Introduction

Insulin-induced hypoglycemia (IIH) is a recurring complication of obligatory strict glycemic management in insulin-dependent diabetes mellitus patients [Cryer 2013, 2014 ]. As vital nerve cell functions such as synaptic transmission are maintained at high-energy expense, hypoglycemic neuroglucopenia can impair neurological function. Central and peripheral metabolic fuel sensors provide a dynamic readout on cellular energy status to the CNS gluco-regulatory network. The ventromedial hypothalamic nucleus (VMN) is a key structural component of the mediobasal hypothalamus, an area of the brain that controls compensatory physiological responses to local and incoming sensory cues of energy insufficiency [Watts and Donovan 2010; Donovan and Watts 2014]. Distinctive VMN neurotransmitters that may implement systemic adjustments to cellular metabolic imbalance include those reported to inhibit [γ-aminobutyric acid (GABA)] or stimulate [nitric oxide (NO); steroidogenic factor-1 (SF-1)] counter-regulation (Chan et al. 2013; Garfield et al. 2014).

Brain astrocytes promote optimal nerve cell function through glio-transmitter signaling, regulation of local micro-circulatory function, and metabolic fuel provision [Stobart and Anderson 2013]. Astrocytes store glucose in the form of the complex polymer glycogen [Nehlig et al. 2004]. This energy reserve is dynamic during normal brain activity and metabolic stasis, and is a vital energy substrate source during states of heightened activity or glucose deficiency, e.g., seizure, sleep deprivation, and hypoglycemia [Gruetter 2003; Brown 2004]. In the CNS and elsewhere, glycogen metabolism is governed by antagonistic actions of glycogen synthase (GS) and glycogen phosphorylase (GP), which respectively catalyze glycogen synthesis and depletion. Our recent observations that pharmacological inhibition of VMN GP activity stimulates expression of the nitrergic neuron marker protein neuronal nitric oxide synthase (nNOS) infer that astrocyte glycogen-derived fuel stream promotes VMN neurometabolic stability [Alhamami et al. 2018]. In male rats, whole hypothalamic glycogen is reported to be substantially diminished 3 h after induction of IIH [Herzog et al. 2008]. Current research investigated the premise that this reduction in stored metabolic fuel may correspondingly intensify and/or suppress activity of the ultra-sensitive energy gauge 5′-adenosine monophosphate-activated protein kinase (AMPK) in corresponding VMN gluco-stimulatory and gluco-inhibitory neurotransmitter neuron populations in hypoglycemic males. Here, animals were pretreated by intracerebroventricular (icv) infusion of the indole carboximide GP inhibitor [R-R*,S*]-5-chloro-N-[2-hydroxy-3-(methoxymethylamino)-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (CP-316,819) via Alzet pump prior to subcutaneous (sc) injection of neutral protamine Hagedorn insulin (INS) or vehicle (V) alone. This and other indole-2-carboxamide GP inhibitors elicit hepatocyte (Martin et al. 1998) and cortical astrocyte (Suh et al. 2007) glycogen accumulation at normal tissue glucose concentrations, but do not avert glycogen breakdown in those cells at lower glucose levels. In the above-referenced work, Suh et al. found that increased cortical/hippocampal tissue glycogen levels owing to systemic CP-316,819 administration, e.g., approximately 250 mg/kg was delivered via divided injections over an 18-h period, prevented neuronal injury in vulnerable brain structures during subsequent IIH, suggesting that enhanced glycogen amassment extends availability of glycogen-derived metabolic fuel. In the present studies, tissue from distinctive hypothalamic metabolic loci, namely the VMN, arcuate hypothalamic nucleus (ARH; another principal mediobasal hypothalamic structure, dorsomedial hypothalamic nucleus (DMN), and lateral hypothalamic area (LHA), was micropunch dissected from brains obtained 4 h after INS injection for HPLC/mass spectrometric measurement of VMN glycogen content to assess effects of CP-316,819 pretreatment on pre-IIH glycogen accumulation and hypoglycemia-associated depletion. VMN tissue was also utilized for combinatory immunocytochemical labeling/laser-catapult microdissection of GABA, nitrergic, or SF-1 neurons for Western blot analysis to determine, for each cell population, expression profiles of total and activated, e.g., phosphorylated AMPK (pAMPK) and relevant biosynthetic enzyme [glutamate decarboxylate65/67 (GAD65/67); neuronal nitric oxide synthase (nNOS)] or neuropeptide transmitter (SF-1) proteins. Neurochemically characterized VMN neurons were also evaluated for expression of the inducible immediate-early transcription factor Fos to determine if cellular transcriptional activity is adjusted in accordance with changes in energy status.

Materials and Methods

Animals

Adult male Sprague-Dawley rats (2–3 months of age) were housed under a 14-h light/10-h dark schedule (lights on at 05.00 h), in groups of two per cage, and allowed ad libitum access to standard laboratory rat chow and water. Animals were accustomed to daily handling before initiation of the study. Experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition, and approved by the University of Louisiana at Monroe Institutional Animal Care and Use Committee.

