured. See Figure 5—figure supplement 1b,c. (g) Plasma glucose in response to CNO and pretreatment with antagonists. n=8 mice. Two-way RM ANOVA (Sidak’s multiple comparison’s test). Time (p<0.0001), Treatment (p=0.03), and Interaction (p=0.0002). (h) Plasma glucagon at 30 min post-CNO (or vehicle) injection. Two-way RM ANOVA with Tukey’s (within treatment) and Sidak’s (between treatments) multiple comparisons. p<0.05=*, ns=not significant. Within treatment, CNO increased glucagon at 30 min versus 0 min (p=0.022). Saline did not (p=0.96). Between treatments, CNO increased glucagon at 30 min versus saline (p=0.001). n=6 mice. (i) In vivo fiber photometry measurements of population GCaMP6 activity in pituitary-projecting SON AVP neurons during A1/C1 neuron inhibition. AAV-DIO-GCaMP6s was injected into the SON and AAV-fDIO-hM4Di-mCherry into the VLM of Avpires-Cre/+::Dbhflp/+ mice. GCaMP6s was then imaged in response to an insulin tolerance test (ITT), following inhibition of the A1/C1 neuron (with CNO at 1 mg/kg), as indicated by the protocol in the lower horizontal bar. See Figure 5—figure supplement 2c. (j) Left: Example population activity in one mouse (as described in (i)) in response to an ITT, following saline or CNO treatment (on different trials). CNO strongly inhibited the response to insulin. Right: Average GCaMP6 signal ((F–F0)/F0) during response to insulin with either saline or CNO pretreatment (n=9 mice). CNO reduces the AVP GCaMP6 signal. t-test, p<0.01=**. (k) Plasma glucagon in response to an ITT in mice described in (i). 30 min before the insulin injection, either saline or CNO was given i.p. Glucagon is represented as fold of basal, where basal is 0 min (just prior to insulin) and the sample was taken at 30 min post-insulin. t-test, p=0.023 (*). n=8 mice. AVP, arginine-vasopressin; SON, supraoptic nucleus.
Fig 2: AVP increases glucagon release and alpha-cell activity ex vivo, in situ, and in vivo.(a) Glucagon secretion from the perfused mouse pancreas in response AVP (10 nM). All data are represented as mean ± SEM. n=5 mice. Extracellular glucose of 3 mM. (b) Islets from GcgCre/+:GCaMP3/+ mice were injected into the anterior chamber of the eye (ACE) of recipient mice (n=5 islets in 5 mice). After >4 weeks, GCaMP3 was imaged in vivo in response to i.v. AVP (10 µg/kg) or saline administration. Saline did not change the GCaMP3 signal. Signal is GCaMP3 fluorescence (F) divided by baseline signal (F0). AVP evoked an increase in calcium activity, typically starting with a large transient. Below: Raster plot of response (normalized F/F0) in different cells (ROIs) with a single islet. (c) Response of alpha-cell to i.v. AVP. Lower panel shows raster plot of response in different cells. (d) Integrated F/F0 (area under curve) response for all alpha-cells in recorded islets (five islets, N=3 mice). The area under the curve was calculated 30 sec before i.v. injection, 30 sec after and 120 sec after. One-way RM ANOVA with Tukey’s multiple comparison test; p<0.01=**. Right: Image of islets (arrows) engrafted in the ACE. (e) Glucagon secretion from isolated mouse islets in response to AVP. One-way ANOVA (p<0.05=*; p<0.01=**; p<0.001=***). n=5–10 wild-type mice in each condition. (f) Glucagon secretion from isolated mouse islets in response to AVP in the presence and absence of the V1bR antagonist SSR149415. One-way ANOVA (p<0.05=*; p<0.01=**). n=5 wild-type mice per condition. (g) Glucagon secretion from islets isolated from human donors, in response to AVP. Paired t-tests, p<0.05=*. n=5 human donors. AVP, arginine-vasopressin; ROIs, regions of interest.
Fig 3: AVP increases action potential firing, Ca2+ activity, and intracellular DAG in alpha-cells in intact islets.(a) Membrane potential (Vm) recording (perforated patch-clamp) of an alpha-cell in response to 100 pM AVP. (b) Frequency-response curve for varying concentrations of AVP (17 alpha-cells, 10 Gcg-GCaMP3 mice). Mixed-effects analysis of variance, Holm-Sidak’s post-hoc (p<0.01=**; p<0.001=***; p=0.073 for 3 mM glucose vs. 10 pM AVP). (c) GCaMP3 signal from an alpha-cell in response to AVP. (d) Box and whisker plot of the frequency of GCaMP3 oscillations in response to AVP. 142–170 alpha-cells, 7 islets, n=7 Gcg-GCaMP3 mice. Recordings in 3 mM glucose. One-way RM ANOVA, p<0.001=***. (e) Frequency of GCaMP3 oscillations in response to 100 pM AVP in the presence and absence of SSR149415 (10 µM). 75–90 alpha-cells, 6 islets, n=5 Gcg-GCaMP3 mice. Recordings in 3 mM glucose. One-way ANOVA (Tukey), p<0.001=***, ns=not significant (p>0.2). (f) Frequency of GCaMP3 oscillations in response to 100 pM AVP in the absence and presence of YM-254890 (0.2 µM). 75–90 alpha-cells, 6 islets, n=5 GcgCre/+:GCaMP3/+ mice. Recordings in 3 mM glucose. One-way RM ANOVA (Tukey’s multiple comparisons test), p<0.05=*, ns=not significant (p>0.3). AVP versus AVP+YM-254890; p=0.99. (g) Heatmap of intracellular diacylglycerol (DAG; upward DAG) signal from single islet cells (dispersed into clusters) in response to AVP. The signal was median filtered and normalized to largest signal in the recording. (h) Area under curve (AUC, normalized to duration) for DAG signal for each experimental condition. 10 recordings, 152 cells, n=3 wild-type mice. One-way RM ANOVA, p<0.05=* (Tukey’s multiple comparisons test). (i) Fluo4 signal from a putative alpha-cell in a human islet in response to AVP (10, 100, and 1000 pM). Recording in 3 mM glucose. (j) Area under curve (AUC, normalized to duration) for Fluo4 signal in each human islet, for each experimental condition. 26 islets, n=4 human donors. One-way ANOVA, p<0.05=* (Sidak). AVP, arginine-vasopressin.
