Hypoglycemic and post‐hypoglycemic patterns of glycogen phosphorylase isoform expression in the ventrolateral ventromedial hypothalamic nucleus: impact of sex and estradiol

Glycogen metabolism shapes ventromedial hypothalamic nucleus (VMN) control of glucose homeostasis. Brain glycogen mass undergoes compensatory expansion post‐recovery from insulin‐induced hypoglycemia (IIH). Current research utilized combinatory high‐resolution microdissection/high‐sensitivity Western blotting to investigate whether IIH causes residual adjustments in glycogen metabolism within the metabolic‐sensory ventrolateral VMN (VMNvl). Micropunch‐dissected tissue was collected from rostral, middle, and caudal levels of the VMNvl in each sex for analysis of glycogen synthase (GS) and glycogen phosphorylase (GP)‐muscle type (GPmm; norepinephrine‐sensitive) and GP‐brain type (GPbb; glucoprivic‐sensitive) isoform expression during and after IIH. Hypoglycemic suppression of VMNvl GS levels in males disappeared or continued after reestablishment of euglycemia, according to sampled segment. Yet, reductions in female VMNvl GS persisted after IIH. Males exhibited reductions in GPmm content in select rostro‐caudal VMNvl segments, but this protein declined in each segment post‐hypoglycemia. Females, rather, showed augmented or diminished GPmm levels during IIH, but no residual effects of IIH on this protein. In each sex, region‐specific up‐ or down‐regulation of VMNvl GPbb profiles during glucose decrements were undetected post‐recovery from IIH. Results provide novel proof of estradiol‐dependent sex‐dimorphic patterns of VMNvl GP variant expression at specific rostro‐caudal levels of this critical gluco‐regulatory structure. Sex differences in persistence of IIH‐associated GS and GPmm patterns of expression after restoration of euglycemia infer that VMNvl recovery from this metabolic stress may involve dissimilar glycogen accumulation in male versus female.


INTRODUCTION
The brain consumes a disproportionate fraction of total body energy to sustain vital high energy-demand nerve cell activities. Iatrogenic insulin (INS)-induced hypoglycemia (IIH) is an unremitting complication of obligatory glycemic management practiced by type I diabetes mellitus patients (Cryer, 2013;2014). Hypoglycemic neuro-glucopenia poses a significant risk of neurological impairment (Auer and Siesjö, 1988;Auer, 2004). Glucoprivation elicits neural-driven counter-regulatory autonomic, neuroendocrine, and behavioral outflow that increases circulating glucose levels. Cellular energy stability is continuously monitored in the ventromedial hypothalamic nucleus (VMN) and other select brain loci by dedicated metabolic-sensory neurons that adjust synaptic transmission in response to excessive or diminished substrate fuel supply (Oomura et al., 1969;Ashford et al., 1990;Adachi et al., 1995Adachi et al., , 1997Silver and Erecińska, 1998). This dynamic readout, together with hormonal indicators of peripheral energy reserves, provides critical input for VMN regulation of glucose homeostasis. The ventrolateral VMN (VMNvl), one of a unique set of sex-dimorphic structures in the brain (Simerly, 2002), contains metabolic-sensory neurons (Cotero and Routh, 2009;Santiago et al., 2016) that participate in gluco-regulation (He et al., 2020).
Brain astrocytes store the complex carbohydrate glycogen as an energy reserve (Stobart and Anderson, 2013). Glycogen metabolism is controlled by the enzymes glycogen synthase (GS) and glycogen phosphorylase (GP), which respectively catalyze glycogen assembly or breakdown. Norepinephrine-sensitive GP-muscle (GPmm) and glucoprivic sensitive GP-brain (GPbb) type isoforms (Nadeau et al., 2018) confer stimulus-specific regulation of brain glycogen. Pharmacological inhibition of VMN glycogen disassembly up-regulates marker protein expression for neurotransmission of signal energy deficiency, which infers that local metabolic-sensory neurons gauge glycogen turnover and associated substrate fuel liberation Briski et al., 2021). The prospect that hypoglycemia may produce sex-specific adjustments in glycogen metabolism within the sex-dimorphic VMNvl has not been addressed. Current studies utilized a combinatory high-resolution microdissection/high-sensitivity Western blot approach to investigate the premise that basal and/or hypoglycemic patterns of VMNvl glycogen metabolic enzyme expression differ between sexes. The VMNvl exhibits high estrogen receptor (ER) content compared to dorsomedial and central divisions (Yang and Shah, 2014). Thus, a corollary objective of this research was to determine if estradiol mediates sex-contingent GS and/or GP protein responses to hypoglycemia in one or both sexes. Here, target proteins were analyzed after VMNvl micropunch dissection from subcutaneous (sc) letrozole (Lz)-or vehicle-injected male and sc estradiol-or oil-implanted ovariectomized (OVX) female rats after INS or vehicle injection for analysis.
