Neuroestradiol regulation of ventromedial hypothalamic nucleus 5′-AMP-activated protein kinase activity and counterregulatory hormone secretion in hypoglycemic male versus female rats

Hypoglycemia activates the ultra-sensitive energy gauge 5′-AMP-activated protein kinase (AMPK) in ventromedial hypothalamic nucleus (VMN) gluco-regulatory neurons. The VMN is exemplified by high levels of expression of the enzyme aromatase, which converts testosterone to estradiol. This study examined the hypothesis that neuroestradiol imposes sex-dimorphic control of VMN AMPK activity during eu- and/or hypoglycemia. VMN tissue corresponding to distinct rostro-caudal segments was obtained by micropunch dissection from testes-intact male and estradiol-replaced ovariectomized female rats that were infused intracerebroventricularly with the aromatase inhibitor letrozole (Lz) before subcutaneous insulin (INS) injection. In euglycemic rats, Lz treatment elevated (male) or decreased (female) middle VMN phosphoAMPK content, with concurrent effects on total AMPK expression. Lz prevented hypoglycemic up-regulation of the mean pAMPK/AMPK ratio in rostral and middle segments of the male VMN, and significantly inhibited this proportion throughout the VMN of hypoglycemic female rats. Lz prevented glucagon secretion in hypoglycemic rats of each sex, and abolished hypoglycemic hypercorticosteronemia in males. Results show that neuroestradiol regulation of VMN AMPK activity during euglycemia is region-specific and gender-divergent, e.g. inhibitory in males versus stimulatory in females. Intra-VMN distribution of hypoglycemia-activated AMPK varies between sexes, but in each sex, locally-generated estradiol is critical for sensor reactivity to this stimulus. Coincident Lz attenuation of VMN AMPK and counter-regulatory hormone responses to hypoglycemia infers a possible cause-and-effect association. Further effort is needed to elucidate the cellular and molecular mechanisms that underlie sex-dimorphic neuroestradiol control of VMN total AMPK and phosphoAMPK expression during distinct metabolic states.

The VMN is a substrate for estradiol regulation of circulating glucose levels [10,11]. In the VMN and elsewhere in the brain, estrogen receptors (ERs) are stimulated by estradiol of dual origin, as this hormone is both acquired from the circulation and produced locally by aromatase conversion of testosterone to estradiol. Brain tissue aromatase protein levels vary according to structure, with highest expression levels observed in the VMN and a limited number of other sites [12][13][14]. The impact of neuroestradiol on VMN AMPK activity has not been established for either sex. In the current study, methodology for intracerebroventricular (icv) infusion of the aromatase inhibitor letrozole (LZ) was used in conjunction with high-resolution microdissection/Western blot techniques in a rat whole-animal model to investigate the premise that neuroestradiol exerts sex-dimorphic control of VMN AMPK protein expression and/or activation and counter-regulatory hormone secretion during systemic glucose availability or scarcity. Our studies show that in each sex, neuroestradiol regulates VMN gluco-regulatory transmission in a region-specific manner during hypoglycemia [15]. Current work analyzed total AMPK as well as activated, e.g. phosphoAMPK (pAMPK) expression in micropunch-dissected tissue samples obtained from rostral, middle, and caudal levels of the VMN to determine if aromatase-dependent hypoglycemic activation of AMPK is co-localized with neuroestradiolsensitive metabolic transmitters in this structure.

Animals
Adult male and female Sprague Dawley rats (3-4 months of age) were housed in groups of 2-3 according to sex, under a 14 h light/10 h dark light cycle (lights on at 05.00 h). Animals had unrestricted access to standard laboratory chow and water, and were acclimated to daily handling. All surgical and experimental protocols were performed in compliance with NIH Guidelines for the Care and Use of Laboratory Animals, 8 th Edition, with approval by the ULM Institutional Animal Care and Use Committee.

