Ventromedial hypothalamic nucleus glycogen regulation of metabolic-sensory neuron AMPK and neurotransmitter expression: role of lactate

Abstract

Astrocyte glycogen is dynamically remodeled during metabolic stability and provides oxidizable l-lactate equivalents during neuroglucopenia. Current research investigated the hypothesis that ventromedial hypothalamic nucleus (VMN) glycogen metabolism controls glucostimulatory nitric oxide (NO) and/or glucoinhibitory gamma-aminobutyric acid (GABA) neuron 5’-AMP-activated protein kinase (AMPK) and transmitter marker, e.g., neuronal nitric oxide synthase (nNOS), and glutamate decarboxylase_65/67 (GAD) protein expression. Adult ovariectomized estradiol-implanted female rats were injected into the VMN with the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) before vehicle or l-lactate infusion. Western blot analysis of laser-catapult-microdissected nitrergic and GABAergic neurons showed that DAB caused lactate-reversible upregulation of nNOS and GAD proteins. DAB suppressed or increased total AMPK content of NO and GABA neurons, respectively, by lactate-independent mechanisms, but lactate prevented drug enhancement of pAMPK expression in nitrergic neurons. Inhibition of VMN glycogen disassembly caused divergent changes in counter-regulatory hormone, e.g. corticosterone (increased) and glucagon (decreased) secretion. Outcomes show that VMN glycogen metabolism controls local glucoregulatory transmission by means of lactate signal volume. Results implicate glycogen-derived lactate deficiency as a physiological stimulus of corticosterone release. Concurrent normalization of nitrergic neuron nNOS and pAMPK protein and corticosterone secretory response to DAB by lactate infers that the hypothalamic-pituitary-adrenal axis may be activated by VMN NO-mediated signals of cellular energy imbalance.

INTRODUCTION

Astrocytes support neurometabolic stability by way of uptake, storage, and metabolism of glucose, the primary energy source to the brain. Glucose acquired by astrocytes is converted to the oxidizable substrate fuel l-lactate (1), which is transferred to neurons by cell type-specific monocarboxylate transporters (2). The astrocyte glycogen shunt, which involves sequential glucose incorporation into and liberation from this complex carbohydrate polymer before entry into the glycolytic pathway, is an active process that accounts for a significant fraction of glucose catabolism in these glia (3, 4). Astrocyte glycogen mass is dynamically remodeled during normal metabolic homeostasis and is a vital source of lactate equivalents during states of heightened neurological activity or glucose deficiency (5).

The hypothalamus controls autonomic and neuroendocrine motor outflow that governs glucose counterregulation (6). The ultrasensitive, evolutionarily conserved energy gauge 5'-AMP-activated protein kinase (AMPK) is activated via phosphorylation in response to increases in AMP/ATP ratio (7, 8). Hypothalamic AMPK provides crucial input on brain cell ATP availability to neural pathways that regulate whole body energy stability (9–11). There, AMPK integrates diverse metabolic and endocrine indicators of energy paucity (ghrelin, corticosterone, thyroxine, adiponectin) or excess (glucose, leptin, insulin) (12) to control food intake, glucose homeostasis, energy expenditure, and body weight (13). Activation of mediobasal hypothalamic (MBH) AMPK is obligatory for optimum glucose counterregulatory responses to insulin-induced hypoglycemia (14, 15). The ventromedial hypothalamic nucleus (VMN), a prominent neuroanatomical constituent of the MBH, is a likely source of AMPK glucoregulatory signaling as hypoglycemia increases AMPK phosphorylation in VMN neurons that express characterized glucoinhibitory (γ-aminobutyric acid; GABA) and glucostimulatory (nitric oxide; NO) neurotransmitters (16, 17).

Lactate is a critical monitored metabolic variable in the MBH, as exogenous infusion of this substrate fuel to this area normalizes counterregulatory outflow in hypoglycemic rats (18). Current research investigated the hypothesis that VMN glycogen metabolism influences local nitrergic and/or GABAergic nerve cell metabolic stability and neurotransmission. Here, the glycogen phosphorylase (GP) inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) was used as a pharmacological tool, in conjunction with combinatory immunocytochemistry/single-cell laser-catapult microdissection and high-sensitivity Western blot techniques, to determine if diminished VMN glycogen disassembly causes lactate-reversible changes in AMPK activity and transmitter marker protein expression in one or both of these neuron populations.