Experimental Design

Model 2001D Alzet pumps (200 μL total volume; 8.0 μL/h/24 h; DURECT Corporation, Cupertino, CA) were filled with V (dimethyl sulfoxide) or the glycogen phosphorylase inhibitor CP-316,819 (CP; R&D Systems, Inc., Minneapolis, MN), then attached via polyvinylchloride catheter tubing to a 28-gauge stainless steel cannula (Alzet Brain Infusion kit-1, prod. no, 0004760; DURECT Corp.). Pumps containing CP were configured to deliver 2.5 (CP-2.5) or 10.0 (CP-10.0) mg/24 h. On study day 1, rats were anesthetized at 09.00 h by intraperitoneal injection of ketamine/xylazine anesthesia (0.1 mL/100 g bw, 90 mg ketamine/10 mg xylazine/ml; Butler Schein, Inc., Melville, NY, USA). A computerized Neurostar GmbH Drill & Microinjection Robot (Tubingen, Germany) was used to implant cannulas into the lateral cerebral ventricle  at the following three-dimensional coordinates: 1.4 mm lateral to midline; 0.8 mm posterior to bregma; 3.5 mm ventral to the brain surface. The attached Alzet pump was positioned sc under the skin of the dorsum of the back. On day 2, groups of V-, CP-2.5 -, or CP-10.0-pretreated animals were injected sc with neutral protamine Hagedorn INS (10.0 U/kg bw; Henry Schein; n = 3 V/INS, n = 3 CP-2.5/INS, n = 3 CP-10.0/INS) or vehicle (sterile diluent; Eli Lilly & Co., Indianapolis, IN; n = 3 V/V; n = 3 CP-2.5/V, n = 3 CP-10.0/V) at 09.00 h, then sacrificed at 13.00 h by microwave fixation (In Vivo Microwave Fixation System, 5 kW; Stoelting Co., Wood Dale, IL). Brains were snap-frozen in liquid nitrogen-cooled isopentane for storage at − 80 °C. Each brain was halved. The ventromedial (VMN; − 1.80 to − 3.24 mm), arcuate (ARH; − 1.80 to − 3.24 mm posterior to bregma), and dorsomedial (DMN; − 2.40 to − 3.24 mm) nuclei and the lateral hypothalamic area (LHA; − 1.80 to − 3.24 mm) were individually micropunch dissected from 200-μm-thick sections cut through the left hemi-hypothalamus, using calibrated Brain Punch set punch tools (prod. no. 57401; Stoelting), and collected into 150 μL 0.02 M Tris buffer, pH 7.2, for tissue glycogen and glucose measurements. Ten-micrometer-thick sections cut through the VMN in the right hemi-hypothalamus were mounted on 1.0 PEN membrane-covered slides (prod. no. 415190-9041-000; Carl Zeiss Microscopy, LLC, Thornwood, NY) in preparation for immunocytochemical labeling of marker proteins of gluco-regulatory neuron populations, e.g., GAD65/67, nNOS, and SF-1. Accuracy of cannula targeting of the LV was verified postmortem by visual examination of tissue sections cut through that structure. Blood was obtained by cardiac puncture for glucose and hormone measurements; plasma was stored at − 20 °C.

Laser-Catapult Microdissection (LCM) and Western Blot Analysis of VMN GAD65/67, nNOS, and SF-1 Neurons

Tissues were fixed with cold acetone, pre-incubated with 0.05 M Tris-buffered saline, pH 7.4 (TBS), containing 0.05% Triton X-100 and either 5.0% normal horse or goat serum, then incubated for 36 h (4 °C) with a rabbit primary antiserum against GAD65/67 (prod. no. ABN904, 1:2000; MilliporeSigma, Burlington, MA) or nNOS (prod. no. NBP1-39681, 1:500; Novus Biologicals, LLC, Littleton, CO) or mouse antibodies against SF-1 (prod. no. PP-N1665-00, 1:500; R&D Systems, Inc., Minneapolis, MN). Sections were sequentially incubated with biotinylated horse anti-mouse IgG (Vectastain Elite ABC HRP kit, mouse IgG; prod. no. PK-6104; Vector Laboratories, Burlingame, CA) or goat anti-rabbit IgG (Vectastain Elite ABC HRP kit, rabbit Ig; prod. no. PK-6101; Vector Lab.) secondary antisera and ABC reagent (1 h each), then exposed to Vector DAB Peroxidase substrate kit reagents (prod. no. SK-4100). Individual labeled neurons exhibiting a visible nucleus and complete labeling of the cytoplasmic compartment were circumdissected from tissues using a Zeiss P.A.L.M. UV-A microlaser (Carl Zeiss Microscopy). For each harvested nerve cell population, triplicate pools of n = 50 heat-denatured cell lysates were created within each treatment group for each protein of interest. Lysates were separated in BioRad TGX 10–12% stain-free gels [Shakya et al. 2018]; gels were activated by UV light (1 min) in a BioRad ChemiDoc TM Touch Imaging System prior to protein transfer (30 V, overnight at 4 °C; Towbin buffer) to 0.45-μm PVDF membranes (prod. no. 88518; ThermoFisherScientific, Waltham, MA). Membranes were blocked (2 h) with TBS containing 0.1% Tween-20 and 2.0% bovine serum albumin prior (36–48 h; 4 °C) incubation in a NextAdvance Blotbot with rabbit primary antisera against AMPKα1/2 (prod. no. 2532, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), pAMPKα1/2 (prod. no. 2531, 1:1000; Cell Signal. Technol.), Fos (prod. no. 4384, 1:1000; Cell Signal. Technol.), GAD65/67 (prod. no. AB1511; EMD Millipore Corporation, Billerica, MA; 1:2000), nNOS (prod. no NBP1-39681, Novus Biologicals, Littleton, CO; 1:2000), or SF-1 (prod. no. PA5-41967, ThermoFisherScientific, Waltham, MA; 1:2000). Membranes were next incubated (1 h) with a goat anti-rabbit antiserum (prod. no. NEF812001EA, 1:5000; PerkinElmer, Boston, MA). Membrane buffer washes and antibody incubations were carried out by Freedom Rocker™ Blotlbot® automation (Next Advance, Inc., Troy NY). After exposure to SuperSignal West Femto maximum-sensitivity chemiluminescent substrate (prod. no. 34096, ThermoFisherScientific), protein band optical density (O.D.) signals were detected and quantified in a Bio-Rad ChemiDoc MP Imaging System equipped with Image Lab™ software. Protein bands were normalized to total protein content of their respective lane. Precision plus protein molecular weight dual color standards (prod. no. 161-0374, Bio-Rad) were included in each Western blot analysis.