VMN tissue micropunch dissection and Western Blot analysis
Each brain was cut into consecutive 100 μm-thick frozen sections through the VMN between −2.00 and −3.3 mm posterior to bregma. For each animal, bilateral micropunches of VMN tissue were taken using a calibrated 0.5 mm hollow punch tool (prod. no. 57401; Stoelting Co., Kiel, WI), as shown in Figure 2, from sections cut at rostral (−2.0 to −2.3 mm), middle (−2.5 to −2.8), and caudal (−3.0 to −3.3 mm) levels of the VMN, and pooled according to region in lysis buffer [18] for Western blot analysis. Accuracy of use of micropunch methodology for collection of distinctive hypothalamic loci of interest, including the VMN, as indicated by marker protein expression, has been verified [19]. In each treatment group, tissue lysate aliquots from individual subjects were combined for rostral, middle, and caudal VMN to create six separate sample pools for each protein of interest for each VMN segment. Tissue sample pools were separated in Bio-Rad TGX 10% stain-free gels (Bio-Rad, Hercules, CA); after electrophoresis, gels were UV light-activated (1 min) in a Bio-Rad ChemiDoc TM Touch Imaging System [20] and proteins were transblotted to 0.45 μm PVDF-Plus membranes (prod. no. 121639; Data Support Co., Panorama City, CA). Membranes were blocked with Tris-buffer saline containing 0.1% Tween-20 and 2.0% bovine serum albumin prior to incubation between 36-42 h (4 °C) with primary polyclonal antisera raised in rabbit against total (activated and inactivated) AMPK α1/2 enzyme protein (prod. no. 2532; 1:2,000; Cell Signaling Technology, Danvers, MA) or the active enzyme form, e.g. phosphorylated AMPK α1/2 -Thr 172 (pAMPK; prod. no. 2535; 1:2,000; Cell Signaling Technol). Membranes were then incubated with a goat anti-rabbit horseradish-peroxidase-labeled secondary antiserum (1:5,000; prod. no. NEF812001EA; PerkinElmer, Waltham, MA), followed by SuperSignal West Femto maximum sensitivity chemiluminescence substrate (prod. no. 34096; ThermoFisherScientific, Waltham, MA). Automated membrane buffer washes and blocking and antibody incubations were performed in a Freedom Rocker™ Blotbot. Protein band optical density (O.D.) measures were normalized to total in-lane protein using Image Lab™ 6.0.0 software (Bio-Rad). Precision plus protein molecular weight dual color standards (prod. no. 161-0374, Bio-Rad) were included in each Western blot analysis.

Statistical analyses
Mean normalized VMN protein O.D. and plasma glucose and hormone data were evaluated by three-way analysis of variance and Student-Newman-Keuls post-hoc test. Differences of p < 0.05 were considered significant. In each figure, statistical differences between specific pairs of treatment groups are denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Results
Male and female rats were infused icv with the aromatase enzyme inhibitor letrozole (Lz) prior to sc vehicle (V) or insulin (INS) injection to investigate the impact of neuroestradiol on rostral VMN AMPK and pAMPK protein expression during eu-or hypoglycemia ( Figure 3). Data presented in Figure  Effects of icv Lz on caudal VMN AMPK and pAMPK expression in eu-or hypoglycemic male and female rats are presented in Figure 5. As shown in Figure 5A Figure 5C, Lz had no significant effect on the caudal VMN pAMPK/AMPK protein ratio in the male, but suppressed this ratio in female rats.

Discussion and conclusion
VMN AMPK is an evident source of metabolic-sensory input to the neural glucostatic network, as hypoglycemia activates this sensor in resident neurons that express neurotransmitters involved in glucose counter-regulation. The VMN is a substrate for estradiol input to that neural circuitry [10]. Evidence for high VMN aromatase expression levels raises the prospect that neuroestradiol may govern local AMPK function. Current studies investigated the hypothesis that aromatase may control VMN AMPK activity during glucose homeostasis and/or hypoglycemia in one or both sexes. Data show that in euglycemic rats, neuroestradiol imposes inhibitory or stimulatory control of mid-level VMN pAMPK expression, according to sex. During hypoglycemia, aromatase activity is a negative stimulus for total AMPK protein content over the length of the female VMN, reflected by diminution of the mean pAMPK/AMPK ratio in each segment. In hypoglycemic males, on the other hand, aromatase increases this ratio through augmentation of pAMPK protein expression. Results show that neuroestradiol signaling is requisite for hypoglycemic hyperglucagonemia in each sex and for elevated corticosterone secretion in hypoglycemic males. Ongoing research seeks to establish whether observed diminution of counter-regulatory outflow in Lz-treated animals is a consequence of drug suppression of hypoglycemic activation of VMN AMPK. Present outcomes emphasize the need for clarification of mechanisms that cause sex-dimorphic aromatase regulation of VMN AMPK activity during euglycemia, and those that underlie hypoglycemia-associated adjustments in neuroanatomical extent (both sexes) and direction (males) of neuroestradiol control of VMN sensor activation.