MATERIALS AND METHODS

Animals

Adult female Sprague–Dawley rats (2–3 mo of age) housed in shoe box cages (2–3 per cage), under a 14-h light/10-h dark lighting schedule (lights on at 05.00 h). Animals were allowed ad libitum access to standard laboratory rat chow (Harlan Teklad LM-485; Harlan Industries, Madison, WI) and tap water and were acclimated to daily handling before experimentation. All animal protocols were conducted in accordance with NIH Guide for Care and Use of Laboratory Animals, Eighth Edition, under approval by the ULM IACUC committee. On study day 1, rats were anesthetized with ketamine/xylazine (0.1 mL/100 g body wt; 90 mg ketamine:10 mg xylazine/mL; Covetrus, Portland, ME), and with a 26-gauge double stainless-steel cannula guide (prod no. C235G-1.2/SPC; Plastics One, Inc., Roanoke, VA) aimed at the VMN [coordinates: 2.85 mm posterior to bregma; 0.6 mm lateral to midline; 9.0 mm below skull surface (19)] by automated stereotaxic surgery. Anesthetized animals were also bilaterally ovariectomized (OVX) and implanted with a subcutaneous (sc) capsule (10 mm/100 g body wt; 0.062 in. i.d., 0.125 in. o.d.) containing 30 µg 17β-estradiol-3-benzoate/mL safflower oil. This steroid replacement regimen yields approximate plasma estradiol concentrations of 22 pg/mL (20), which replicate circulating hormone levels characteristic of 4-day estrous cycle metestrus in ovary-intact female rats (21). After surgery, rats were injected with enrofloxacin (Enroflox 2.27%; 10 mg/kg bw im) and ketoprofen (3 mg/kg body wt sc), then transferred to individual cages.

Experimental Design

Animals were divided into three groups (n = 4 rats/group). At 0.900 h on day 7, fed rats were injected using a double internal injection cannula (C235I-1.2/SPC; Plastics One; 1.0-mm projection) with vehicle (V) alone [sterile 0.9% saline (SAL); group 1] or DAB [150 pM (22)] in SAL, in a total volume of 0.5 µL, at a rate of 0.25 µL/min over a 2-min period. After treatment, the internal cannula was kept in place for 60 s. Beginning at 9.10 h, animals were infused with V (groups 1 and 2) or l-lactate [100 nM; group 3 (22)], in an infusion volume and rate similar to that described above. Rats were sacrificed at 10:00 h for brain and blood collection. The current experimental design evaluated protein expression in VMN metabolic-sensory neurons harvested from brains collected 1 h after intra-VMN administration of the glycogen phosphorylase inhibitor DAB. This time point was selected based upon prior work that demonstrated noradrenergic regulation of VMN metabolic-sensory nerve cell transmitter marker protein profiles and energy sensor activity, within that timeframe, involves control of astrocyte glycogen metabolic enzyme expression and l-lactate signal volume (23). Each brain was snap-frozen in liquid nitrogen-cooled isopentane for storage at −80°C, while plasma was stored at −20°C.