Transitory exposure to physiological conditions leading to energy imbalance, e.g., hypoxia, glucoprivation, and sleep deprivation, purportedly causes adaptive post-exposure augmentation of whole-brain glycogen content (Brucklacher et al., 2002;Kong et al., 2002;Choi et al., 2003). There has been speculation that post-hypoglycemic expansion of the glycogen reserve may adversely attenuate counter-regulation (Herzog et al., 2008;Duarte et al., 2017;Öz et al., 2017). This view has remained controversial, in part, due to a lack of methods of requisite neuroanatomical resolution for analysis of glycogen metabolism within sites where metabolic sensory neurons reside. The current project afforded the novel opportunity to examine whether regulatory effects of IIH on VMNvl glycogen metabolic enzyme protein expression coincide with plasma glucose decrements and/or are demonstrable following reestablishment of euglycemia in one or both sexes.
All surgical and experimental protocols were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Ani-mals, 8 th Edition, under approval by the ULM Institutional Animal Care and Use Committee.

Statistics
Mean normalized VMNvl segmental protein OD and plasma glucose measures were evaluated within each sex by two-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
Current research investigated the premise that insulin-induced hypoglycemia effects on VMNvl glycogen metabolic enzyme expression occur by estradiol-dependent mechanisms in one or both sexes, and that altered expression of one or both GP isoforms may persist in distinctive rostro-caudal segments of the VMNvl after recovery from hypoglycemia. Here, combinatory high-resolution microdissection/high-sensi-tivity Western blotting was used to measure GP, GPmm, and GPbb protein expression profiles in rostral, middle, and caudal regions of the VMNvl in male rats pretreated with Lz or V, as well as OVX female rats implanted with estradiol or oil.
Data presented in Table I show that INS-injected male and female rats exhibited reductions in plasma glucose concentrations compared to V-injected controls at both +1 and +3.5 h post-treatment. In each sex, restoration of glucose profiles to the normal range occurred between +3.5 and +7 h. INS effects on circulating glucose levels were not different between groups of males that were pretreated with Lz versus V, or between groups of estradiol-versus oil-implanted OVX female rats.
Rostral, middle, and caudal VMNvl tissue was collected from Lz-or V-pretreated male (   ic rostro-caudal levels of the VMNvl during hypoglycemia. Compared to euglycemic controls, post-hypoglycemic patterns of GS expression were diminished throughout the male VMNvl as well as in the female rostral and caudal VMNvl. Fig. 3 depicts effects of INS injection on VMNvl GPmm protein expression during and after diminution of plasma glucose concentrations. Euglycemic male rats exhibited a reduction in VMNvl GPmm profiles in the rostral (Fig. 3A) and caudal (Fig. 3F) VMNvl following Lz administration. In this sex, INS significantly inhibited rostral and caudal VMNvl GPmm content both during and after glucose decrements. While INS-injected males showed no change in middle VMNvl GPmm levels during hypoglycemia, this protein profile was subsequently reduced after reestablishment of plasma glucose levels to the normal range. Lz pretreatment prevented hypoglycemia-associated decline in rostral VMNvl GPmm content, but did not prevent diminution of this protein profile at +7 h. Post-hypoglycemic decrements in the male rat middle VMNvl GPmm were also refractory to Lz. Yet, Lz-pretreated males had modified patterns of GPmm expression in the caudal VMNvl during and after hypoglycemia. Data illustrated in Figs 3B, 3D, and 3F reveal divergent INS effects on VMNvl GPmm levels in estradiol-implanted OVX female rats, as this protein was up-regulated in the rostral segment (+3.5 h), but down-regulated at middle and caudal levels (+1 h). In contrast, INS injection did not influence VMNvl GPmm profiles in oil-implanted OVX female rats. In the presence of estradiol, post-hypoglycemic GPmm expression patterns over the length of the VMNvl were equivalent to those in non-INS-injected controls. Outcomes document uniform versus bi-directional effects of hypoglycemia on VMNvl GPmm content in male versus female, respectively, and identify variation of post-hypoglycemic VMNvl GPmm profiles between sexes.