Data here provide novel evidence that aromatase regulates baseline AMPK activity within a distinct rostro-caudal segment of the male and female VMN, where direction of control is sexspecific. As ERs occur over the length of the VMN in each sex, it is unclear how this regulatory action is confined to the mid-VMN. A conceivable explanation is that aromatase production and/or activity is elevated in that segment compared to others, resulting in relatively higher neuroestradiol tissue levels. Conversely, neuroestradiol yield may be equivalent throughout the VMN, but input via ERs may vary by region due to dissimilar magnitude of ER expression or post-receptor signaling in AMPK-expressing neurons. Since basal middle VMN aromatase protein profiles are similar in the two sexes [15], bi-directional effects of neuroestradiol on pAMPK expression in that location, e.g. inhibition in males versus augmentation in females could reflect, in part, differences in absolute and proportional expression of ER variants, including classical (ER-alpha and ER-beta) and membrane (G protein-coupled ER-1; GPER/GPR30) ERs, that may regulate AMPK activity in that location. This notion remains speculative until verified by additional research. Lz administration did not affect baseline plasma glucose or counter-regulatory hormone profiles despite adjusted VMN pAMPK expression, which infers that sensor activity in the mid-VMN does not regulate glucostasis, or alternatively, that altered metabolic sensory signaling in that site does not override metabolic cues that are unaffected by this icv drug treatment.
Implementation here of an icv route of Lz administration raises the possibility that demonstrable drug effects on VMN AMPK activity may result from, to some extent, diminished aromatase activity within and outside the VMN. Indeed, the VMN is extensively interconnected with other forebrain structures that participate in gluco-regulation, including the lateral hypothalamic area and arcuate, dorsomedial, and paraventricular hypothalamic nuclei. However, evidence for high VMN aromatase expression profiles supports the view that treatment outcomes are largely reflective of Lz inhibition of local enzyme action. Current research did not evaluate Lz effects on regional VMN aromatase enzyme activity or tissue estradiol concentrations as quantitative methods of requisite sensitivity for analysis of these parameters in small-volume tissue samples obtained from region-based VMN microdissection were not available. In the brain, aromatase is reportedly expressed mainly or exclusively in neurons [23][24][25][26]. As that published work did not include analysis of the VMN, further work is needed to characterize the VMN cell type (s) that produce neuroestradiol, and to establish how aromatase expression in that (those) cell (s) may be regulated.
Results show that during hypoglycemia, aromatase is a negative stimulus for total AMPK protein expression in the rostral, middle, and caudal VMN in female rats. As net AMPK levels in those segments did not differ after V or INS injection of V-infused females, this inhibitory tone is evidently offset by other regulatory signals at the time examined here. Since only a single time point, e.g. +1 hr post-injection was examined here, it is unclear if neuroestradiol suppression of total AMPK this sex depends upon duration of hypoglycemia. A possible physiological outcome of decreased total AMPK expression is augmentation of the cellular pAMPK/AMPK ratio, i.e. enzyme specific activity. On the other hand, diminished AMPK availability could, depending upon magnitude of decline, eventually limit enzyme mass available for activation by phosphorylation. Phosphorylation is a rapid post-translational modification that generates an appropriate acute response to hypoglycemia, whereas decrements in total AMPK protein expression may possibly serve as a more protracted adaptive response. Conversely, in the male, Lz pretreatment diminished rostral and caudal VMN AMPK levels during hypoglycemia, indicating that neuroestradiol is a positive stimulus of this protein.