VMN Glucoregulatory Neuron Laser-Catapult Microdissection and Western Blotting

A series of 10-μm-thick frozen sections was cut over the length of the VMN and mounted on polyethylene naphthalate membrane-coated slides (Carl Zeiss Microscopy, LLC, White Plains, NY). After acetone fixation, washing with phosphate-buffered saline, pH 7.5, containing 0.1% Tween 20, and blocking with 2.5% normal horse serum, tissues were incubated for 48 h at 4°C with primary antisera against glutamate decarboxylase_65/67 (GAD) (prod. no. ABN904, 1:1,000; Millipore Sigma, Burlington, MA) or neuronal nitric oxide synthase (nNOS) (prod. no. NBP1-39681, 1:1,000; Novus Biologicals, LLC, Littleton, CO), as described (24). Sections were next incubated with ImmPRESS Universal PLUS polymer kit horse anti-mouse/anti-rat secondary antibodies (prod. no. MP-7800; Vector Laboratories, Burlingame, CA). GAD- or nNOS-immunoreactive (ir) neurons were visualized using ImmPACT DAB EqV peroxidase substrate kit reagents (prod. no. SK-4103; Vector Lab.). For each animal, numbers of VMN nNOS- or GAD-ir neurons per tissue section were averaged from cell counts made from sections obtained at rostral, middle, and caudal levels of the VMN; mean counts of each cell population were established for each treatment group. Immunolabeled neurons were individually dissected from sections using a Zeiss P.A.L.M. UV-A microlaser IV and collected into lysis buffer (2.0% sodium dodecyl sulfate, 0.05 M dithiothreitol, 10.0% glycerol, 1.0 mM EDTA). For each treatment group, cell lysates from individual subjects were combined to create triplicate sample pools (n = 50 NO or GABA nerve cell lysates per pool) for each target protein before separation on BioRad TGX 10% stain-free gels (prod. no. 1610183, Bio-Rad Laboratories Inc., Hercules CA). After electrophoresis, gels were UV light-activated (1 min) in a Bio-Rad ChemiDoc TM Touch Imaging System before transblotting (30 V, overnight at 4°C; Towbin buffer) to 0.45-μm PVDF membranes (ThermoFisherScientific; Waltham, MA) (25). After blocking with Tris-buffer saline (TBS), pH 7.4, containing 0.1% Tween-20 and 2% bovine serum albumin, membranes were incubated overnight (4°C) with rabbit primary antibodies against AMPKα_1/2 (prod. no. 2532S; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA), phosphoAMPKα_1/2 (pAMPK; prod. no. 2535S; 1:2,000; Cell Signaling Technology, Inc.), GAD (prod. no. ABN904, 1:10,000; Millipore Sigma), or nNOS (prod. no. NBP1-39681, 1:2,000; Novus Biol.). Membranes were then incubated with peroxidase-conjugated goat anti-rabbit (prod. no. NEF812001EA, 1:5,000; PerkinElmer, Waltham, MA) secondary antibodies before exposure to Supersignal West Femto Maximum Sensitivity Substrate (prod. no. 34096; ThermoFisher Scientific, Rockford, IL). Membrane washes and antibody incubations were performed using Freedom Rocker Blotbot automation (Next Advance, Inc., Troy NY). Chemiluminescence band optical density (O.D.) values for target proteins were normalized to total protein quantified in the sample lane, e.g., the lane in which that protein was electrophoresed, using Bio-Rad proprietary stain-free imaging gel technology and ChemiDoc MP instrumentation with Image Lab 6.0.0 software, as described at http://www.bio-rad.com/en-us/applications-technologies/stain-free-imaging-technology?ID=NZ0G1815. This superior method for Western blot normalization distinctly diminishes data variability through improved measurement accuracy and precision (26, 27). Precision plus protein molecular weight dual color standards (prod. no. 161-0374, Bio-Rad) were included in each Western blot analysis.

Plasma Glucose, Counterregulatory Hormone, and Free Fatty Acid Analyses

Circulating glucose levels were determined using an ACCU-CHEK guide glucometer (Roche Diagnostic Corporation, Indianapolis, IN) (28). 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, as described (22, 28). Free fatty acid (FFA) levels were analyzed as previously reported (prod. no. MAK044; Sigma Aldrich, St. Louis, MO) (22, 28).

Statistics

Mean normalized protein O.D. measures, mean plasma glucose, hormone, and FFA values, and mean nNOS- or GAD-ir-positive neurons were analyzed by one-way analysis of variance and Student–Newman–Keuls post hoc test. Differences of P < 0.05 were considered significant.

RESULTS

Figure 1 depicts combinatory VMN nitrergic and GABAergic neuron immunocytochemical labeling and laser-catapult microdissection. Individual nerve cells in VMH tissue sections were identified by nNOS- (1A) or GAD- (1B) immunoreactivity (-ir) before laser-catapult harvesting (left-hand column); representative labeled neurons are indicated by blue arrows. Middle and right-hand columns illustrate actions, including sequential positioning of a continuous laser cut (shown in green in 1B and 2B) surrounding individual nerve cells that result in separate removal of each neuron without destruction of surrounding tissue and minimal inclusion of adjacent tissue (1C and 2C). Data presented in Fig. 2 indicate that mean numbers of nNOS- (Fig. 2A) or GAD-ir (Fig. 2B) VMN neurons were not different between treatment groups.

