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. 2020 Mar 15:504:110703.
doi: 10.1016/j.mce.2020.110703. Epub 2020 Jan 11.

Sex-specific estrogen regulation of hypothalamic astrocyte estrogen receptor expression and glycogen metabolism in rats

Mostafa M H Ibrahim  1 , Khaggeswar Bheemanapally  1 , Paul W Sylvester  1 , Karen P Briski  2
Affiliations

Sex-specific estrogen regulation of hypothalamic astrocyte estrogen receptor expression and glycogen metabolism in rats

Mostafa M H Ibrahim et al. Mol Cell Endocrinol. .

Abstract

Brain astrocytes are implicated in estrogenic neuroprotection against bio-energetic insults, which may involve their glycogen energy reserve. Forebrain estrogen receptors (ER)-alpha (ERα) and -beta (ERβ) exert differential control of glycogen metabolic enzyme [glycogen synthase (GS); phosphorylase (GP)] expression in hypoglycemic male versus female rats. Studies were conducted using a rat hypothalamic astrocyte primary culture model along with selective ER agonists to investigate the premise that estradiol (E2) exerts sex-dimorphic control over astrocyte glycogen mass and metabolism. Female astrocyte GS and GP profiles are more sensitive to E2 stimulation than the male. E2 did not regulate expression of phospho-GS (inactive enzyme form) in either sex. Data also show that transmembrane G protein-coupled ER-1 (GPER) signaling is implicated in E2 control of GS profiles in each sex and alongside ERα, GP expression in females. E2 increases total 5'-AMP-activated protein kinase (AMPK) protein in female astrocytes, but stimulated pAMPK (active form) expression with equivalent potency via GPER in females and ERα in males. In female astrocytes, ERα protein was up-regulated at a lower E2 concentration and over a broader dosage range compared to males, whereas ERβ was increased after exposure to 1-10 nM versus 100 pM E2 levels in females and males, respectively. GPER profiles were stimulated by E2 in female, but not male astrocytes. E2 increased astrocyte glycogen content in female, but not male astrocytes; selective ERβ or ERα stimulation elevated glycogen levels in the female and male, respectively. Outcomes imply that dimorphic astrocyte ER and glycogen metabolic responses to E2 may reflect, in part, differential steroid induction of ER variant expression and/or regulation of post-receptor signaling in each sex.

Keywords: 5′-AMP-activated protein kinase; Calcium/calmodulin-dependent protein kinase kinase-beta; Estrogen receptor-alpha; G protein-coupled estrogen receptor; Glycogen phosphorylase; Glycogen synthase.

