Article Figures & Data
Figures
- Figure 1.
Interaction of the CNS GLP system with ARC Kiss1 in OVX mice. GLP-ir fibers (red) come in close apposition, with an average of 22% of ARC kisspeptin-ir cells (green). A, Maximal projection at 40×. B, Maximal projection at 63×. C, 1-μm plane at 63×. Scale bars = 10 μm. n = 5 animals, four sections per animal. D, Representative gel of single-cell RT-PCR demonstrating that a subpopulation (20%) of ARC Kiss1 cells from OVX mice express Glp1r mRNA. n = 5 animals, 16–33 cells per animal. The expected sizes for the PCR products are 120 bp for Kiss1 and 148 bp for Glp1r. MM, molecular marker; –RT, Kiss1-GFP cell reacted without reverse transcriptase; tissue controls (+, –), basal hypothalamic RNA reacted with (+) or without (–) RT. E, Dual ISH demonstrating coexpression of Glp1r (red) and Kiss1 (green) mRNA in the ARC (51 of 240 cells, 21.3% coexpression). In this example, an OVX animal showed robust Kiss1 mRNA expression in neurons intermingled with a larger population of Glp1r + neurons in the ARC. F, Inset of E. At higher magnification, a subpopulation of ARC Kiss1 neurons express robust and detectable mRNA signal for Glp1r. Filled black arrows, high Glp1r expression; open arrows, low Glp1r expression. n = 4 animals. Scale bars = 100 μm. 3V, third ventricle.
- Figure 2.
Electrophysiological recordings in brain slices demonstrating effects of GLP-1R signaling on ARC Kiss1 cells of OVX mice. A, Current clamp recordings in brain slices from OVX mice demonstrate that ARC Kiss1 cells treated with liraglutide showed membrane depolarization and increased action potential firing. *p < 0.05, **p < 0.01 vs. RMP, one-way RM-ANOVA with Bonferroni’s post hoc test; ††p < 0.01, 100 vs. 300 nm liraglutide, one-way RM-ANOVA with Bonferroni’s post hoc test. B, Current clamp recordings performed in the presence of presynaptic blockers demonstrate that liraglutide caused a membrane depolarization in ARC Kiss1 cells of OVX mice. **p < 0.01 RMP vs. TTX+CNXQ+AP5+liraglutide 300 nm, one-way RM-ANOVA with Bonferroni’s post hoc test; ††p < 0.01, 100 vs. 300 nm liraglutide, one-way RM-ANOVA with Bonferroni’s post hoc test. ##p < 0.01, TTX+CNXQ+AP5 vs. TTX+CNXQ+AP5+liraglutide 300 nm, one-way RM-ANOVA with Bonferroni’s post hoc test; n = 23 cells from 16 animals. ∼60% of ARC Kiss1 cells respond to liraglutide.
- Figure 3.
Electrophysiological recordings in brain slices demonstrating effects of GLP-1R signaling on ARC Kiss1 cells of OVX+E2 and male mice. A, B, Current clamp recordings in ARC Kiss1 cells from brain slices treated with the long-acting GLP-1R agonist, liraglutide, showed membrane depolarization in both OVX+E2 (A; 52% of cells responded) and males (B; 60% of cells responded). ***p < 0.001, ****p < 0.0001 vs. RMP, one-way RM-ANOVA with Bonferroni’s post hoc test; †††p < 0.001, 100 vs. 300 nm liraglutide, one-way RM-ANOVA with Bonferroni’s post hoc test. C, The magnitude of depolarization was greater in males compared with OVX+E2 females at 100- and 300-nm concentrations. *p < 0.05, OVX+E2 vs. males, one-way ANOVA with Bonferroni’s post hoc test. n = 13 male and 27 OVX +E2 mice.
- Figure 4.
Effects of fasting in OVX mice on brainstem preproglucagon expression and on GLP-1R agonism to restore fasting-suppressed LH levels. A, Brainstem preproglucagon (Gcg) expression was assessed using qPCR and was decreased after a 48-h fast (***p < 0.001, unpaired t-test). B, C, To determine whether GLP-1R agonism prevents LH inhibition during calorie restriction, liraglutide (30 nmol/kg) was administered subcutaneously twice a day at the start of a 48-h fast. Saline-fasted and liraglutide-fasted animals display decreased body weight compared with saline-fed controls (B; **p < 0.01, two-way ANOVA with Bonferroni’s post hoc test). Saline-fasted and liraglutide-fasted animals displayed significantly lower levels of LH compared with saline-fed controls (C; ****p < 0.0001, one-way ANOVA with Tukey’s post hoc test). n = 7–8 animals per group.
- Figure 5.
Effect of chronic ICV Ex-9 on ARC gene expression and plasma LH in OVX mice. A, B, One week after OVX, C57BL/6 mice received an ICV infusion of saline or Ex-9 for 6 d (7.5 nmol/d). Ex-9 caused a significant increase in ARC expression of Agrp (A; **p < 0.01, unpaired t-test) but did not alter ARC expression of Kiss1 (B). C, Plasma LH levels were similar in saline- and Ex-9–treated mice. n = 9 animals per group.
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