Effects of estradiol on lactoprivic signaling of the hindbrain upon the contraregulatory hormonal response and metabolic neuropeptide synthesis in hypoglycemic female rats

Abstract

Background: Caudal dorsomedial hindbrain detection of hypoglycemia-associated lactoprivation regulates glucose counter-regulation in male rats. In females, estradiol (E) determines hypothalamic neuroanatomical and molecular foci of hindbrain energy sensor activation. This study investigated the hypothesis that E signal strength governs metabolic neuropeptide and counter-regulatory hormone responses to hindbrain lactoprivic stimuli in hypoglycemic female rats.

Methods: Ovariectomized animals were implanted with E-filled silastic capsules [30 (E-30) or 300 μg (E-300)/mL] to replicate plasma concentrations at estrous cycle nadir versus peak levels. E-30 and E-300 rats were injected with insulin or vehicle following initiation of continuous caudal fourth ventricular L-lactate infusion.

Results: Hypoglycemic hypercorticosteronemia was greater in E-30 versus E-300 animals. Glucagon and corticosterone outflow was correspondingly fully or partially reversed by hindbrain lactate infusion. Insulin-injected rats exhibited lactate-reversible augmentation of norepinephrine (NE) accumulation in all preoptic/hypothalamic structures examined, excluding the dorsomedial hypothalamic nucleus (DMH) where hindbrain lactate infusion either suppressed (E-30) or enhanced (E-300) NE content. Expression profiles of hypoglycemia-reactive metabolic neuropeptides were normalized (with greater efficacy in E-300 animals) by lactate infusion. DMH RFamide-related peptide-1 and -3, arcuate neuropeptide Y and kisspeptin, and ventromedial nucleus nitric oxide synthase protein responses to hypoglycemia were E dosage-dependent.

Conclusions: Distinct physiological patterns of E secretion characteristic of the female rat estrous cycle elicit differential corticosterone outflow during hypoglycemia, and establish both common and different hypothalamic metabolic neurotransmitter targets of hindbrain lactate deficit signaling. Outcomes emphasize a need for insight on systems-level organization, interaction, and involvement of E signal strength-sensitive neuropeptides in counter-regulatory functions.

Keywords: Estradiol; Nitric oxide synthase; Norepinephrine; Pre-pro-kisspeptin; RFamide-related peptide-1/-3, neuropeptide Y; l-lactate.

Figure 1. Effects of Hindbrain Lactate Repletion on Blood Glucose and Counter-Regulatory Hormone Concentrations in Insulin (INS)-Injected Estradiol (E)-Implanted Ovariectomized (OVX) Adult Female Rats

OVX rats were implanted with sc capsules filled with E at a dose of 30 (E-30) [left-hand side; bars 1-3] or 300 (E-300) [right-hand side; bars 4-6] ug/mL to replicate estrous cycle baseline versus peak plasma hormone levels. Animals were subsequently injected sc with INS or vehicle (V) after initiation of caudal fourth ventricular (CV4) infusion of L-lactate (L) or the vehicle artificial cerebrospinal fluid (V) alone. Data depict mean circulating glucose (Panel 1A), glucagon (Panel 1B), corticosterone (Panel 1C), and leptin (Panel 1D) 6.B) levels ± S.E.M. in groups (n=5 rats/group) of E-30 and E-300 rats treated as follows: CV4 V/sc V (V/V; solid white bars), CV4 V/sc INS (V/INS; solid grey bars), and CV4 L/sc INS (L/INS; cross-hatched grey bars). *p<0.05; versus V/V; **p<0.05; versus V/I; #p<0.05; versus E-30 V/V.

Figure 2. Effects of Hindbrain Lactate Infusion on Hypoglycemic Patterns of Norepinephrine (NE) Accumulation in Forebrain Gluco-Regulatory- and Reproduction-Relevant Structures in E-Implanted OVX Female Rat Brain

At the conclusion of 2 hour infusion of V or L to the CV4, groups of V/V-, V/INS-, and L/INS-treated E-30 and E-300 rats were sacrificed for micro-punch dissection of rostral preoptic area (rPO; Panel 2A), anteroventral periventricular nucleus (AVPV; Panel 2B), medial preoptic nucleus (MPN; Panel 2C), arcuate hypothalamic nucleus (ARH; Panel 2D), dorsomedial hypothalamic nucleus (DMH; Panel 2E), lateral hypothalamic area (LHA; Panel 2F), paraventricular hypothalamic nucleus (PVH; Panel 2G), and ventromedial hypothalamic nucleus (Panel 2H) tissues from each left hemi-forebrain for ELISA analysis of NE content. Data in each Panel illustrate mean tissue NE content ± S.E.M. for groups (n=5/group) of V/V- (solid white bars), V/INS- (solid grey bars), and L/INS- (crosshatched grey bars) treated E-30 [bars 1-3] and E-300 [bars 4-6] rats. *p<0.05; versus V/V; **p<0.05; versus V/INS; #p<0.05; versus E-30 V/V; +p<0.05; versus INS-injected E-30 rats.

