Fig 1: N38 cells are glucose inhibited and responses are blunted by recurrent glucose deprivation.(A) Schema of experimental protocol for repeated glucose deprivation of N38 cells in vitro by treatment with media containing 25 mM glucose (standard culture conditions) or 2.5 mM glucose (glucose deprivation). (B) Quantification of GHRH release from N38 cells after no (0x) or single (1x) or repeated glucose deprivation (3x and 5x; n = 3–4 experiments, in triplicate). *P = 0.04, F[3, 61] = 3.854, 1-way ANOVA with Tukey’s multiple-comparisons test. (C) Quantification of Ghrh expression in N38 cells after no (0x) or single (1x) or repeated glucose deprivation (3x and 5x) (n = 3–4 experiments, in triplicate). **P = 0.004, ?2[3] = 11.61, Kruskal-Wallis test with Dunn’s multiple-comparisons test. (D) Time-resolved calcium responses using calcium indicator Fluo-4 (F/F0, color scale) of 53 N38 cells without previous glucose deprivation (1 cell/row) with 25 mM, 5 mM, and 2.5 mM glucose treatment. (E) Quantification of peak fluorescence (F/F0) with 25 mM, 5 mM, and 2.5 mM glucose treatment in N38 cells without previous glucose deprivation (4 studies, 51–307 cells). ***P = 0.0004 25 mM vs. 5 mM; ###P = 0.0008 5 mM vs. 2.5 mM; ****P < 0.0001, 25 mM vs. 2.5 mM, ?2[2] = 56.2, Kruskal-Wallis test with Dunn’s multiple-comparisons test. (F) Quantification of peak fluorescence (F/F0) with no (0x) or single (1x) or repeated glucose deprivation (3x and 5x) in N38 cells (4 studies, 170–307 cells). ****P < 0.0001, ?2[3] = 188.2, Kruskal-Wallis test with Dunn’s multiple-comparisons test. Each dot represents data from individual cells and data are displayed as mean ± SEM.
Fig 2: Repeated glucose deprivation disrupts inputs into GHRH neurons and activates microglia.(A) Confocal analyses (left) and 3D reconstruction (right) of dendritic spines on Lucifer yellow–filled ARC GHRH-GFP neurons after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration. Scale bar: 5 µm. (B) Quantification of dendritic spines on filled ARC GHRH-GFP neurons after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration. *P = 0.03 1x vs. 5x, ?2[2] = 7.305, Kruskal-Wallis test with Dunn’s multiple-comparisons test, n = 5–11/group. (C) Confocal analysis (left) and 3D model (right) of SST terminals contacting ARC GHRH-GFP neurons. Scale bar: 30 µm. (D) Quantification of SST terminals contacting ARC GHRH-GFP neurons after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration. *P = 0.02 0x vs. 5x, F[2, 15] = 4.87, 1-way ANOVA with Tukey’s multiple-comparisons test, n = 4–7/group. (E) Confocal analyses of ARC IBA1-positive microglia after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration at low and high magnification. Scale bars: 100 µm for left 3 panels, 50 µm for right panel. (F) Quantification of IBA1 intensity by IHC in the ARC after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration. *P = 0.01 0x vs. 5x, **P = 0.0003 1x vs. 5x, F[2, 36] = 9.612, 1-way ANOVA with Tukey’s multiple-comparisons test, n = 7–20/group. (G) Cumulative intensity distribution of IBA1 intensity. **P = 0.028 0x vs. 5x, Kolmogorov-Smirnov test n = 7–20/group. (H) Quantification of IBA1-positive cells in the ARC after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration. **P = 0.003, ***P = 0.0004, F[2, 13] = 15.35, 1-way ANOVA with Tukey’s multiple-comparisons test, n = 5–6/group. Each dot represents results from individual animals and data are displayed as mean ± SEM.
