Effects of short-term food deprivation on catecholamine and metabolic-sensory biomarker gene expression in hindbrain A2 noradrenergic neurons projecting to the forebrain rostral preoptic area: Impact of negative versus positive estradiol feedback

Abstract

Hindbrain A2 noradrenergic neurons assimilate estrogenic and metabolic cues. In female mammals, negative- versus positive-feedback patterns of estradiol (E) secretion impose divergent regulation of the gonadotropin-releasing hormone (GnRH)-pituitary-gonadal (HPG) neuroendocrine axis. Current research used retrograde tracing, dual-label immunocytochemistry, single-cell laser-microdissection, and multiplex qPCR methods to address the premise that E feedback modes uniquely affect metabolic regulation of A2 neurons involved in HPG control. Ovariectomized female rats were given E replacement to replicate plasma hormone levels characteristic of positive (high-E dose) or negative (low-E dose) feedback. Animals were either full-fed (FF) or subjected to short-term, e.g., 18-h food deprivation (FD). After FF or FD, rostral preoptic area (rPO)-projecting A2 neurons were characterized by the presence or absence of nuclear glucokinase regulatory protein (nGKRP) immunostaining. FD augmented or suppressed mRNAs encoding the catecholamine enzyme dopamine-beta-hydroxylase (DβH) and the metabolic-sensory biomarker glucokinase (GCK), relative to FF controls, in nGKRP-immunoreactive (ir)-positive A2 neurons from low-E or high-E animals, respectively. Yet, these transcript profiles were unaffected by FD in nGKRP-ir-negative A2 neurons at either E dosage level. FD altered estrogen receptor (ER)-alpha and ATP-sensitive potassium channel subunit sulfonylurea receptor-1 gene expression in nGKRP-ir-positive neurons from low-E, but not high-E animals. Results provide novel evidence that distinct hindbrain A2 neuron populations exhibit altered versus unaffected transmission to the rPO during FD-associated metabolic imbalance, and that the direction of change in this noradrenergic input is controlled by E feedback mode. These A2 cell types are correspondingly distinguished by FD-sensitive or -insensitive GCK, which correlates with the presence versus absence of nGKRP-ir. Further studies are needed to determine how E signal volume regulates neurotransmitter and metabolic sensor responses to FD in GKRP-expressing A2 neurons.

Keywords: AMPK, adenosine 5′-monophosphate-activated protein kinase; DVC, dorsal vagal complex; Dopamine-beta-hydroxylase; DβH, dopamine-beta-hydroxylase; E, estradiol; ERα, estrogen receptor-alpha; ERβ, estrogen receptor-beta; Estradiol; FD, food-deprivation; FF, full-fed; Food deprivation; GCK, glucokinase; GKRP, glucokinase regulatory protein; Glucokinase; Glucokinase regulatory protein; GnRH, gonadotropin-releasing hormone; HPG, hypothalamic-pituitary-gonadal; KATP, ATP-sensitive potassium channel; LH, luteinizing hormone; NE, norepinephrine; O.D., optical density; OVX, ovariectomy; SUR-1, sulfonylurea receptor-1; Single-cell quantitative multiplex PCR; TH, tyrosine hydroxylase; pAMPK, phospho-AMPK; rPO, rostral preoptic area.

Fig. 1

Identification of Rostral Preoptic Area (rPO)-Projecting A2 Noradrenergic Neurons by Dual Cytoplasmic Retrobead Epifluorescence and Tyrosine Hydroxylase (TH)-Immunoreactivity (-ir) and Their Sub-Categorization According to Presence versus Absence of Nuclear Glucokinase Regulatory Protein (nGKRP) Immunolabeling. Ovariectomized (OVX) rats were implanted with a subcutaneous (sc) estradiol (E)-filled silastic capsule [30 ug E/mL (E-30) versus 300 ug E/mL (E-300)], then injected into the rPO with Retrobead retrograde tracer prior to 18 h food deprivation (FD) or uninterrupted feeding (full-fed, i.e., FF controls). Hindbrain A2 neurons were identified in situ by tyrosine hydroxylase (TH)-immunoreactivity (-ir) (Panel I); TH-ir-positive cells exhibiting red cytoplasmic tracer epifluorescence (Panel II) were individually laser-catapult-microdissected for single-cell multiplex qPCR following sub-categorization based upon the presence or absence of nGCKR-ir (Panel III). The encircled area in Panels I, II, and III contains two representative cytoplasmic TH-ir- and Retrobead epifluorescence-positive hindbrain A2 neurons (one indicated by a yellow arrow, the other by a red arrow) that exhibit immunostaining for nGKRP-ir. Panel IV presents a merged image of cytoplasmic and nuclear staining patterns. The inset to each Panel, upper right-hand corner, shows an encircled TH-ir- and Retrobead epifluorescence-positive neuron that lacks nGKRP-ir.

