Chronic estrogen affects TIDA neurons through IL-1β and NO: effects of aging

in Journal of Endocrinology
Correspondence should be addressed to P S MohanKumar: psmohan@uga.edu
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Women are chronically exposed to estrogens through oral contraceptives, hormone replacement therapy or environmental estrogens. We hypothesized that chronic exposure to low levels of estradiol-17β (E2) can induce inflammatory and degenerative changes in the tuberoinfundibular dopaminergic (TIDA) system leading to reduced dopamine synthesis and hyperprolactinemia. Young (Y; 3–4 months) and middle-aged (MA; 10–12 months) Sprague–Dawley rats that were intact or ovariectomized (OVX) were either sham-implanted or implanted with a slow-release E2 pellet (20 ng E2/day for 90 days). To get mechanistic insight, adult 3- to 4-month-old WT, inducible nitric oxide synthase (iNOS) and IL-1 receptor (IL-1R) knockout (KO) mice were subjected to a similar treatment. Hypothalamic areas corresponding to the TIDA system were analyzed. E2 treatment increased IL-1β protein and nitrate levels in the arcuate nucleus of intact animals (Y and MA). Nitration of tyrosine hydroxylase in the median eminence increased with E2 treatment in both intact and OVX animals. There was no additional effect of age. This was accompanied by a reduction in dopamine levels and an increase in prolactin in intact animals. E2 treatment increased nitrate and reduced dopamine levels in the hypothalamus and increased serum prolactin in WT mice. In contrast, the effect of E2 on nitrate levels was blocked in IL-1R-KO mice and the effect on dopamine and prolactin were blocked in iNOS KO animals. Taken together, these results show that chronic exposure to low levels of E2 decreases TIDA activity through a cytokine-nitric oxide-mediated pathway leading to hyperprolactinemia and that aging could promote these degenerative changes.

 

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    (A) Serum E2 levels (mean ± s.e.; pg/mL) in reproductively intact young (3–4 months) and middle-aged (10–12 months) sham (YIS and MIS, respectively; n = 9 and 8 respectively) rats, rats treated with slow-release E2 pellets for 90 days (YIE and MIE; n = 9 per group). ** indicates significant difference (P < 0.01) and *** indicates P < 0.001. (B) Serum E2 levels in ovariectomized young (3–4 months) and middle-aged (10–12 months) sham (YOS and MOS, respectively; n = 9 per group) rats and rats implanted with slow-release E2 pellets for 90 days (YOE and MOE; n = 9 per group). * indicates significant difference from YOS and MOS (P < 0.05).

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    (A) IL-1β mRNA gene expression (fold change) in the arcuate nucleus (AN) of MIS, YIE and MIE rats were normalized to gene expression in YIS rats with a ∆∆Ct value of 1.0. (B) IL-1β mRNA gene expression (fold change) in the arcuate nucleus of MOS, YOE and MOE rats normalized to YOS rats with a ∆∆Ct value of 1.0. (C) IL-1β mRNA gene expression (fold change) in the substantia nigra (SN) of intact young and middle-aged rats (sham and E2 implanted) normalized to young intact sham (YIS) rats with a ∆∆Ct value of 1.0. (D) IL-1β mRNA gene expression fold change in the substantia nigra (SN) of ovariectomized young and middle-aged rats (sham and E2 implanted) normalized to young ovariectomized sham (YOS) rats with a ∆∆Ct value of 1.0. * indicates significant difference from YOS (P < 0.05). n = 6 per group.

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    (A) Interleukin 1-beta (IL-1β) concentrations (pg/µg protein; mean ± s.e.) in the arcuate nucleus of sham-implanted YIS and MIS rats, and rats treated implanted with E2 pellets (YIE and MIE). * indicates significant difference from YIS (P < 0.05); ** indicates significant difference from both YIS and MIS (P < 0.01). n = 6 per group for YIS, MIS and MIE. YIE had n = 8. (B) IL-1β concentrations in the arcuate nucleus of ovariectomized young and middle-aged sham (YOS and MOS, respectively) rats, and rats implanted with E2 pellets (YOE and MOE). n = 6 per group. (C) IL-1β concentrations in the substantia nigra of intact sham (YIS and MIS) rats, and young and aging rats implanted with E2 pellets (YIE and MIE). (D) IL-1β concentrations the substantia nigra of ovariectomized sham (YOS and MOS) and E2 implanted animals (YOE and MOE). n = 6 per group.

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    (A) iNOS mRNA gene expression (fold change) in the arcuate nucleus (AN) of MIS, YIE and MIE rats were normalized to YIS rats with a ∆∆Ct value of 1.0. * indicates significant difference between MIS and YIS and between MIE and YIE (P < 0.05). ** indicates significant difference between MIE and YIS (P < 0.01). n = 6 for YIS and MIE; n = 8 for MIS and n = 7 for YIE. (B) iNOS mRNA gene expression (fold change) in the arcuate nucleus of MOS, YOE and MOE rats normalized to YOS rats with a ∆∆Ct value of 1.0. (C) iNOS mRNA gene expression (fold change) in the substantia nigra (SN) of intact young and middle-aged rats (sham and E2 implanted) normalized to young intact sham (YIS) rats with a ∆∆Ct value of 1.0. (D) iNOS mRNA gene expression fold change in the substantia nigra (SN) of ovariectomized young and middle-aged rats (sham and E2 implanted) normalized to young ovariectomized sham (YOS) rats with a ∆∆Ct value of 1.0. * indicates significant difference from YOS (P < 0.05). n = 6 per group for graphs B–D.