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Figures

Figure 1:
Figure 1:
Effects of 17β-Estradiol (E2) or Selective Estrogen Receptor (ER) Agonists on Glycogen Synthase (GS) Expression in Female or Male Rat Hypothalamic Astrocyte Primary Cultures. After incubation of female [Panel 1A; p < 0.0001] or male [Panel 1B; p < 0.0001] rat hypothalamic astrocytes with varying (100 pM – 100 nM; gray bars) E2 concentrations or media lacking E2 (white bar), cell lysates were analyzed by Western blotting for GS protein expression. Data depict E2 effects on mean normalized GS protein optical density (O.D.) measures ± S.E.M. Panels 1C [female; p < 0.0001] and 1D [male; p < 0.0001] illustrate effects of E2 (solid gray bar) versus the selective ERα agonist PPT (diagonal-striped gray bar), the ERβ agonist DPN (horizontal-striped gray bar), membrane impermeable E2-BSA conjugate (cross-hatched gray bar), or the GPER agonist G1 (vertical-striped gray bar) on GS protein profiles. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 2:
Figure 2:
E2 versus ER Agonist Regulation of Phospho-GS (pGS) Expression in Female or Male Hypothalamic Astrocytes. Data depict mean normalized pGS protein O.D. measures ± S.E.M. for female [Panel 2A; p = 0.56] or male [Panel 2B; p = 0.5106] astrocytes exposed to graded E2 dosages. Panels 2C and 2D correspondingly show E2 versus ER agonist effects on female [p < 0.0001] or male [p = 0.6641] astrocyte pGS content. **p < 0.01; ***p < 0.001.
Figure 3:
Figure 3:
Effects of E2 versus ER Agonist Treatment on Female or Male Hypothalamic Astrocyte Glycogen Phosphorylase (GP) Expression. Data depict mean normalized astrocyte GP O.D. values ± S.E.M. following incubation of female [Panel 3A; p < 0.0001] or male [Panel 3B; p = 0.003] astrocytes with graded E2 concentrations. Panels 3C and 3D show effects of E2 versus selective ER agonist exposure on female [p = 0.02] and male astrocyte [p = 0.005] GP content. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4:
Figure 4:
Effects of E2 versus ER Agonist Treatment on Female or Male Hypothalamic Astrocyte ER-Alpha (ERα) Protein Expression. Data depict mean normalized female [Panel 4A; p = 0.0002] or male [Panel 4B: p = 0.05] astrocyte ERα O.D. values ± S.E.M. after graded E2 exposure. Panels 4C and 4D show effects of E2 versus ER agonist exposure on female [p < 0.0001] or male [p = 0.3383] astrocyte ERα protein profiles. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5:
Figure 5:
Effects of E2 versus ER Agonist Incubation on Female or Male Hypothalamic Astrocyte ER-Beta (ERβ) Protein Expression. Data depict mean normalized female [Panel 5A; p = 0.001] or male [Panel 5B; p < 0.0001] astrocyte ERβ O.D. measures ± S.E.M. after treatment with graded E2 dosages. Panels 5C and 5D illustrate effects of E2 versus selective ER agonists on female [p = 0.017] or male [p < 0.0001] astrocyte ERβ protein expression. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 6:
Figure 6:
E2 versus Selective ER Agonist Regulation of Transmembrane G Protein-Coupled ER (GPER) Expression in Female or Male Hypothalamic Astrocytes. Data show mean normalized GPER O.D. measures ± S.E.M. for female [Panel 6A; p =0.02] or male [Panel 6B; p =0.6498] astrocytes incubated with graded concentrations of E2. Panel versus selective ER agonists on female hypothalamic astrocytes [p = 0.14], and 6D: Effect of E2 versus selective ER agonists on male hypothalamic astrocytes [p < 0.0001]. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7:
Figure 7:
Effects of E2 versus Selective ER Agonist Treatment on 5’-AMP-Activated Protein Kinase (AMPK) Expression in Female or Male Hypothalamic Astrocytes. Data depict mean normalized AMPK O.D. measures ± S.E.M. for female [Panel 7A; p = 0.03] or male [Panel 7B; p = 0.61] astrocytes incubated with varying E2 doses. Panels 7C and 7D show effects of E2 versus selective ER agonists on female [p = 0.53] or male [p = 0.08] hypothalamic astrocyte AMPK content. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 8:
Figure 8:
Effects of E2 or ER Agonist Treatment on Female or Male Astrocyte Phospho-AMPK (pAMPK) Expression. Data depict mean normalized pAMPK O.D. measures ± S.E.M. for female [Panel 8A; p = 0.0002] or male [Panel 8B; p = 0.0002] astrocytes incubated with graded E2 concentrations. Panels 8C and 8D illustrate effects of E2 versus selective ER agonists on female [p < 0.0001] or male [p < 0.0001] astrocyte pAMPK protein expression. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 9:
Figure 9:
Effects of E2 versus ER agonist Treatment on Calcium/Calmodulin-Dependent Protein Kinase Kinase-Beta (CAMKKβ) Expression in Female or Male Hypothalamic Astrocytes. Data depict mean normalized CAMKKβ O.D. values ± S.E.M. for female [Panel 9A; p = 0.10] or male [Panel 9B; p < 0.0001] astrocytes incubated with graded doses of E2. Panels 9C and 9D illustrate effects of E2 versus selective ER agonists on female [p = 0.92] or male [p = 0.0013] astrocyte CAMKKβ content. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 10:
Figure 10:
Effects of E2 versus Selective ER Agonists on Protein Phosphatase-1 (PP1) Profiles in Female or Male Hypothalamic Astrocytes. Data depict mean normalized PP1 O.D. measures ± S.E.M. for female [Panel 10A; p = 0.65] or male [Panel 10B; p = 0.3960] astrocytes treated with graded E2 dosages. Panels 10C and 10D show effects of E2 versus ER agonist incubation on female [p = 0.003] or male [p =0.0066] PP1 protein profiles. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 11:
Figure 11:
Effects of E2 versus Selective ER Agonists on Female or Male Astrocyte Glycogen Content. Panels 11A and 11 B depict treatment effects on mean cellular glycogen levels in female [p = 0.0006] or male [p = 0.006] astrocytes, respectively. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 12.
Figure 12.
Estradiol (E2) and Estrogen Receptor (ER) Agonist Regulation of Female versus Male Hypothalamic Astrocyte Glycogen Metabolic Enzyme and ER Protein Expression. Tables on right (female) and left (male) sides denote minimum E2 dosages that significantly augmented astrocyte GS, GP, ERα, ERβ, GPER, and pAMPK protein expression in each sex. Sex dimorphic effects of E2 and individual ER agonists on astrocyte ER variant profiles and glycogen content are presented below illustrations of female versus male astrocytes.
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