Fig. 2

Effects of Short-Term Food Deprivation (FD) on Dopamine-Beta-Hydroxylase (DβH) and TH Gene Expression in A2 Noradrenergic Neurons Innervating the rPO of Estradiol-Implanted OVX Female Rats. Graphs depict mean normalized DβH mRNA levels +/- S.E.M. measured in cytoplasmic epifluorescence-positive, cytoplasmic TH-ir-positive, nGKRP-ir-positive A2 neurons (Fig. 2A) or cytoplasmic epifluorescence-positive, cytoplasmic TH-ir-positive, nGKRP-ir-negative (Fig. 2B) A2 neurons collected from FF/E-300 (solid white bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), FD/E-300 (striped white bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), FF/E-30 (solid gray bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), or FD/E-30 (striped gray bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons) treatment groups. Data were analyzed by two-way ANOVA and Student-Neuman-Keuls post-hoc test, using GraphPad Prism (Volume 8) software; statistical results are presented in Supplementary Table 1. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 3

Effects of High (E-300) versus Low (E-30) Estradiol Dosing on Glucokinase (GCK) and Sulfonylurea Receptor-1 (SUR-1) mRNA Responses to FD in rPO-Projecting A2 Noradrenergic Neurons. Data show mean normalized GCK or SUR-1 mRNA levels +/- S.E.M. measured in cytoplasmic epifluorescence-positive, cytoplasmic TH-ir-positive, nGKRP-ir-positive A2 neurons (Fig. 3A and 3C) or cytoplasmic epifluorescence-positive, cytoplasmic TH-ir-positive, nGKRP-ir-negative (Figs. 3B and 3D) A2 neurons collected from FF/E-300 (solid white bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), FD/E-300 (striped white bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), FF/E-30 (solid gray bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), or FD/E-30 (striped gray bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons) treatment groups. Data were analyzed by two-way ANOVA and Student-Neuman-Keuls post-hoc test, using GraphPad Prism (Volume 8) software; statistical results are presented in Supplementary Table 1. *p < 0.05; * *p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 4

Effects of FD on Estrogen Receptor-Alpha (ERα) and Estrogen Receptor-Beta (ERβ) Gene Expression in rPO-Projecting A2 Noradrenergic Neurons from High E- versus Low E-Treated OVX Rats. Results depict show mean normalized ERα or ERβ mRNA levels +/- S.E.M. in cytoplasmic epifluorescence-positive, cytoplasmic TH-ir-positive, nGKRP-ir-positive A2 neurons (Fig. 4A and 4 C) or cytoplasmic epifluorescence-positive, cytoplasmic TH-ir-positive, nGKRP-ir-negative (Figs. 4B and 4D) A2 neurons collected from FF/E-300 (solid white bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), FD/E-300 (striped white bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), FF/E-30 (solid gray bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons), or FD/E-30 (striped gray bars; n = 4 nGKRP-ir-positive A2 nerve cells; n = 4 nGKRP-ir-negative A2 neurons) treatment groups. Data were analyzed by two-way ANOVA and Student-Neuman-Keuls post-hoc test, using GraphPad Prism (Volume 8) software; statistical results are presented in Supplementary Table 1. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5

Effects of FD on Plasma Glucose Levels, rPO Gonadotropin-Releasing Hormone-1 (GnRH-1) Content, and Plasma Luteinizing Hormone (LH) Concentrations in High E- versus Low E-Treated OVX Rats. Data illustrate FD effects on mean plasma glucose levels (Fig. 5 A; n = 5/group), mean normalized rPO GnRH-1 tissue content (Fig. 5B; n = 3 lysate pools/group) or mean plasma LH levels (Fig. 5 C; n = 5 group) +/- S.E.M. for the following treatment groups: FF/E-300 (solid white bars), FD/E-300 (striped white bars), FF/E-30 (solid gray bars), or FD/E-30 (striped gray bars) treatment groups. Data were analyzed by two-way ANOVA and Student-Neuman-Keuls post-hoc test, using GraphPad Prism (Volume 8) software; statistical results are presented in Supplementary Table 1. *p < 0.05; * *p < 0.01; * **p < 0.001; * ** *p < 0.0001.