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. 2016 Aug 20;5(10):823-833.
doi: 10.1016/j.molmet.2016.08.002. eCollection 2016 Oct.

Estrogens modulate ventrolateral ventromedial hypothalamic glucose-inhibited neurons

Ammy M Santiago  1 , Deborah J Clegg  2 , Vanessa H Routh  3
Affiliations

Estrogens modulate ventrolateral ventromedial hypothalamic glucose-inhibited neurons

Ammy M Santiago et al. Mol Metab. .

Abstract

Objective: Brain regulation of glucose homeostasis is sexually dimorphic; however, the impact sex hormones have on specific neuronal populations within the ventromedial hypothalamic nucleus (VMN), a metabolically sensitive brain region, has yet to be fully characterized. Glucose-excited (GE) and -inhibited (GI) neurons are located throughout the VMN and may play a critical role in glucose and energy homeostasis. Within the ventrolateral portion of the VMN (VL-VMN), glucose sensing neurons and estrogen receptor (ER) distributions overlap. We therefore tested the hypothesis that VL-VMN glucose sensing neurons were sexually dimorphic and regulated by 17β-estradiol (17βE).

Methods: Electrophysiological recordings of VL-VMN glucose sensing neurons in brain slices isolated from age- and weight-matched female and male mice were performed in the presence and absence of 17βE.

Results: We found a new class of VL-VMN GI neurons whose response to low glucose was transient despite continued exposure to low glucose. Heretofore, we refer to these newly identified VL-VMN GI neurons as 'adapting' or AdGI neurons. We found a sexual dimorphic response to low glucose, with male nonadapting GI neurons, but not AdGI neurons, responding more robustly to low glucose than those from females. 17βE blunted the response of both nonadapting GI and AdGI neurons to low glucose in both males and females, which was mediated by activation of estrogen receptor β and inhibition of AMP-activated kinase. In contrast, 17βE had no impact on GE or non-glucose sensing neurons in either sex.

Conclusion: These data suggest sex differences and estrogenic regulation of VMN hypothalamic glucose sensing may contribute to the sexual dimorphism in glucose homeostasis.

Keywords: 17β-estradiol; 17βE, 17β-estradiol; AICAR, aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AMP-activated kinase; AMPK, AMP-activated protein kinase; ARC, arcuate nucleus; BSA-17βE, bovine serum albumin-conjugated 17βE; CC, compound C; ER, estrogen receptor; GABA, γ-aminobutyric acid; GE, glucose-excited; GI, glucose-inhibited; Glucose excited neurons; Glucose inhibited neurons; HRP, horse radish peroxidase; IR, input resistance; MPP, methylphenolpyrazole; NGS, non-glucose sensing; PHTPP, phenyltrifluoromethylpyrazolophenol; POMC, pro-opiomelanocortin; PVDF, polyvinylidene difluoride; SF-1, steroidogenic factor; Sexual dimorphism; TTX, tetrodotoxin; VL-VMN, ventrolateral VMN; VMH, ventromedial hypothalamus; VMN, ventromedial hypothalamic nucleus; Ventromedial hypothalamic nucleus; Vm, membrane potential.