Figure 1.

Laser-catapult microdissection of immunolabeled ventromedial hypothalamic nucleus (VMN) nitric oxide (NO) or γ-aminobutyric acid (GABA) neurons. VMN neurons were identified in situ for neuronal nitric oxide (nNOS)- [top row; 1A] or glutamate decarboxylase_65/67 (GAD)- [bottom row; 2A] immunoreactivity (-ir); representative nNOS- or GAD-ir-positive neurons are indicated by blue arrows. Areas shown in 1A and 2A were rephotographed after positioning of a continuous laser track (depicted in green) around individual nNOS-ir [1B; blue arrow] or GAD-ir neurons [2B; blue arrow] and subsequent ejection of each cell by laser pulse [1C and 2C]. Note that this microdissection technique causes negligible destruction of surrounding tissue and minimal inclusion of adjacent tissue.

Figure 2.

A and B: comparison of mean VMN neuronal nitric oxide synthase (nNOS)- or glutamate decarboxylase_65/67 (GAD)-immunopositive nerve cell numbers among treatment groups. For each animal, numbers of nNOS- or GAD-ir neurons per tissue section were averaged from counts made at rostral, middle, and caudal levels of the VMN; mean values for each cell population were established for each treatment group. Statistical analysis of mean treatment group cell counts revealed no significant effects of DAB with or without l-lactate on numbers of demonstrable neurons in either nerve cell type. DAB, 1,4-dideoxy-1,4-imino-d-arabinitol; ir, immunoreactivity; L, l-lactate; V, vehicle; VMN, ventromedial hypothalamic nucleus.

Figure 3 depicts the impact of intra-VMN delivery of DAB on nitrergic neuron nNOS (Fig. 3A), AMPK (Fig. 3B), and pAMPK (Fig. 3C) protein expression. Results show that DAB treatment caused significant upregulation of nNOS content compared with controls [DAB/V vs. V/V], and that this stimulatory effect was abolished by l-lactate administration to the VMN [F(2,6) = 34.87; P < 0.0001]. NO nerve cell AMPK content was decreased to a similar extent in DAB/V and DAB/lactate treatment groups below mean control values [F(2,6) = 5.71; P = 0.013]. DAB stimulation of pAMPK protein levels in these neurons was averted by lactate [F(2,6) = 11.31; P = 0.0007]. The mean nitergic nerve cell pAMPK/AMPK ratio (Fig. 3D) was significantly elevated following DAB treatment but was normalized in animals exposed to DAB plus lactate [F(2,6) = 6.02; P = 0.009].

Figure 3.

Effects of l-lactate on patterns of nNOS, AMPK, and pAMPK protein expression in VMN nitric oxide neurons in rats treated by intra-VMN administration of the glycogen phosphorylase inhibitor 4-dideoxy-1,4-imino-d-arabinitol (DAB). Groups of rats (n = 4/group) were infused (2 min) into the VMN with vehicle (V) or DAB (150 pM), then infused 10 min later with vehicle (V) or l-lactate (L; 100 nM). For each animal, VMN nNOS-immunopositive neurons were collected by laser-catapult microdissection; in each treatment group, aliquots of NO nerve cell lysate from individual subjects were combined to create triplicate samples for Western blot analysis of individual target proteins. Data show mean nNOS (A), AMPK (B), and pAMPK (C) protein optical density (O.D.) values ± SE for the following treatment groups: V/V (solid white bars); DAB/V (solid gray bars); DAB/Lactate (diagonal-striped gray bars). Treatment effects on the mean pAMKP/AMPK ratio in VMN nitrergic neurons are shown in D. *P < 0.05, **P < 0.01, ***P < 0.001. AMPK, 5′-AMP-activated protein kinase; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; pAMPK, phosphorylated 5′-AMP-activated protein kinase; VMN, ventromedial hypothalamic nucleus.