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    (A) Nitrate concentrations (µM/µg protein; mean ± s.e.) in the arcuate nucleus (AN) of intact young (3–4 months) and middle-aged (10–12 months) sham (YIS and MIS, respectively) rats, and rats treated with slow-release E2 pellets for 90 days (YIE and MIE). * indicates significant difference between E2 treated and sham-implanted rats (P < 0.05). n = 7 for YIS; n = 8 for YIE; n = 6 for MIS and MIE groups. (B) Nitrate concentrations in the arcuate nucleus of ovariectomized young and aging animals that were sham-implanted or implanted with E2 pellets. n = 6 per group. (C) Nitrate concentrations in the substantia nigra (SN) of intact young and middle-aged sham (YIS and MIS, respectively) rats, and rats treated with slow-release E2 pellets (YIE and MIE). n = 6 per group. (D) Nitrate concentrations in the substantia nigra of ovariectomized young and aging animals that were sham-implanted or implanted with E2 pellets. n = 6 per group.

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    (A) The ratio of nitrotyrosine (NO-TH) to tyrosine hydroxylase (TH) in the median eminence (ME) of intact young (3–4 months) and middle-aged (10–12 months) sham (YIS and MIS, respectively) rats, rats treated with slow-release E2 pellets (YIE and MIE). * indicates significant difference from YIS (P < 0.05). n = 6 per group. (B) The ratio of nitrotyrosine to tyrosine hydroxylase in the median eminence of ovariectomized young and middle-aged sham (YOS and MOS, respectively) rats, and rats treated with slow-release E2 pellets (YOE and MOE). * indicates significant difference from YOS and MOS groups (P < 0.05). n = 6 per group. (C) Representative Western blot depicting differences in the protein expression of tyrosine hydroxylase and nitrated tyrosine hydroxylase in the median eminence from different treatment groups.

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    (A) Dopamine levels (mean ± s.e.; pg/μg protein) in the median eminence (ME) of reproductively intact young (3–4 months) and middle-aged (10–12 months) sham (YIS and MIS, respectively) rats and rats treated with slow-release E2 pellets (YIE and MIE). * indicates significant difference between MIE and YIE groups (P < 0.05); ** indicates P < 0.01 between MIS and YIE and *** indicates P < 0.001 between YIS and YIE. YIS: n = 6; YIE and MIE: n = 7 per group and MIS: n = 6. (B) Dopamine levels in the median eminence of ovariectomized rats. * indicates significant difference from YOE (P < 0.05). (C) Dopamine levels in the caudate putamen (CP) of intact young and middle-aged sham (YIS and MIS, respectively) rats and rats treated with slow-release E2 pellets for 90 days (YIE and MIE). (D) Dopamine levels in the caudate putamen (CP) of ovariectomized rats that were sham-implanted or treated with slow-release E2 pellets for 90 days (YOE and MOE). n = 6 per group for graphs B–D.

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    (A) Serum prolactin levels (mean ± s.e.; ng/mL) in young intact (YIS: n = 9) and middle-aged (MIS: n = 8) sham rats and rats treated with slow-release E2 pellets for 90 days (YIE and MIE: n = 10 and 9 respectively). * indicates significant difference from YIS (P < 0.05) and ** indicates P < 0.01 from sham-implanted rats. (B) Serum prolactin levels in OVX young (YOS) and middle-aged (MOS) sham rats and rats implanted with slow-release E2 pellets for 90 days (YOE and MOE). n = 9 per group.

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    Nitrate concentrations (µM/µg protein, mean ± s.e.) in the hypothalamic arcuate nucleus of reproductively intact young (3–4 months) wild type and IL-1R(−/−) KO mice that were sham-implanted or implanted with slow-release E2 pellets for 90 days. * indicates significant difference from WT sham group (P < 0.05) and ** indicates P < 0.01 from IL-1R−/− groups. WT sham: n = 7; WT-E2: n = 8; IL-1R−/− n = 6 per group.

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    (Panel A) DA concentrations (pg/μg protein, mean ± s.e.) in the hypothalamus and (panel B) serum PRL (ng/mL ± s.e.) in wild type and iNOS(−/−) KO mice that were either sham-implanted or implanted with slow-release E2 pellets for 90 days. * indicates significant difference (P < 0.05); ** indicates P < 0.01 and *** indicates P < 0.001. WT: n = 8 per group; iNOS−/−: n = 7 per group.

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    Schematic representing changes in the arcuate nucleus and median eminence in response to E2 exposure and aging that culminates in hyperprolactinemia. Changes in the arcuate nucleus and median eminence are represented in separate boxes. Glia and TIDA neuronal cell bodies in the arcuate nucleus are represented by circles in which possible ongoing alterations are listed. Courtesy: Stephanie Pfeiffer, Educational Resource Center, UGA. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0274.

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