Fig 3: Repeated glucose deprivation with insulin blunts GHRH neuron activation.(A) Schema of experimental protocol for repeated glucose deprivation with i.p. administration of insulin in vivo. (B) Blood glucose levels (**P < 0.0036, ***P < 0.0002, ?2[2] = 18.23, Kruskal-Wallis ANOVA with Dunn’s multiple-comparisons test, n = 9–10/group). (C) Plasma glucagon levels (*P = 0.04, ?2[2] = 5.3, Kruskal-Wallis ANOVA with Dunn’s multiple-comparisons test, n = 7–12/group). (D) Quantification of fos-positive and GHRH-positive cells in the ARC after vehicle (0x) or single (1x) or repeated (5x) i.p. insulin administration, n = 5–14/group. (E) Quantification of dendritic spines on ARC GHRH-GFP neurons after vehicle (0x) or single (1x) or repeated (5x) i.p. insulin administration. *P = 0.04 1x vs. 0x; ****P < 0.0001 1x vs. 5x, ?2[2] = 25.26 Kruskal-Wallis test with Dunn’s multiple-comparisons test, n = 17–21/group. (F) Quantification of SST-immunoreactive terminals contacting ARC GHRH-GFP neurons after vehicle (0x) or single (1x) or repeated (5x) i.p. insulin administration, n = 3/group. (G) Analysis of plasma growth hormone after vehicle (0x) or single (1x) or repeated (5x) i.p. insulin administration. *P = 0.049, F[2, 26] = 2.41, 1-way ANOVA with Tukey’s multiple-comparisons test, n = 9–10/group.
Fig 4: GHRH neuron activation by acute glucose deprivation is impaired by repeated glucose deprivation.Confocal images and quantification of FISH after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration. (A) ARC cfos; n = 4–6/group. (B) Cfos and Ghrh; ****P < 0.0001 vs. 0x and vs. 5x F[2, 12] = 42.1, n = 4–6/group. Arrowheads indicate neurons’ expression of both Fos and Ghrh. (C) Gck and Ghrh in the ARC; *P = 0.013 vs. 0x and **P = 0.008 vs. 5x, F[2, 11] = 8.94, n = 4–5/group. (D) Ghrh in the ARC; *P = 0.04 vs. 0x and **P = 0.02 vs. 5x, F[2, 11] = 6.34, n = 4–5/group. One-way ANOVA with Tukey’s multiple-comparisons test. Scale bars: 100 µm (main images), 30 µm (insets). Each dot represents results from individual animals and data are displayed as mean ± SEM.
Fig 5: Mdivi-1 preserves the response to low glucose after repeated glucose deprivation.(A) Mitochondrial length in N38 cells after no (0x) or single (1x) or repeated glucose deprivation (3x and 5x; n = 3 experiments, 110–168 mitochondria/group) with and without mdivi-1 treatment. ****P < 0.0001, F[7, 1082] = 35.33, 1-way ANOVA with Tukey’s multiple-comparisons test. (B) Peak fluorescence (F/F0) with 25 mM, 5 mM, and 2.5 mM glucose treatment in N38 cells with and without previous glucose deprivation, with and without mdivi-1 treatment. ****P < 0.0001 5x with mdivi-1 vs. 5x at 2.5 mM glucose; **P = 0.003 5x with mdivi-1 vs. 5x at 5 mM glucose; *P = 0.01 5x vs. 0x at 2.5 mM glucose. Significant effect of mdivi-1 treatment P < 0.0001, F[3, 1482] = 9.187, 2-way ANOVA with Tukey’s multiple-comparisons test, n = 68–147 cells/group. (C) Blood glucose after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration or repeated 2DG administration with mdivi-1 treatment (5x MD). ***P = 0.0004, *P = 0.02, ?2[3] = 15.67, Kruskal-Wallis test with Dunn’s multiple-comparisons test, n = 7–11/group. (D) c-fos+Ghrh+ cells after vehicle (0x) or single (1x) or repeated (5x) i.p. 2DG administration or repeated 2DG administration with mdivi-1 treatment (5x MD). *P = 0.04 0x vs. 1x; #P = 0.01 0x vs. 5x MD, Welch’s F[3, 7.869] = 8.338, Welch’s ANOVA test with Dunnett’s multiple-comparisons test, n = 4–9/group. Each dot represents data from individual cells or animals and data are displayed as mean ± SEM.
Supplier Page from Aviva Systems Biology for GHRH ELISA Kit (Mouse) (OKEH03121)
Specificity: Natural and recombinant Mouse Somatoliberin