As shown in Fig. 4, DAB brought about a lactate-reversible augmentation of VMN GABAergic nerve cell GAD expression (Fig. 3A) [F(2,6) = 32.53; P < 0.0001]. This drug treatment stimulated AMPK profiles but did not alter pAMPK levels in these neurons. DAB effects on AMPK expression were equivalent in DAB/V and DAB/lactate treatment groups. As shown in Fig. 4D, mean GABAergic neuron pAMPK/AMPK ratio values declined in an equivalent extent in response to DAB plus V versus DAB plus lactate [F(2,6) = 5.83; P = 0.039].

Figure 4.

VMN GABAergic nerve cell GAD, AMPK, and pAMPK protein responses to intra-VMN delivery of DAB/vehicle versus DAB/l-lactate. Data show mean GAD (A), AMPK (B), and pAMPK (C) protein optical density (O.D.) values ± SE for the following treatment groups: V/V (solid white bars); DAB/V (solid gray bars); DAB/lactate (diagonal-striped gray bars). Effects of DAB with or without l-lactate on the GABA nerve cell pAMPK/AMPK ratio are presented in D. *P < 0.05, **P < 0.01, ***P < 0.001. AMPK, 5′-AMP-activated protein kinase; DAB, 4-dideoxy-1,4-imino-d-arabinitol; GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase_65/67; L, l-lactate; pAMPK, phosphorylated 5′-AMP-activated protein kinase; V, vehicle; VMN, ventromedial hypothalamic nucleus.

Figure 5 illustrates effects of DAB administration to the VMN followed by local delivery of lactate on plasma glucose, counterregulatory hormone, and FFA concentrations. As shown in Fig. 5A, mean circulating glucose levels were unaffected by this treatment [F(2,6) = 1.90; P = 0.23]. Data in Fig. 5B indicate that DAB caused a significant increase in corticosterone secretion, an effect that was prevented by lactate [F(2,6) = 7.29; P = 0.006]. Plasma glucagon levels were significantly diminished as a result of DAB; this inhibitory response was not altered by lactate [F(2,6) = 16.21; P = 0.0002]. Circulating FFA concentrations were refractory to treatment with DAB with or without lactate [F(2,6) = 0.26; P = 0.776].

Figure 5.

Effects of lactate on plasma glucose, counterregulatory hormone, and free fatty acid (FFA) concentrations in DAB-treated rats. Data show mean plasma glucose (A), corticosterone (B), glucagon (C), and FFA (D) measures ± SE for V/V- (solid white bars), DAB/V- (solid gray bars), or DAB/Lactate- (diagonal-striped gray bars) treated rats. **P < 0.01, ***P < 0.001. DAB, 4-dideoxy-1,4-imino-d-arabinitol; L, l-lactate; V, vehicle.

DISCUSSION

Astrocyte-derived lactate sustains nerve cell mitochondrial aerobic respiration. The glycogen reserve maintained in this glial cell compartment is actively remodeled during metabolic stability (3) and is the main alternative to blood glucose as a source of this substrate fuel (29–31). Lactate supply within the MBH impacts glucose counterregulation (18). Here, high-neuroanatomical resolution microdissection tools were used to address the premise that glycogen-derived fuel provision regulates VMN metabolic-sensory nerve cell AMPK activity and metabolic effector neurotransmission. Results show that the GP inhibitor DAB may upregulate VMN glucostimulatory nitrergic and glucoinhibitory GABAergic signaling through diminution of lactate stream. VMN NO and GABA neurons exhibited divergent adjustments, e.g., down- versus upregulation of total AMPK protein expression in response to DAB, responses that were refractory to lactate. Yet, glycogen-derived lactate may control AMPK activity in the former but not latter neuron population. Inhibition of VMN glycogen disassembly stimulated corticosterone secretion by means to reduction of lactate supply but inhibited glucagon release by lactate-independent mechanisms. Outcomes show that VMN glycogen metabolism controls local glucoregulatory transmission by means of lactate signal volume. Additional work is warranted to determine if lactate-reversible DAB upregulation of nitrergic neuron nNOS and pAMPK profiles is causally related, and if NO signaling mediates drug-associated hypercorticosteronemia. There is also a need for clarification of mechanisms by which VMN glycogen metabolism controls NO and GABA neuron total AMPK expression and pancreatic glucagon secretion.