Current data provide unique proof that neuroestradiol signaling is requisite for hypoglycemic activation of VMN AMPK in each sex. Interestingly, neuroanatomical patterns of VMN AMPK activation differed between sexes, as sensor activity was increased in the male rostral and middle VMN versus rostral, middle, and caudal female VMN. The neurotransmitters GABA and NO act within VMN neural circuities to correspondingly suppress or amplify counter-regulatory hormone secretion during hypoglycemia [27,28]. Western blot analysis of pooled lysates of nitrergic and GABAergic neurons collected at regular intervals over the rostro-caudal extent of the male VMN revealed elevated pAMPK/AMPK protein ratios in each nerve cell population at a post-insulin injection time point similar to that used here [8,9]. Present outcomes point to a need for a functional mapping approach to establish the neuroanatomical distribution of GABA and NO neurons within the VMN that exhibit neuroestradiol-dependent adjustments in neurotransmission during hypoglycemia, and to determine if hypoglycemia-sensitive cells of each transmitter type express aromatase and/or AMPK. In the hypoglycemic male, aromatase stimulation of mid-VMN pAMPK expression contrasts with negative effects of this signal on sensor activity during euglycemia. The mechanisms that cause this bidirectional metabolic state-specific neuroestradiol action remain to be clarified. It is possible that neuroestradiol may inhibit or stimulate AMPK depending upon tissue concentrations, and that local production may vary between states of glucose homeostasis versus deprivation. On the other hand, nutrient, endocrine, and neurochemical inputs to AMPK-expressing cells may modulate their receptivity to neuroestradiol. A similar premise could likely explain how hypoglycemia expands neuroestradiol regulation of AMPK activity beyond the middle VMN segment.
Electrophysiological studies have documented the presence of dedicated metabolic-sensory neurons in the VMN that supply a dynamic cellular energy readout by increasing ('glucose-inhibited') or decreasing ('glucose-excited') their synaptic firing as ambient energy substrate levels fall [29,30]. Recent studies involving genetic manipulation of VMN AMPK α1 and −2 subunit gene expression affirm that both regulatory subunits function to promote increased electrical activity of local 'glucose-inhibited' metabolic-sensory neurons during hypoglycemia, but do not regulate 'glucoseexcited' nerve cell firing [31]. Thus, current documentation of sex differences in neuroanatomical localization of hypoglycemia-associated up-regulation of pAMPK α1 and −2 subunit proteins in the VMN infer that 'glucose-inhibited' neurons occur throughout the rostro-caudal length of the female VMN, but are restricted to rostral and middle segments of this structure in males. Current results also provide novel evidence that 'glucose-inhibited' nerve cell reactivity to hypoglycemia may be neuroestradiol-dependent.
Present findings that Lz treatment prevented hypoglycemic augmentation of glucagon secretion in both sexes, and abolished hypoglycemic hypercorticosteronemia in males uniquely implicate neuroestradiol in neural mechanisms governing counter-regulation. MBH AMPK signaling is required for optimal counter-regulatory outflow [6,7]. Evidence here for parallel Lz effects on VMN pAMPK expression and counter-regulatory hormone secretion in hypoglycemic animals suggests that these treatment outcomes may be causally associated. Nevertheless, the prospect that neuroestradiol may act outside the VMN to control counter-regulatory responses to hypoglycemia cannot be disregarded. Notably, forebrain aromatase apparently regulates hypoglycemic hypercorticosteronemia in male, but not female rats. We previously reported that icv ER antagonist administration alters corticosterone secretion in INS-injected rats of each sex [18,32]. Current data suggest that the forebrain neural circuitry that controls hypoglycemic patterns of corticosterone release in females may exhibit discriminative sensitivity to systemic-versus brain-derived estradiol, by mechanisms that remain unclarified at this time.
In summary, present research establishes a role for forebrain aromatase in VMN AMPK sensor and counter-regulatory hormone responses to hypoglycemia in the rat. In each sex, neuroestradiol regulation of pAMPK expression occurs more broadly within the VMN during hypo-versus euglycemia. Outcomes show that in males, brain-derived estradiol imposes energy state-dependent control of sensor activation; further effort is required to determine how this directional, e.g. negativeto-positive shift in aromatase action between eu-and hypoglycemia occurs.