Effects of INS injection on male versus female VMNvl GPbb protein content are presented in Fig. 4. Euglycemic males injected with Lz exhibited significant augmentation of caudal (Fig. 4E), but not rostral (Fig. 4A) or middle (Fig. 4C) VMNvl GPbb expression compared to V controls. INS injection of male rats resulted in either stimulation (rostral and middle VMNvl) or suppression (caudal VMNvl) of GPbb content by 1 h post-treatment. These region-based responses were abolished by Lz. In males sacrificed at 7 h after INS injection, GPbb profiles in all VMNvl segments did not differ from controls. Baseline GPbb levels were significantly lower in rostral (Fig. 4B) and middle (Fig. 4D) VMNvl segments of estradiol-versus oil-implanted rats. In response to insulin, estradiol-, but not oil-im-planted female rats exhibited up-or down-regulated GPbb expression in rostral (+1 and +3.5 h) and middle (+1 h) VMNvl regions, respectively. INS had primarily no impact on post-hypoglycemic VMNvl GPbb content in estradiol-or oil-implanted animals. Data indicate that region-specific up-or down-regulation of VMNvl GPbb expression coinciding with diminished plasma glucose concentrations is discontinued after recovery from hypoglycemia.

DISCUSSION
Glycogen metabolism affects whole-VMN gluco-regulatory transmitter signaling Briski et al., 2021). Sex-dimorphic VMNvl sensitivity to estradiol, due to developmental hormonal imprinting, manifests as sex-specific control of several behavioral and physiological functions. VMNvl metabolic-sensory neurons influence blood glucose levels (He et al., 2020). Here, VMNvl tissue was micropunch-dissected in segmental fashion to investigate whether estradiol-dependent mechanisms mediate sex-specific patterns of glycogen metabolic enzyme protein expression in distinctive locations over the longitudinal axis of this structure during and/or after hypoglycemia. Outcomes document estradiol-dependent VMNvl region-specific GS inhibition during glucose decrements, a response that persisted post-hypoglycemia in some (male) or all (female) of those segments. INS-associated diminution of GPmm profiles occurred in hypoglycemic animals of each sex, and also in recovered male, but not female rats. VMNvl GPbb profiles were up-or down-regulated according to rostro-caudal segment during, but not after plasma glucose decline. Results infer that during hypoglycemia, AMP-driven glycogen breakdown is prioritized relative to norepinephrine input in discrete regions of the VMNvl. Hypoglycemia may likely impose lingering inhibitory effects on VMNvl glycogen synthesis in each sex and on noradrenergic stimulation of glycogen breakdown in this structure in males. Further effort is warranted to characterize the molecular mechanisms that underlie common versus unique patterns of VMNvl glycogen enzyme expression in the two sexes, and to determine how glucoprivic-versus neurotransmitter-mediated glycogen disassembly may affect gluco-regulatory function over the length of the VMNvl.
In each sex, gonad-derived estradiol is a positive stimulus for rostral and caudal VMNvl GS expression during euglycemia, but is involved in hypoglycemic inhibition of that protein in those same locations. These findings likely infer that hypoglycemia sup- presses glycogen synthesis at selective levels along the longitudinal axis of the VMNvl. There remains a need to clarify whether this switch in direction of estradiol control of rostral and caudal VMNvl GS may result, in part, from dissimilar astrocyte ER expression due to differences in glucose uptake during normo-versus hypoglycemia. Regional differences in VMNvl GS reactivity to hypoglycemia may be contingent, somewhat, upon relative numbers of one or more distinctive nuclear or membrane ER receptor variants. There is also the possibility that control of astrocyte sensitivity to estradiol input by other regulatory signals may vary between rostro-caudal segments. It is notable that in each sex, the onset of hypoglycemia-associated suppression of GS protein diverged between rostral (+3.5 h) versus caudal (+1 h) VMNvl. A sizable fraction of glucose acquired by astrocytes from blood is diverted to the glycogen shunt, which involves glucose monomer incorporation into this polymer, then release prior to entry into the glycolytic pathway. Attenuation of glycogen assembly during hypoglycemia is an effective strategy for energy conservation as ATP expenditure is required for glycogen assembly. It is thus possible that latency between onset of hypoglycemia and diminution of glycogen synthesis may be governed, to some extent, by local nerve cell energy requirements. Persistent down-regulation of GS profiles in the male and female caudal VMNvl following recovery from hypoglycemia may similarly reflect continuance of heightened neuronal energy demands in that segment. Further research is required to examine how estradiol signaling may integrate with putative cues on neuronal metabolic stability to control GS expression and glycogen formation.