The current study utilized the female rat as experimental model since our previous work provided novel evidence that in that sex, norepinephrine, the critical hindbrain transmitter that conveys information from hindbrain metabolic sensory neurons to the hypothalamus, controls VMN metabolic-sensory transmitter signaling via regulation of lactate stream (23). Evidence here for lactate-reversible DAB regulation of transmitter marker protein expression in pure VMN nitrergic and GABAergic nerve cell samples shows that glycogen-derived energy substrate stream governs neurotransmission by both neuron populations. Although it was predicted that NO and GAD proteins, corresponding markers for glucostimulatory or glucoinhibitory transmitter release, would be respectively increased or decreased by DAB, each protein profile was upregulated by this treatment, as summarized in Fig. 6. These data plausibly infer that diminished glycogen-derived lactate supply activates nitrergic neurons to signal metabolic deficiency. Meanwhile, the unanticipated observation of DAB-associated GAD protein upregulation supports the possibility that augmented glycogen mass due to GP inhibition may be a stimulus for GABAergic nerve cell communication of stored energy abundance. Disparities between current and earlier work in which no effect of DAB on micropunch-dissected VMN GAD content was observed (32) likely reflect, in part, dissimilar time frames between drug injection time and sacrifice, e.g., +2 h in earlier work; differential neuroanatomical resolution of dissection tools, namely, collection of pure neuron population versus whole tissue samples; and/or sex differences in glycogen regulation of GABAergic transmission. As DAB was administered here to euglycemic rats, it can be speculated that GABA neurons may monitor astrocyte glycogen accumulation relative to direct substrate fuel, e.g., lactate and glucose uptake. Thus, hypoglycemia-associated reductions in availability of the latter energy substrate to GABA cells might conceivably influence the direction of GABA transmitter response to decreased glycogen turnover. Current outcomes highlight the need for ongoing studies to determine if VMN glucoregulatory neurons exhibit dissimilar responses to physiological and pharmacological paradigms in which, on one hand, deceleration of glycogen disassembly is a consequence of depletion of this energy reserve, or alternatively, GP activity is inhibited to expand glycogen mass. It should be noted that since the current study design did not include confirmation of efficiency of the DAB treatment protocol utilized here for inhibition of VMN GP enzyme activity nor assessment of magnitude of change in VMN tissue glycogen content following drug delivery, alternate explanations for observed drug effects on target protein expression, including off-target actions, cannot not be overlooked.

Figure. 6.

Summary presentation of effects of DAB with or without l-lactate on VMN nitrergic and GABAergic nerve cell transmitter marker protein expression and pAMPK/AMPK ratio. AMPK, 5′-AMP-activated protein kinase; DAB, 4-dideoxy-1,4-imino-d-arabinitol; GABA, γ-aminobutyric acid; L, l-lactate; NO, nitric oxide; pAMPK, phosphorylated 5′-AMP-activated protein kinase; V, vehicle; VMN, ventromedial hypothalamic nucleus.

Present studies affirm that VMN nitrergic and GABAergic neurons express AMPK (16, 33) and reveal here that glycogen metabolism regulates total protein expression in each nerve cell populations, as well as AMPK phosphorylation in NO neurons. DAB effects on AMPK content were nerve cell type-specific, as this protein was corresponding decreased or increased in NO and GABA neurons. As neither response was averted by lactate, ongoing efforts will be needed to identify the intercellular mechanism(s) of communication that mediate effects of deceleration of astrocyte glycogen breakdown on total AMPK expression in these neuron populations. Data show that DAB caused lactate-reversible augmentation of pAMPK protein in nitrergic, but not GABAergic neurons; correlation of this stimulatory response with net suppression of NO neuron AMPK protein profiles infers that GP inhibition may amplify enzyme-specific activity in this cell population. This supposition is bolstered by evidence here that DAB treatment augments the mean pAMPK/AMPK ratio for VMN nitrergic neurons. Conversely, DAB inhibition of this ratio in GABA neurons suggest that specific activity is diminished due to reductions in glycogen turnover. Although previous research showed that net VMN tissue pAMPK is increased in response to local DAB administration (32), current data emphasize that glycogen regulation of sensor phosphorylation is nerve cell type-specific. It remains to be determined if coincident upregulation of nitrergic neuron nNOS and pAMPK expression in drug-treated animals is indicative of a causal relationship between these protein responses. It is presumed that immunocytochemical processing before nerve cell collection, involving brief light fixation, antiserum incubation, and exposure to enzyme substrate, would likely have a negligible impact on Western blot analysis of target proteins, as prior work showed that following in situ identification of VMN neurons by labeling for GAD- or nNOS-ir, we were able to subsequently quantify those proteins in immunoblots (24).