Glycogen HPLC/Mass Spectrometric Analysis

Micropunched VMN, ARH, DMN, and LHA tissues were heated to 95 °C (1 h) and homogenized by ultrasonification (30 s). Supernatants were stored at − 80 °C. Supernatant aliquots were hydrolyzed by incubating 20 μL with 10 μL each of 0.5 mg/mL amyloglucosidase and 0.1 M sodium acetate for 2 h, then heating to 100 °C (5 min), followed by cooling to room temperature. Supernatant glycogen concentrations were determined by reverse-phase HPLC in a Hitachi LaChrom Elite® System (Hitachi America, Ltd., Tarrytown, NY), by modification of published methods (Bai et al. 2015; Fuller et al. 2012; Honda et al. 1989). Hydrolyzed and non-hydrolyzed sample aliquots were derivatized with 100 μL 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent supplemented with 0.3 M NaOH. After acidification with 400 μL 0.75% formic acid, derivatized samples were extracted with chloroform, vacuum concentrated to remove chloroform and formic acid, transferred to fresh tubes, frozen at − 80 °C, and lyophilized. Lyophilized samples were diluted to 1.0 mL with acetonitrile/0.01 M triethylamine (65:35) v/v; 20 μL aliquots was then separated on a Zorbax ODS (4.6 cm × 250 mm, 5 μm), with an acetonitrile/0.005 M triethylamine (65:35) v/v mobile phase, at a flow rate of 1 mL/min. Wavelength of detection was 245 nm. Tissue glycogen concentrations, as calculated by subtracting from hydrolysis-derived total glucose concentrations, were determined using a calibration curve, where y = 0.2463x + 0.887, R2 = 0.9998. Glycogen and D-glucose concentrations were expressed as ng/mL and μg/mL respectively. Prepared D-glucose-PMP and HPLC eluent containing D-glucose-PMP were analyzed in a QTRAP® LC-MS/MS 3200 System. One microgram per milliliter of D-glucose-PMP was diluted with HPLC-grade methanol prior to injection and eluted with acetonitrile/0.005 M triethylamine (65:35) v/v over consecutive 1–3-min intervals. HPLC eluent was evaporated at 45 °C for 60 min, frozen to − 80 °C, lyophilized at − 55 °C, and reconstituted with methanol prior to injection. Confirmatory mass spectrometric spectra showed peaks for D-glucose-PMP at 511.3 m/z and D-glucose-PMP+Na+ at 533.2 m/z prior to and after HPLC.

Glucose and Counter-Regulatory Hormone Measurements

Blood glucose levels were determined using an ACCU-CHECK Aviva plus glucometer (Roche Diagnostic Corporation, Indianapolis, IN) [Kale et al. 2006]. Plasma corticosterone (ADI-900-097; Enzo Life Sciences, Inc., Farmingdale, NY) and glucagon (EZGLU-30K, EMD Millipore, Billerica, MA) concentrations were determined using commercial ELISA kit reagents [Alhamami et al. 2018].

Statistical Analyses

Mean chromatogram area under the curve measures of tissue free or total glucose and normalized Western blot protein O.D., blood glucose, plasma glucagon, and plasma corticosterone values were evaluated between treatment groups by two-way analysis of variance and the Student-Newman-Keuls post hoc test. Differences of p < 0.05 were considered significant.

Results

Figure 1 depicts HPLC measures of mean tissue glycogen content of VMN (panel a [F(5,12) = 28.66; p < 0.0001]; Table 1), ARH (panel b [F(5,12) = 7.08; p = 0.0003]), LHA (panel c [F(5,12) = 55.90; p < 0.0001]), and DMN (panel d [F(5,12) = 26.89; p = 0.0002]) tissue obtained from vehicle- versus CP-pretreated eu- and hypoglycemic male rats. Data show that IIH did not alter glycogen levels in each structured evaluated. Administration of the low CP dose, e.g., 2.5 mg (CP-2.5), elevated basal glycogen [CP-2.5/V versus V/V] in the LHA only, whereas the higher drug dosage (10.0 mg; CP-10.0) increased glycogen accumulation [CP-10.0/V versus V/V] in each hypothalamic site. INS-injected animals pretreated with CP-2.5 exhibited a decline in basal VMN and LHA glycogen levels [CP-2.5/INS versus CP-2.5/V], while those given the CP-10.0 dosage showed significant amplification of glycogen amassment beyond baseline values in each hypothalamic location [CP-10.0/INS versus CP-10.0/V]. These data show that CP caused dose-dependent expansion of glycogen content of the VMN and other hypothalamic metabolic loci, and that high-dose CP allowed further enhancement of this energy reserve during hypoglycemia.