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Figures

Supplemental Figure 1
Supplemental Figure 1
Solution change, vehicle-only, and pharmacological drug-only controls. (A) Pooled data scatter plot of %ΔIR of VL-VMN neurons during control solution change from both females (n = 13) and males (n = 6). (B) Quantification of %ΔIR of VL-VMN neurons in response to vehicle controls pooled from both sexes. Numbers above columns indicate the n for each group. (C, D) Quantification of %ΔIR in response to MPP, PHTPP, AICAR and CC in 2.5 mM glucose for nonadapting GI (C) and AdGI neurons (D) from females only. Numbers above columns indicate the n number for each group. Shaded boxes represent 2xSTD of control solution change experiments (see Supplemental Figure 1A). Treatments exhibiting %ΔIR responses within this shaded area were deemed as “no effect.” *p < 0.05 via one-sample student t-test with a theoretical mean = 0. AICAR: AMPK agonist (0.5 mM), CC: AMPK antagonist (10 μM), G: mM glucose, ER: estrogen receptor, IR: input resistance, MPP: ERα antagonist (10 μM), PHTPP: ERβ antagonist (1 μM), Res: 2.5G from general reservoir, Syr: 2.5G from treatment syringe, Vm: membrane potential.
Supplemental Figure 2
Supplemental Figure 2
Basal resting Vm and IR in VL-VMN glucose sensitive cells from both sexes. (A, B) Scatter plot of resting Vm (A) and IR (B) in 2.5 mM glucose for VL-VMN NGS (♀n = 26, ♂n = 7), GE(♀n = 25, ♂n = 7), nonadapting GI (♀n = 47, ♂n = 18) and AdGI (♀n = 47, ♂n = 10) neurons from both sexes. *p < 0.05 versus female nonadapting GI neurons via unpaired student t-test; all other comparisons were not statistically significant.
Supplemental Figure 3
Supplemental Figure 3
Characterization of consecutive glucose challenges and the secondary glucose response in VL-VMN AdGI neurons. (A, B) Scatter plot of %ΔIR in AdGI neurons from females (A; n = 19) and males (B; n = 6). n.s: not significant via paired students t-test (C, D) Quantification of %ΔVm and %ΔIR in response to 0.1 mM glucose in AdGI exhibiting or not exhibiting a secondary glucose response in females (C, n = 18 of 51) and males (D, n = 6 of 10). G: mM glucose, IR: input resistance, Vm: membrane potential.
Figure 1
Figure 1
VL-VMN glucose sensing neuron subtypes. (A–D) Representative consecutive whole cell current-clamp recordings of a female NGS, (A) GE, (B) nonadapting GI, (C) and adapting GI (D) neurons. Figure 1D Inset: Magnification of a secondary glucose response following solution change to 2.5 mM glucose. Glucose changes are schematically displayed above each recording; dashed grey line represents resting Vm. G: mM glucose, IR: input resistance, Vm: membrane potential.
Figure 2
Figure 2
VL-VMN nonadapting GI neurons are inherently sexually dimorphic and 17βE sensitive. (A) Representative voltage responses to a hyperpolarizing pulse for nonadapting GI neurons from both sexes. Vm was normalized to 2.5 mM glucose to emphasize changes in IR. (B) Quantification of %ΔVm and %ΔIR in response to 0.1 mM (♀n = 48, ♂n = 18) glucose in nonadapting GI neurons from both sexes. (C) Quantification of %ΔIR in response to 0.1 mM glucose in the presence and absence of 17βE (100 nM) for nonadapting GI neurons from females (n = 9) and males (n = 6). n.s: not significant via unpaired students t-test. (D) Quantification of %ΔIR in response to 0.1 mM glucose in the presence and absence of TTX (n = 5) and 17βE (n = 5). Columns with different letters are significantly different from each other as determined by repeated measures one-way ANOVA followed by Tukey post-hoc tests. (E, F) Quantification of %ΔIR in response to 0.1 mM glucose in the presence and absence 17βE and AICAR, (E, n = 4), MPP (F, n = 5) or PHTPP (F, n = 6). *p < 0.05 via unpaired students t-test. (G) Quantification of %ΔIR in response to 0.1 mM glucose and 2.5 mM glucose+17βE in nonadapting GI neurons from females (n = 12) and males (n = 5). n.s: not significant via unpaired students t-test; p < 0.05 via paired students t-test. (H) Representative 17βE-sensitive V-I relationship in 2.5 mM glucose in female nonadapting GI neurons (n = 9). The 17βE-sensitive conductance in 2.5 mM glucose reversed near the K+ equilibrium potential (EK+ = −99 mV) for our solutions. (I) Quantification of %ΔIR in response to 2.5G+17βE (n = 4) in the presence and absence of TTX. *p < 0.05 via paired students t-test. 17βE:17β-Estradiol (100 nM), AICAR: AMPK agonist (0.5 mM), G: mM glucose, IR: input resistance, MPP: ERα antagonist (10 μM), PHTPP: ERβ antagonist (1 μM), TTX: tetrodotoxin (voltage-gated Na+ channel blocker; 500 nM), Vm: membrane potential.