The current experimental design did not involve analysis of Fos protein expression in GABA or nitrergic neurons as the project goal was to evaluate glycogen metabolism regulation of specific protein markers for transmitter signaling and energy sensor activity in these cell populations. We successfully utilized Fos immunocytochemistry, in several earlier studies, as a valuable neuroanatomical mapping tool to identify individual brain structures characterized by net augmentation of neuron AP-1-mediated transcriptional activity due to exposure to a discrete physiological/pharmacological stimulus. However, a limitation of Fos measurement is that it does not identify the specific gene(s) that is(are) upregulated.

Outcomes provide novel evidence for divergent VMN glycogen metabolic regulation of the glucose counterregulatory hormones corticosterone and glucagon. Here, DAB caused significant, opposing changes in corticosterone (increased) versus glucagon (decreased) secretion. Proof of the efficacy of exogenous lactate administration to avert the former hormone response infers that the hypothalamic-pituitary-adrenal (HPA) axis can be activated by diminished astrocyte provision of this energy fuel within the VMN. Coincident reversal of DAB-induced NO neuronal nNOS expression and hypercorticosteronemia by lactate bolsters the possibility that glucostimulatory nitrergic neurons may function within local VMN circuitries that impose brain glycogen regulation of HPA activity. The inhibitory glucagon secretory response to DAB reported here was unexpected. It is speculated that diminution of this hormone profile may function as a signal of enhanced neuroglial metabolic stability due to augmentation of VMN tissue glycogen concentrations, a premise that supported by evidence that drug effects on glucagon release are refractory to lactate. As noted above, glucagon outflow may be affected differently under circumstances where glycogen breakdown is slowed due to exhaustion of glycogen mass versus targeted regulation of glycogen turnover to expand this reserve. As plasma glucose levels were measured here at a single time point after intracranial drug administration, the possibility cannot be overlooked that DAB-treated animals might have exhibited significant change in this profile over all or a portion of the one hour span of time between treatment and sacrifice.

Perspectives and Significance

Current evidence for glycogen regulation of AMPK total protein expression or phosphorylation in characterized VMN glucoregulatory neurons bolsters the view that this energy reserve is a critical monitored metabolic variable affecting neural control of glucostasis. Estradiol acts on neural substrates, including the VMN, to regulate plasma glucose (34). The female lifespan involves transition between distinct reproductive states, e.g., juvenile quiescence, fecundity, and reproductive senescence that are characterized by unique patterns of ovarian estradiol secretion. A promising future avenue of research would entail investigation of how diverse physiological patterns of estrogen output, such as low prepubertal estrogen secretion, dynamic fluctuation of estradiol release over the menstrual/estrous cycle, elevated ovarian hormone levels associated with pregnancy, and significant age-related decline in estrogen output impact glycogen control of VMN metabolic sensing and transmitter signaling during glucose sufficiency or shortage.

GRANTS

This work was supported by National Institutes of Health Grant DK-109382.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.P.B. conceived and designed research; K.B., M.M.H.I., and A.A. performed experiments; K.B., M.M.H.I., and A.A. analyzed data; K.B., M.M.H.I., and A.A. interpreted results of experiments; K.B., M.M.H.I., and A.A. prepared figures; M.M.H.I. and K.P.B. drafted manuscript; M.M.H.I. and K.P.B. edited and revised manuscript; K.P.B. approved final version of manuscript.