Fig. 1
figure 1

Effects of intracerebroventricular (icv) infusion of the glycogen phosphorylase inhibitor [R-R*,S*]-5-chloro-N-[2-hydroxy-3-(methoxymethylamino)-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (CP-316,819; CP) on microdissected hypothalamic tissue glycogen content in insulin-induced hypoglycemic (IIH) male rats. Groups of rats were infused into the left lateral cerebral ventricle with vehicle (V) versus CP (2.5 or 10.0 mg) over a 24-h period prior to subcutaneous (sc) injection of neutral protamine Hagedorn insulin (INS; 10.0 U/kg bw) or V (sterile diluent), and sacrificed by microwave fixation 4 h post-injection. The ventromedial (VMN; panel a), arcuate (ARH; panel b), and dorsomedial (DMN; panel d) hypothalamic nuclei and lateral hypothalamic area (LHA; panel c) were individually microdissected from the left hemi-hypothalamus for HPLC/mass spectrometric analysis of tissue glycogen content. Data indicate mean tissue glycogen levels + S.E.M. for the following treatment groups: (1) V-infused/V-injected (V/V; solid white bars); (2) V-infused/INS-injected (V/I; diagonal-striped white bars); (3) low-dose (2.5 mg) CP-infused/V-injected (CP-2.5/V; solid light gray bars); (4) low-dose CP-infused/INS-injected (CP-2.5/INS; diagonal-striped light gray bars); (5) high-dose (10.0 mg) CP-infused/V-injected (CP-10.0/V; solid dark gray bars); (6) high-dose CP-infused/INS-injected (CP-10.0/INS; diagonal-striped dark gray bars). *p < 0.05; **p < 0.01; ***p < 0.001

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Table 1 Summary of graded intracerebroventricular (icv) CP-319,819 (CP) dosage effects on ventromedial hypothalamic nucleus (VMN) glycogen/glucose content and metabolic neuron 5′-AMP-activated kinase (AMPK) activity and neurotransmitter protein expression during eu- versus hypoglycemia
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Data in Fig. 2 illustrate how hypoglycemic site-specific adjustments in tissue free glucose levels are impacted by CP pretreatment. As shown in panel a, VMN glucose content was significantly decreased in the V/INS group [F(5,12) = 7.38; p = 0.0003]. Pretreatment with CP-2.5 or CP-10.0 doses correspondingly elevated or had no impact on glucose levels; INS injection to CP-pretreated rats reduced VMN tissue glucose (Table 1). Panels b (ARH [F(5,12) = 7.24; p = 0.0004]), c (LHA [F(5,12) = 14.30; p < 0.0001]), and d (DMN [F(5,12) = 12.09; p < 0.0001]) show that hypoglycemia also suppressed glucose levels in the ARH, LHA, and DMN. Baseline ARH and LHA, but not DMN tissue glucose was elevated by low-dose CP pretreatment, whereas high-dosage delivery diminished basal LHA and DMN glucose. CP-2.5 pretreatment prevented hypoglycemia-associated suppression of tissue free glucose in the ARH and DMN. ARH, LHA, and DMN glucose levels were refractory to hypoglycemia following CP-10.0 administration.

Fig. 2
figure 2

a–d Effects of icv CP pretreatment on hypoglycemic microdissected tissue glucose content in hypoglycemic male rats. *p < 0.05; **p < 0.01; ***p < 0.001

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Results presented in Fig. 3 and summarized in Table 1 show effects of pretreatment with graded CP doses on hypoglycemic patterns of GAD65/67, Fos, AMPK, and pAMPK protein expression in laser-microdissected VMN GAD65/67-ir neurons. Results in panels a and b indicate that GABAergic nerve cell GAD65/67 [F(5,12) = 5.71; p = 0.002] and Fos [F(5,12) = 10.25; p = 0.0002] protein profiles were both significantly increased in response to IIH. GAD nerve cell AMPK (panel c [F(5,12) = 4.24; p = 0.01]) and pAMPK (panel d [F(5,12) = 6.12; p = 0.003]) levels were correspondingly unaffected or augmented in V/INS versus V/V groups. Low-dose CP pretreatment elevated baseline GAD65/67 and pAMPK protein expression [CP-2.5/V versus V/V], whereas the high drug dosage did not affect any protein examined [CP-10.0/V versus V/V]. After CP-2.5 pretreatment, IIH significantly diminished GAD65/67, Fos, and pAMPK expression relative to baseline [CP-2.5/INS versus CP-2.5/V]. Conversely, animals pretreated with CP-10.0 exhibited no difference in GAD65/67, Fos, or pAMPK protein profiles in INS- versus V-injected groups [CP-10.0/INS versus CP-10.0/V]. These data show that low CP dose caused IIH-reversible augmentation of VMN GABA neuron AMPK activity and GAD65/67 protein expression, while the higher dose abolished hypoglycemic augmentation of these profiles.