Figure 3
Figure 3
VL-VMN adapting GI (AdGI) neurons are not inherently sexually dimorphic but are 17βE sensitive. (A) Quantification of %ΔVm and %ΔIR in response to 0.1 mM glucose in both sexes (♀n = 47, ♂n = 9). (B) Comparison of %ΔIR in response to 0.1 mM glucose in nonadapting GI and AdGI neurons from females (n = 45 GI, 47 AdGI) and males (n = 17 GI, 9 AdGI). ***p < 0.001 via unpaired student t-test. (C) Quantification of a secondary glucose response (%ΔVm and %ΔIR) in AdGI neurons from both sexes (♀n = 18, ♂n = 6). *p < 0.05 via unpaired student t-test. (D–F) Quantification of %ΔIR in response to 0.1 mM glucose in the presence and absence of 17βE (D, n = 5), and AICAR (E, n = 5), MPP (F, n = 6) or PHTPP (F, n = 5). 17βE: 17β-estradiol, AICAR: AMPK activator (0.5 mM), *p < 0.05 via paired student t-test; **p < 0.01, via paired student t-test. G: mM glucose, IR: input resistance, MPP: ERα antagonist (10 μM), PHTPP: ERβ antagonist (1 μM), Vm: membrane potential. (G) Representative action potentials generated in female nonadapting GI and AdGI neurons. Dashed grey line represents Vm. (H, I) Quantification of action potential threshold (H; n = 42 GI, 74 AdGI events from n = 3 neurons each) and afterhyperpolarization magnitude (I; n = 38 GI, 43 AdGI events from n = 3 neurons each) in 0.1 mM glucose for nonadapting GI and AdGI neurons. ***p < 0.001 via unpaired student t-test. AHP: afterhyperpolarization, AP: action potential, IR: input resistance, Vm: membrane potential.
Figure 4
Figure 4
VL-VMN GE neurons are neither sexually dimorphic nor 17βE sensitive. (A) Representative voltage responses to a hyperpolarizing pulse for GE neurons from both sexes. Vm was normalized to 2.5 mM glucose to emphasize changes in IR. (B) Quantification of %ΔVm and %ΔIR in response to 0.1 mM glucose in VL-VMN GE neurons from both sexes (♀n = 25, ♂n = 7). (C, D) Quantification of %ΔVm and %ΔIR in response to 0.1 mM glucose in the presence and absence of 17βE in females (C, n = 4) and males (D, n = 3). 17βE: 17β-estradiol, G: mM glucose, IR: input resistance, Vm: membrane potential.
Figure 5
Figure 5
VL-VMN nonadapting GI neurons and AdGI utilize an AMPK-dependent glucose sensing mechanism. (A) Representative glucose-sensitive V-I relationship in nonadapting GI neurons (left; ♀n = 13, ♂n = 7) and AdGI neurons (right; ♀n = 6, ♂n = 3). The glucose-sensitive conductance reversed near the K+ (EK+ = −99 mV) equilibrium potential for our solutions. (B) Quantification of %ΔVm and %ΔIR in response to 0.1 mM glucose in the presence and absence of CC in female nonadapting GI neurons (left; n = 6) and female AdGI neurons (right; n = 6). (C) Quantification of %ΔVm and %ΔIR in response to 0.1 mM glucose in of female AdGI neurons in the presence and absence of TTX (n = 7) *p < 0.05 via paired student t-test. AMPK: AMP-activated kinase, CC: Compound C (AMPK antagonist; 10 μM), G: mM glucose, IR: input resistance, TTX: tetrodotoxin (voltage-gated Na+ channel antagonist; 500 nM), Vm: membrane potential.
Figure 6
Figure 6
In females, 17β-estradiol modulates whole VMH phospho (p)-AMPK levels via a membrane-bound ER. (A) Representative western blot images for pT172-AMPK, tAMPK, and β-actin. (B–E) Quantification of relative fold expression of whole VMH pAMPK (B, C) and total (t)-AMPK (D, E) in the presence and absence of 17βE or BSA-17βE. Numbers above columns indicate the n of each group. Columns with different letters are significantly different from each other as determined by two-way ANOVA followed by Bonferroni post-hoc tests. 17βE: 17β-estradiol (100 nM), AMPK:AMP-activated kinase, BSA-17βE: bovine serum albumin-conjugated 17βE (100 nM), ER: estrogen receptor, G: mM glucose, VMH: ventromedial hypothalamus.
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