Figure 3. Effects of Hindbrain Lactate Infusion on Rostral Preoptic Area (rPO) Gonadotropin-Releasing Hormone-I (GnRH-I), Anteroventral Periventricular Nucleus (AVPV) PrePro-Kisspeptin, and Medial Preoptic Nucleus (MPN) Glutamate Decarboxylase _65/67 (GAD_65/67) Protein Responses to Insulin-Induced (IIH) in E-Implanted OVX Female Rat Brain

Micropunched rPO (Panel 3A), AVPV (Panel 3B), and MPN (Panel 3C) tissues from right hemi-forebrains of E-30 and E-300 rats treated by V/V, V/INS, or L/INS were analyzed by Western blot for GnRH-I, prepro-kisspeptin, and GAD_65/67 proteins, respectively. Protein band optical densities (O.D.) were quantified with Syngene Genetool 4.01 software and expressed relative to α-tubulin. Bars depict illustrate mean normalized protein O.D. measures ± S.E.M. for groups (n=5/group) of V/V- (solid white bars), V/INS- (solid grey bars), and L/INS- (cross-hatched grey bars) treated E-30 [bars 1-3] and E-300 [bars 4-6] rats. *p<0.05; versus V/V; **p<0.05; versus V/INS; #p<0.05; versus E-30 V/V.

Figure 4. Effects of Hindbrain Lactate Infusion on Arcuate Hypothalamic Nucleus (ARH) Neuropeptide Y (NPY), PrePro-Kisspeptin, and Pro-Opiomelanocortin (POMC) Protein Responses to Insulin-Induced (IIH) in E-Implanted OVX Female Rat Brain

Right hemi-forebrain – derived ARH tissue from E-30 and E-300 rats treated by V/V, V/INS, or L/INS was analyzed by Western blot for NPY (Panel 4A), prepro-kisspeptin (Panel 4B), and POMC (Panel 4C). Bars show mean normalized protein O.D. measures ± S.E.M. for groups (n=5/group) of V/V- (solid white bars), V/INS- (solid grey bars), and L/INS- (crosshatched grey bars) treated E-30 [bars 1-3] and E-300 [bars 4-6] rats. *p<0.05; versus V/V; **p<0.05; versus V/INS; #p<0.05; versus E-30 V/V.

Figure 5. Effects of Hindbrain Lactate Infusion on IIH-Associated Patterns of Dorsomedial Hypothalamic Nucleus (DMH) RFAmide-Related Peptide-1 (RFRP-1) and -3 (RFRP-3) and Ventromedial Hypothalamic Nucleus (VMH) GAD_65/67 and Neuronal Nitric Oxide Synthase (nNOS) Protein Expression in E-Implanted OVX Female Rat Brain

Right forebrain DMH and VMH tissues were analyzed by Western blot for RFRP-1 (Panel 5A), RFRP-3 (Panel 5B), GAD_65/67 (Panel 5C), and nNOS (Panel 5D) proteins. Bars show mean normalized protein O.D. measures + S.E.M. for groups (n=5/group) of V/V- (solid white bars), V/INS- (solid grey bars), and L/INS- (cross-hatched grey bars) treated E-30 [bars 1-3] and E-300 [bars 4-6] rats. *p<0.05; versus V/V; **p<0.05; versus V/INS; #p<0.05; versus E-30 V/V.

Figure 6. Effects of Hindbrain Lactate Infusion on IIH-Associated Patterns of Lateral Hypothalamic Area (LHA) Orexin-A (ORX-A) and Melanin-Concentrating Hormone (MCH) and Paraventricular Hypothalamic Nucleus (PVH) Corticotropin-Releasing Hormone (CRH) Protein Expression in E-Implanted OVX Female Rat Brain

Right forebrain LHA and PVH tissues were analyzed by Western blot for ORX-A (Panel 6A), MCH-3 (Panel 6B), and CRH (Panel 6C) proteins. Bars show mean normalized protein O.D. measures + S.E.M. for groups (n=5/group) of V/V- (solid white bars), V/INS- (solid grey bars), and L/INS- (cross-hatched grey bars) treated E-30 [bars 1-3] and E-300 [bars 4-6] rats. *p<0.05; versus V/V; **p<0.05; versus V/INS; #p<0.05; versus E-30 V/V.