Fig. 3
figure 3

Effects of CP pretreatment on IIH patterns of glutamate decarboxylase65/67 (GAD65/67), Fos, 5′-adenosine monophosphate-activated protein kinase (AMPK), and phosphoAMPK (pAMPK) protein expression in VMN GABAergic neurons. GAD65/67-immunoreactive neurons located in the right hemi-hypothalamic VMN were laser-catapult microdissected for stain-free Western blot analysis of GAD65/67 (panel a), Fos (panel b), AMPK (panel c), and pAMPK (panel d) protein expression in V- or CP-pretreated eu- versus hypoglycemic male rats. For each protein of interest, triplicate pools of n = 50 heat-denatured cell lysates were created within each treatment group. Target protein band optical densities (O.D.) were detected and quantified in a Bio-Rad ChemiDoc MP Imaging System equipped with Image Lab™ software; target protein O.D. values were normalized to total protein content of individual lanes. Data depict mean normalized protein O.D. values + S.E.M. for groups of animals pretreated by icv V or CP (2.5 or 10.0 mg/24 h) infusion prior to sc INS or V injection. *p < 0.05; **p < 0.01; ***p < 0.001

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Figure 4 depicts effects of hypoglycemia with or without CP pretreatment on VMN SF-1 nerve cell SF-1 (panel a [F(5,12) = 10.24; p = 0.0001]), Fos (panel b [F(5,12) = 31.67; p < 0.0001]), AMPK (panel c [F(5,12) = 10.65; p < 0.0001]), and pAMPK (panel d [F(5,12) = 15.46; p < 0.0001) protein expression. Data show that SF-1 protein profiles were refractory to IIH, whereas pAMPK protein levels were increased. Low-dose CP pretreatment suppressed basal SF-1, Fos, AMPK, and pAMPK expression; none of these profiles was modified in response to INS injection [CP-2.5/INS versus CP-2.5/V]. Pretreatment with CP-10.0 also diminished Fos, AMPK, and pAMPK protein levels, but SF-1 expression was not different from V-pretreated controls [CP-10.0/V versus V/V]. The CP-10/INS group exhibited no change in any protein profile compared to CP-10.0/V animals. Outcomes indicate that CP pretreatment either decreased (low CP dose) or had no impact (high CP dose) on VMN SF-1 nerve cell SF-1 transmitter protein expression, and that both dosages averted hypoglycemic activation of AMPK in these neurons.

Fig. 4
figure 4

Hypoglycemic patterns of steroidogenic factor-1 (SF-1), Fos, 5′-AMPK, and pAMPK protein expression in male rat VMN SF-1 neurons; impact of graded CP dosage pretreatment. Laser-catapult microdissected VMN SF-1-immunoreactive neurons were evaluated by stain-free Western blotting to determine effects of CP on SF-1 (panel a), Fos (panel b), AMPK (panel c), and pAMPK (panel d) protein responses to IIH. Data depict mean normalized protein O.D. values + S.E.M. for V/V (solid white bars), V/I (diagonal-striped white bars), CP-2.5/V (solid light gray bars), CP-2.5/INS (diagonal-striped light gray bars), CP-10.0/V (solid dark gray bars), and CP-10.0/INS (diagonal-striped dark gray bars) treatment groups. *p < 0.05; **p < 0.01; ***p < 0.001

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Data in Fig. 5 illustrate hypoglycemic patterns of VMN nitrergic nerve cell protein expression in male rats pretreated with CP or vehicle. Results indicate that nNOS (panel a [F(5,12) = 33.57; p < 0.0001]), Fos (panel b [F(5,12) = 14.09; p < 0.0001]), AMPK (panel c [F(5,12) = 33.90; p < 0.0001]), and pAMPK (panel d [F(5,12) = 14.49; p < 0.0001]) protein profiles were unaffected by IIH. Low-dose CP pretreatment elevated baseline expression of each measured protein. IIH amplified nNOS expression in CP-2.5- versus vehicle-pretreated groups, while suppressing Fos, AMPK, and pAMPK profiles [CP-2.5/INS versus CP-2.5/V]. High-dose CP further increased baseline nNOS profiles related to CP-2.5 rats [CP-10.0/V versus CP-2.5/V], stimulated Fos protein expression to a magnitude equivalent to the CP-2.5 group, but did not modify baseline AMPK or pAMPK protein expression [CP-10.0/V versus V/V]. CP pretreatment caused hypoglycemic intensification (CP-2.5) or inhibition (CP-10.0) of nNOS expression relative to CP-2.5/V and CP-10.0 groups, respectively. IIH decreased Fos protein levels in both CP pretreatment groups. Nitrergic neuron AMPK and pAMPK levels were both reduced in CP-2.5/INS versus CP2.5/V groups. In contrast, CP-10.0 pretreatment resulted in hypoglycemic repression of pAMPK, but not AMPK levels compared to baseline [CP-10.0/INS versus CP-10.0/V]. Results show that CP caused dose-proportionate augmentation of nNOS protein expression in euglycemic animals, and either enhanced (low dose) or repressed (high dose) this protein profile during hypoglycemia. IIH decreased pAMPK protein expression below basal levels in CP-10.0, but not CP-2.5-pretreated rats.