Current data document sex-specific hypoglycemic regulation of stimulus-specific glycogen disassembly in the VMNvl. The norepinephrine-sensitive GP variant GPmm was inhibited by INS in different segments in each sex, as this protein was decreased at rostral and caudal levels in males, but in middle and caudal segments in the female. Females also showed coincident augmentation of rostral VMN GPmm content. Diminution or augmentation of GPmm expression likely results in decreased or elevated norepinephrine-stimulated glycogen disassembly, respectively. This premise remains speculative as tools for assessment of hypoglycemic effects on phosphorylation, e.g. activation of this GP isoform are currently unavailable. Outcomes also show that, in each sex, hypoglycemia simultaneously augmented or inhibited glucoprivic-sensitive GPbb expression in select VMNvl segments. These results infer that AMP-driven glycogen breakdown may be correspondingly enhanced or reduced in those locations during this metabolic stress. Confirmation of this premise will require region-specific analysis of phosphorylation state of GPbb protein expressed during hypoglycemia. The data summary provided in Table II suggests that in hypoglycemic male rats, select VMNvl regional glycogen stores may be protected from noradrenergic disassembly, and at the same time undergo enhanced (rostral segment) or attenuated (caudal segment) AMP-mediated mobilization. Thus, in the former site, glycogen disassembly may be selectively driven by energy deficiency, whereas glycogen mass may be preserved in the latter region despite neuro-glucopenia. In contrast, the female VMNvl is evidently characterized by regional enhancement (rostral segment) or diminution (middle segment) of glycogen sensitivity to both noradrenergic and glucoprivic breakdown. The possibility that hypoglycemic up-or down-regulation of VMNvl GPbb expression, by segment, may reflect, to some extent, elevated or decreased energy demands needs of local neurons will require further investigation. There is ample justification for additional effort to verify effects of GPbb-mediated acceleration of glucose liberation on metabolic-sensory function. Importantly, outcomes addressed in Table II emphasize estradiol involvement in sex-specific patterns of GP variant expression during hypoglycemia. A possible explanation for these divergent GP responses between sexes may involve, in part, dissimilar of ER expression profiles (Ibrahim et al., 2020). Current studies provide novel evidence for residual effects of hypoglycemia on VMNvl GPmm, but not GPbb variant expression. Notably, post-hypoglycemic GPmm down-regulation occurred in male, but not female rats; the mechanisms that this sex difference remain unclear. Persistent reductions in this protein would likely attenuate glycogen breakdown in the absence of neuro-glucopenia. Down-regulated GPmm, coincident with normal (rostral VMNvl) or diminished (middle and caudal VMNvl) patterns of GS expression at +7 h after INS injection, plausibly facilitates glycogen enhancement in the former site, yet may stabilize glycogen mass in other regions through attenuation of glycogen shunt activity. Implications of putative post-hypoglycemia glycogen expansion in the male rostral VMNvl on metabolic transmitter signaling require additional consideration. Female rats exhibited suppression of rostral and caudal VMNvl GS expression after recovery from hypoglycemia, alongside reestablishment of GPmm and GPbb profiles to control levels. Post-hypoglycemia glycogen synthesis and glycogen shunt activity are thus likely impeded in those areas. As both GP isoforms were up-regulated in the female rat rostral VMNvl during hypoglycemia, glycogen depletion in that segment occurred may not be reversed within the post-hypoglycemia interval evaluated here. Functional ramifications of possible sex-dimorphic adaptations in rostral VMNvl glycogen accumulation (increased in male, decreased in female) after recovery from hypoglycemia warrant further attention.
In summary, current research offers unique evidence for estradiol-dependent sex-dimorphic VMNvl glycogen metabolic enzyme protein expression during and after exposure to hypoglycemia. Results emphasize, for each sex, rostro-caudal region-specific control of glycogen synthesis and stimulus-specific glycogen mobilization. Further research is needed to investigate potential relationships between local adjustments in glycogen metabolism and nerve cell energy stability during and after hypoglycemia.