Fig 4: WPOI progression is not associated with the serum levels of the OXTR ligand.Graphical representation of analysis of serum (a) OXT and (b) AVP levels from healthy, leukoplakia and (c, d) OSCC patients with WPOI 1–3 (n = 64) and 4–5 (n = 45) types as indicated and as determined by ELISA (n = 180). P = two-tailed t test. e, f Graphical representation of analysis of serum OXT and AVP levels from patients with WPOI 1–3 (n = 28) and 4–5 (n = 68) types in a validation group. P = two-tailed t test. g, h Graphical representation of analysis of OXT/AVP levels from patients with/without LNM (n = 85) and postoperative metastasis (n = 93). P = two-tailed t test. i Graphical representation of survival data from WPOI 4–5 OSCC patients stratified according to OXT level (low: n = 34 and high: n = 34). j Correlative index of OXTR, nuclear ERK5 and serum OXT levels from OSCC patient tissue (n = 96) by IHC and ELISA analysis using Pearson’s correlation test. k, l Graphical analysis of Lmax (invasion in 3D collagen matrix) of HN6 in heterotypic spheroids treated with OXT or AVP. n = 4/group, P = two-tailed t test. m Proposed working model depicting the mechanism of OXTR autoactivation-mediated ERK5 nucleus translocation for maintaining the function and phenotype of OXTRHigh CAFs in WPOI 4–5 type stroma. Results are shown as mean and standard deviation (SD). Source data are provided as a Source data file.
Fig 5: Insulin-induced hypoglycemia enhances population activity of AVP neurons in the supraoptic nucleus (SON), driving glucagon secretion AAV-DIO-hM3Dq-mCherry was injected bilaterally into the SON of Avpires-Cre/+ mice.CNO (3 mg/kg) or vehicle was injected i.p. In the same cohort (different trial), LY2409021 (5 mg/kg) or SSR149415 (30 mg/kg) was injected (i.p.) 30 min prior to CNO. See Figure 1—figure supplement 1. (a) Blood glucose measurements from (a). Two-way RM ANOVA (Tukey’s). CNO 0 min versus CNO at 15, 30, and 60 min; p<0.05=*, p<0.01=**. Comparison of CNO versus Saline, CNO+LY2409021 or CNO+SSR149415 at 30 min; p<0.01=††. Time, p<0.0001; Treatment, p=0.0006; Interaction, p<0.0001. n=6 mice. (b) Terminal plasma copeptin 30 min following saline or CNO injection. Mann-Whitney t-test (p=0.0025, **). n=15–18 mice. (c) Plasma glucose during an insulin tolerance test (ITT; 0.75 U/kg) in n=5 wild-type mice. (d) Plasma glucagon following an ITT. n=5 wild-type mice. Paired t-test, p<0.01=**. (e) Glucagon secretion from the perfused mouse pancreas. Glucagon released during all time points in 8 mM glucose was not significantly different from 4 mM glucose (all p>0.8). Right: area under curve. Paired t-test, ns=not significant. (f) Glucagon secretion from isolated mouse islets during 60 min static incubation at indicated glucose concentrations. n=7 wild-type mice. One-way ANOVA with Tukey post-hoc. 4 mM versus 8 mM glucose, p=0.99. (g) Measurements of population GCaMP6s activity in pituitary-projecting AVP neurons in the SON. Inset: Expression of GCaMP6s in AVP neurons in the SON. Arrow=tip of the optic fiber. (h) Simultaneous in vivo fiber photometry of AVP neuron activity (GCaMP6) and continuous glucose monitoring (black line) in response to an ITT (1 U/kg). Dashed gray line indicates the time of insulin injection. (i) Same animal as in (j), but for saline vehicle injection (dashed gray line). (j) GCaMP6s signal (normalized) in response to insulin (n=6 mice) or saline vehicle (n=6). Plasma copeptin at 30 min following saline or insulin. Mann-Whitney U-test, p=0.021. n=15–18 mice. AVP, arginine-vasopressin.
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