Fig. 5
figure 5

Effects of icv CP infusion on VMN nitrergic nerve cell neuron nitric oxide synthase (nNOS), Fos, 5′-AMPK, and pAMPK protein responses to IIH. VMN nNOS-immunoreactive neurons were laser-microdissected for stain-free Western blot analysis of nNOS (panel a), Fos (panel b), AMPK (panel c), and pAMPK (panel d) protein expression. Data depict mean normalized protein O.D. values + S.E.M. for the following treatment groups: V/V, V/I, CP-2.5/V, CP-2.5/INS, CP-10.0/V, and CP-10.0/INS. *p < 0.05; **p < 0.01; ***p < 0.001

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Figure 6 illustrates effects of CP pretreatment on circulating glucose, glucagon, and corticosterone concentration at 4 h after INS injection of male rats. As shown in panel a, blood glucose levels were significantly decreased to an equivalent extent among INS groups irrespective of pretreatment [F(5,12) = 25.90; p < 0.0001]. Plasma glucagon levels were significantly increased in vehicle-pretreated hypoglycemic animals [V/INS versus V/V; F(5,12) = 10.85; p = 0.001]. CP pretreatment either elevated (low-dose CP) or had no impact (high-dose CP) on circulating levels of this hormone in euglycemic animals. INS injection of CP-pretreated rats either decreased glucagon secretion relative to baseline [CP-2.5/INS versus CP-2.5/V] or did not modify hormone release [CP-10.0/INS versus CP-2.5/V]. Plasma corticosterone levels did not vary among treatment groups [F(5,12) = 3.52; p < 0.07]. These results indicate that CP exerts dose-dependent effects on basal and hypoglycemic patterns of glucagon, including normalization of glucagon secretion after high-dosage pretreatment. On the other hand, CP pretreatment did not modify corticosterone secretion.

Fig. 6
figure 6

Effects of CP pretreatment on glycemic and glucagon and corticosterone secretory responses to INS injection. Data depict mean circulating glucose (panel a), glucagon (panel b), and corticosterone (panel c) levels + S.E.M. in groups of vehicle- or CP-pretreated eu- versus hypoglycemic male rats. *p < 0.05; **p < 0.01; ***p < 0.001

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Discussion

Current research utilized combinatory micropunch dissection/HPLC/mass spectrometric and immunocytochemistry/laser-catapult microdissection/Western blot techniques to examine the hypothesis that male rats treated by intracerebroventricular infusion of the GP inhibitor CP-316,819 (CP), with the goal of increasing VMN glycogen mass, will exhibit diminished VMN gluco-stimulatory and/or enhanced gluco-inhibitory transmitter signaling during eu- and/or hypoglycemia, alongside reduced AMPK activation. Data show that CP caused dose-dependent expansion of glycogen content of the VMN and other hypothalamic metabolic loci, and that high-dose CP allowed further enhancement of this fuel depot during IIH. In each location, excluding the VMN, hypoglycemia-associated suppression of tissue free glucose levels was averted by the higher drug dose. Results provide unique evidence for metabolic sensor expression in VMN GABA, SF-1, and nitrergic neurons, and suggest that these cells may monitor distinguishing correlates of neurometabolic instability. Among laser-microdissected VMN neurotransmitter cell groups investigated here, hypoglycemic activation of AMPK occurred in only GABA and SF-1 cells and coincided with altered transmitter signaling in the former cell population alone. Results show that VMN glycogen augmentation correlates with attenuated hypoglycemic upregulation of GAD and SF-1 neuron AMPK activity and GABA signaling, as well as diminished nitrergic cell AMPK activity during IIH. These outcomes infer that expansion of the glycogen fuel reservoir can enhance VMN gluco-regulatory nerve cell energy stability and impact neurotransmission by one or more distinctive populations during this metabolic stress. Further studies are needed to investigate VMN metabolic transmitter involvement in CP-induced suppression of hypoglycemic hyperglucagonemia.

HPLC/mass spectrometric data analysis shows that tissue glycogen levels in distinctive micropunch-dissected hypothalamic metabolic loci, including the VMN, were equivalent in hypoglycemic versus euglycemic male rats 4 h after INS injection, whereas free D-glucose levels were significantly reduced in each location. Discrepancies between current outcomes versus earlier reports of hypoglycemia-associated hypothalamic glycogen depletion likely reflect, in part, differences in analytical methodology, including the high-resolution tissue dissection approach used here for assessment of discrete hypothalamic nuclei/areas versus whole hypothalamus and utilization of HPLC as opposed to fluorescence spectroscopy, and/or timing of sacrifice after INS therapy. As work here focused on a single time point after induction of IIH, it is possible that in one or more hypothalamic loci investigated here, patterns of glycogen mobilization may vary over time after INS administration and diminution of this astrocyte energy reserve may commence in some locations after more prolonged exposure to hypoglycemia. It also should be noted that equivalent measures of glycogen content may mask differences in glycogen turnover and liberation of glucosyl units for nerve cell utilization. Current data disclose dosage-dependent effects of CP on pre-hypoglycemic glycogen amassment, with significant augmentation occurring in each hypothalamic structure after provision of the higher, e.g., 10.0 mg, dose. Hypoglycemia-associated adjustments in tissue glycogen were inverse in direction in CP-2.5- versus CP-10.0-pretreated males, as the former group showed a decline in the VMN and LHA glycogen, whereas the latter exhibited glycogen augmentation in each location after INS injection. We interpret site-specific glycogen diminution in the CP-2.5/INS group as an indicator of breakdown of this reserve; yet, an alternative but more remote prospect is that glycogen turnover may be equivalent to CP-2.5/V rats due to GS downregulation. Further expansion of glycogen mass in CP-10.0/INS versus CP-10.0/V animals may signify amplified inhibition of GP and/or intensified GS enzyme activity by as-yet-unidentified mechanisms. Our presumption that GP enzyme activity was inhibited under current experimental conditions, albeit in a CP dosage-dependent manner, was not confirmed as methods of requisite sensitivity for measurement of activity in small volume tissue samples, such as those utilized here, are currently unavailable. Data disclose that CP-2.5 elevated basal D-glucose levels in the VMN, ARH, and LHA, but not DMN. CP-10.0 pretreatment did not alter VMN or ARH glucose content in euglycemic animals, but significantly diminished LHA and DMN tissue glucose levels. The latter site-specific reductions in glucose may reflect heightened incorporation of this monomer into glycogen, intensified glucose utilization in energy and non-energy pathways, and/or decreased glucose uptake from the circulation. Indeed, it is unclear if and how local reductions in hypothalamic glycogen turnover may impact rates of glucose acquisition into distinctive neural structures. Whereas low-dose CP pretreatment prevented IIH-associated diminution of glucose levels in the ARH and DMN, the higher drug dose stabilized glucose levels in each structure, excepting the VMN, during hypoglycemia. These outcomes support the need for further research to investigate potential therapeutic value of preservation of neural tissue steady-state glucose equilibrium.

