Brain regulation of systemic glucose homeostasis involves a dedicated, expansive neural circuitry that extends throughout the central neuroaxis (Watts and Donovan, 2010). The hypothalamus, the hierarchic visceral motor center of the brain, oversees autonomic, neuroendocrine, and behavioral motor outflow to control glucose dietary intake, cellular uptake and utilization, de novo synthesis, and storage as glycogen (Tu et al., 2022). The ventromedial hypothalamic nucleus (VMN), a prominent bilateral constituent of the mediobasal hypothalamus, operates as a critical sensory and integrative component of the neural glucostatic network. Dedicated metabolic-sensory neurons in the VMN and other select brain loci provide a dynamic cellular energy readout by augmenting (‘glucose-inhibited’; GI) or reducing (‘glucose-excited’; GE) synaptic firing as ambient energy substrate levels fall (Oomura et al., 1969), (Ashford et al., 1990), (Silver and Erecińska, 1998). Neurotransmitter effectors of ventromedial hypothalamic energy imbalance include the amino acid γ-aminobutyric acid (GABA) and labile gas nitric oxide (NO), which respectively suppress (Chan et al., 2006; Chan et al., 2008; Chan and Sherwin, 2013) or stimulate (Fioramonti et al., 2010) counterregulatory hormone secretion. VMN neurons express hypoglycemia-sensitive gene transcripts that encode protein markers for these counterregulatory neurochemicals, namely glutamate decarboxylase (GAD65/67) and neuronal nitric oxide synthase (nNOS) (Ali et al., 2022; Roy et al., 2023a; Roy et al., 2023b). VMN GABAergic and nitrergic neurons monitor intrinsic energy status as the ultra-sensitive ATP gauge 5’-AMP-activated protein kinase (AMPK) (Hardie et al., 2012; Hardie et al., 2016; Hardie and Lin, 2017) is expressed in both nerve cell populations, wherein it is activated in response to hypoglycemia (Briski et al., 2020; Ibrahim et al., 2020).

Astrocytes engage in metabolic coupling with neurons, providing oxidizable substrate fuels to support energy production in the latter cell compartment (Stobart and Anderson, 2013; Jha and Morrison, 2018). As a critical component of the blood-brain barrier, astrocytes control glucose entry into the brain (Chowen et al., 2019; Beard et al., 2022). Glucose is retained within astrocytes, as in other cell types, and directed to metabolic pathways by phosphorylation (Hassan et al., 2014). Astrocyte glucose-6-phosphate (Glc-6-P) is used either for glycogen mass expansion or conversion via glycolysis to the trafficable energy fuel L-lactate (Laming et al., 2000; Waitt et al., 2017; Dienel, 2019). Our studies show that the glycogen shunt, involving sequential assembly/disassembly of glucosyl monomers prior to lactate manufacture, is a monitored metabolic variable that shapes VMN counterregulatory neurotransmission (Bheemanapally et al., 2021a). Lactate evidently functions as this regulatory signal in its dual capacity as a usable metabolic fuel and volume transmitter ligand for the plasma membrane G protein-coupled receptor GPR81 (Roy et al., 2023a; Mahmood et al., 2019). Peripheral glucose-exporting cell types contain the hydrolase glucose-6-phosphatase-alpha (Glc-6-Pase-α), which dephosphorylates Glu-6-P within the endoplasmic reticulum (ER) lumen (Nordlie and Sukalski, 1985; Pan et al., 1998; Chen et al., 2001; Chou et al., 2002; Martin et al., 2002). Astrocytes are distinguished from other brain cell types by expression of Glu-6-Pase, specifically the beta isoform (Ghosh et al., 2005). Glu-6-Pase-β is purported to augment brain astrocyte ER accumulation of free glucose (Müller et al., 2018). The prospect that astrocyte endogenous glucose production may affect VMN counterregulatory neurochemical signaling has not yet been addressed.

Current research investigated the premise that under conditions of systemic glucose sufficiency and/or deficiency, VMN Glc-6-Pase-β activity may regulate astrocyte glycogen and glucose metabolism and net tissue accumulation alongside counterregulatory neurotransmitter protein marker expression and systemic counterregulatory hormone profiles in the adult male rat. Our experimental design implemented in vivo gene knockdown, combinative in situ immunocytochemistry/laser-catapult-microdissection, total in-lane protein-normalized Western blot, and HPLC-electrospray ionization-mass spectrometry (LC-ESI-MS) tools within the context of a validated whole-animal model for insulin (INS)-induced hypoglycemia (IIH) (Paranjape and Briski, 2005). Recent studies infer that astrocyte glycogen and glucose metabolism may vary between functionally distinctive dorsomedial (VMNdm) versus ventrolateral (VMNvl) divisions of the VMN (Bheemanapally and Briski, 2024). Procurement here of pure VMNdm or VMNvl astrocyte cell samples, owing to high-neuroanatomical resolution microdissection capabilities, allowed investigation of Glc-6-Pase-β gene silencing effects on astrocyte glucose transporter/sensor glucose transporter-2 (GLUT2), the rate-limiting glycolytic pathway enzyme/glucose sensor glucokinase (GCK), and glycogen metabolic enzyme, i.e. glycogen synthase (GS), glycogen phosphorylase-brain type (GPbb), glycogen phosphorylase-muscle type (GPmm) protein expression profiles in each VMN division. Selective harvesting of GABAergic or nitrergic neurons from the VMNdm versus VMNvl enabled analysis of Glc-6-Pase-β gene knockdown effects on transmitter marker, AMPK, and activated, i.e. phosphorylated AMPK (pAMPK) protein levels in these distinctive counterregulatory neurotransmitter neuron populations according to VMN division. In light of recent evidence for that the neuropeptide transmitter growth hormone-releasing hormone (Ghrh) shapes counterregulatory hormone secretion and is co-expressed with GABA and NO (Sapkota et al., 2023), current studies addressed the premise that Glc-6-Pase-β may control expression of this neurochemical in one or both neuron populations according to VMN division. Lastly, LC-ESI-MS analytical methodology was used in conjunction with VMN tissue micropunch dissection to determine Glc-6-Pase-β gene silencing effects on VMNdm versus VMNvl tissue glucose and glycogen content (Bheemanapally et al., 2020).