GABA neurotransmission in the male rat ventromedial hypothalamus is viewed as a signal of energy sufficiency as local GABA concentrations are elevated alongside diminished counter-regulatory hormone, e.g., glucagon and epinephrine secretion, and this hormone outflow is inhibited by GABA receptor blockade (Chan et al. 2006; Zhu et al. 2010). Accordingly, our prediction here was that IIH would suppress VMN GAD65/67 protein expression. Yet, data here show that this protein profile was increased in GAD-immunoreactive (-ir) neurons harvested 4 h post-INS injection. These contrary outcomes are likely due, in part, to differential neuroanatomical-level resolution of dissection techniques and analytical endpoints, namely HPLC analysis of mediobasal hypothalamic GABA neurochemical content versus Western blot analysis of GABA biosynthetic enzyme protein expression in VMN GAD65/67 neurons. The current approach affords discriminative focus on VMN GABA neurons, separate from those located in the neighboring ARH, which justifiable in light of disparities between VMN and ARH pertaining to neurotransmitter cytoarchitecture, neuroanatomical connectivity within the brain, and receptivity to nutrient and endocrine signals of metabolic stability/instability. On the other hand, it should be considered that measures of GAD65/67 protein profiles may not be an accurate indicator of GABAergic transmission over the duration of IIH. The possibility remains that VMN GABAergic cells comprise a distinctive subset of mediobasal hypothalamic GABA neurons that is uniquely activated over the duration of hypoglycemia or at specific time points after onset.

Current work provides novel proof that IIH causes transcriptional activation, e.g., increased Fos expression in VMN GABA neurons, along with upregulated pAMPK protein profiles, indicating that signaling under current hypoglycemic conditions is likely prompted by energy deficiency. The CP-2.5, but not CP-10.0 dosage stimulated AMPK activity and Fos and GAD65/67 protein expression in these cells in euglycemic rats. These data infer that putative heightened VMN GABA release in the CP-2.5/V group may be driven by diminished energy stability, plausibly due to reduced glycogen mobilization and associated generation of L-lactate, the oxidizable end-product of astrocyte glycolysis, rather than glucose shortage since tissue glucose levels were equivalent to V/V controls. INS injection of CP-2.5-pretreated animals reversed the above responses, suggesting that glycogen breakdown correlates with improved GABA cell energy stability. Unlike the lower CP dose, the higher drug dose did not alter baseline GAD65/67, Fos, or pAMPK expression in GABA neurons and, moreover, prevented adjustments in these profiles during hypoglycemia. These results infer that drug-enhanced glycogen accumulation may amplify GABA neuron-positive energy state versus CP-2.5/V rats and, moreover, prevent energy imbalance in these cells during hypoglycemia. The possible mechanisms that mediate this putative dose-specific neuroprotective treatment effects on GABA transmission, including beneficial effects of normalized VMN tissue glucose content, require further investigation.

Present data provide novel evidence for hypoglycemic activation of AMPK in laser-microdissected VMN SF-1-ir, whereas Fos and SF-1 protein profiles were unchanged. SF-1 refractivity to IIH may reflect an AMPK-mediated permissive rather than active impact of this neurotransmitter signal on neural outflow at 4 h after initiation of this metabolic stress. CP-2.5/V animals showed a decline in AMPK, pAMPK, Fos, and SF-1 profiles, whereas the CP-10.0/V group exhibited reductions in the aforementioned proteins excepting the latter. Current work does not illuminate the mechanisms by which CP reduces baseline total and phosphorylated AMPK and Fos protein expression in SF-1 neurons. CP-mediated deterrence of hypoglycemic augmentation of pAMPK expression implies that downregulated sensor function and/or stimulus-transcription coupling may afford, by means as-yet-unknown, protection against energetic instability in VMN SF-1 neurons during IIH.

Outcomes show that putative gluco-stimulatory VMN nitrergic neurons displayed no change in AMPK, pAMPK, Fos, or nNOS protein expression in response to hypoglycemia, implying that nitric oxide signaling under current circumstances may reflect cellular energy stability despite decreased blood glucose levels. CP pretreatment elicited dose-proportionate augmentation of nNOS protein levels in nNOS-ir neurons, results that align with prior evidence that pharmacological inhibition of VMN GP by 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) increases VMN nNOS expression [Alhamami et al. 2018]. Parallel effects of graded CP on neuronal nNOS protein and VMN glycogen levels in euglycemic rats imply that incremental augmentation of NO signaling, which correlates with increased Fos-activated genomic activity, may reflect, in part, magnitude of shortage of glycogen-derived substrate fuel stream and/or degree of expansion of glycogen mass. Nitrergic nerve cell AMPK and pAMPK protein profiles were increased in parallel by low-dose CP, whereas both proteins were refractory to the higher CP dosage, inferring that drug-associated augmentation of euglycemic nNOS signaling may be instigated by AMPK-independent cues of energy imbalance. Contrary to vehicle pretreatment, CP-pretreated animals showed bi-directional, dose-specific changes in nitrergic cell nNOS protein expression during IIH, adjustments that were inverse in direction to hypoglycemia-associated diminution (low-dose CP) versus augmentation (high-dose CP) of VMN glycogen content. While AMPK and pAMPK proteins were both decreased in CP-2.5/INS versus CP-2.5/V groups, pAMPK, but not AMPK profiles were reduced by IIH in CP-10.0/INS compared to CP-10.0/V. These data imply that hypoglycemic patterns of NO release following glycogen amassment correspondingly reflect AMPK-insensitive negative versus AMPK-sensitive positive change in energy state in low- versus high-dose-pretreated groups. The factors that may increase nitrergic neuron energy instability despite glycogen mobilization in CP-2.5/INS animals remain to be elucidated.

Circulating glucose concentrations were similar in CP- versus V-pretreated euglycemic animals and were decreased to a similar extent among INS treatment groups. CP pretreatment either augmented (low dose) or had no impact (high dose) on basal plasma glucagon concentrations. Circulating levels were elevated at + 4 h of IIH; CP/INS animals exhibited either a reduction (low dose) or no change (high dose) in this hormone profile compared to their matched CP/V group. Plasma corticosterone levels did not differ significantly between treatment groups at the time point evaluated here. Maintenance of euglycemia despite hyperglucagonemia in CP-2.5/V animals suggests that augmented glucagon release may be inadequate to elevate blood glucose levels above the physiological range, or alternatively, that potential stimulatory effects may be offset by simultaneous CP-initiated mechanisms that reduce this metabolic fuel profile in the circulation. Similarly, normalized glucagon output in hypoglycemic CP-pretreated rats did not exacerbate glucose decrements, implying that other counter-regulatory functions may be correspondingly enhanced. Parallel adjustments in VMN GAD65/67 protein expression and glucagon secretion across treatment groups support the intriguing possibility that local GABA signaling may be a key positive effector of glucagon responses to IIH at this time point.

The singular focus of this project on a 4-h post-INS injection interval does not preclude the possibility that one or more VMN neurotransmitter neuron populations investigated here may exhibit discrepant AMPK and/or biosynthetic enzyme/neurotransmitter responses at time points prior to or after + 4 h of IIH. This notion is bolstered by evidence that unlike current outcomes, whole VMN GAD65/67 and nNOS protein profiles are respectively down- or upregulated 1 h after INS treatment (Ali et al. 2019; Napit et al. 2019). While the status of VMN GABA or nitrergic nerve cell AMPK activity at that earlier time point was not assessed, prospective temporal variations in GABA and NO transmission after IIH onset may likely reflect, in part, dynamic changes in metabolic fuel stream, including glycogen-derived lactate, cellular adaptations to diminished substrate supply, and volume of signaling by hormonal surrogates of peripheral energy reserves. Current outcomes using graded icv CP dosages provide novel proof that CP-associated pre-hypoglycemic glycogen augmentation correlates with diminished AMPK activation IIH, supporting glycogen conferral of neuroprotection against neurometabolic instability. Implementation of an expanded CP dose range may aid in the identification of cellular and molecular mechanisms that mediate drug effects on VMN tissue glycogen and glucose content under eu- versus hypoglycemic conditions and those that underlie VMN metabolic sensory neuron reactivity to glycogen metabolism.

In summary, present studies show that icv CP infusion promotes dose-dependent site-specific augmentation of pre-hypoglycemic glycogen content in the male rat hypothalamus, as well as further expansion of this energy reserve during ensuing IIH. CP also caused dose-contingent deterrence of hypoglycemic diminution of tissue glucose levels in multiple hypothalamic loci. Outcomes provide novel proof of AMPK expression in multiple VMN gluco-regulatory nerve cell populations, but that hypoglycemic activation of this sensor is restricted to GABA and SF-1 neurons and coincides with altered GABA, but not SF-1 signaling. CP-pretreated euglycemic rats exhibited dose-dependent divergent adjustments in GAD65/67 and SF-1 protein profiles and dose-proportionate augmentation of nNOS protein. In hypoglycemic rats, CP pretreatment reversed (low dose) or prevented (high dose) AMPK activation and GAD65/67 expression in GABA neurons, prevented AMPK activation in SF-1 cells, and either stimulated (low dose) or inhibited (high dose) nNOS protein and decreased pAMPK profiles (high dose) in nitrergic neurons. Current evidence that high CP dose-augmented VMN glycogen amassment correlates with diminished gluco-regulatory nerve cell AMPK activation during IIH suggests that expansion of this fuel reservoir enhances cellular energy stability during this metabolic stress. Further research is needed to ascertain if this neuroprotection of neurometabolic state involves provision of glycogen-derived substrate fuel, despite increased glycogen accumulation, and/or astrocyte glio-transmitter upregulation of nerve cell energy metabolism involving alternative metabolic fuels.