Estrogen-dependent hypersensitivity to diabetes-evoked cardiac autonomic dysregulation: Role of hypothalamic neuroinflammation

Abstract

Aims: To investigate if autonomic dysregulation is exacerbated in female rats, subjected to diabetes mellitus (DM), via a paradoxical estrogen (E₂)-evoked provocation of neuroinflammation/injury of the hypothalamic paraventricular nucleus (PVN).

Main methods: We measured cardiac autonomic function and conducted subsequent PVN neurochemical studies, in DM rats, and their respective controls, divided as follows: male, sham operated (SO), ovariectomized (OVX), and OVX with E₂ supplementation (OVX/E₂).

Key findings: Autonomic dysregulation, expressed as sympathetic dominance (higher low frequency, LF, band), only occurred in DM E₂-replete (SO and OVX/E₂) rats, and was associated with higher neuronal activity (c-Fos) and higher levels of TNFα and phosphorylated death associated protein kinase-3 (p-DAPK3) in the PVN. These proinflammatory molecules likely contributed to the heightened PVN oxidative stress, injury and apoptosis. The PVN of these E₂-replete DM rats also exhibited upregulations of estrogen receptors, ERα and ERβ, and proinflammatory adenosine A1 and A2a receptors.

Significance: The E₂-dependent autonomic dysregulation likely predisposes DM female rats and women to hypersensitivity to cardiac dysfunction. Further, upregulations of proinflammatory mediators including adenosine A1 and A2 receptors, TNFα and DAPK3, conceivably explain the paradoxical hypersensitivity of DM females to PVN inflammation/injury and the subsequent autonomic dysregulation in the presence of E₂.

Keywords: Adenosine; Autonomic dysregulation; Diabetes; Estrogen; Hypothalamus.

Fig. 1.

Mean blood pressure (MAP), measured at the conclusion of the study, along with spectral indices of heart rate variability in the following diabetic (DM) and respective control groups (n=5 rats each): male, ovariectomized (OVX), sham operated (SO), and OVX with E₂ supplementation (OVX/E₂). (A) MAP (mmHg). (B) low-frequency (LFnu) (0.25–0.75 Hz) bands. (C) High-frequency (HFnu) (0.75–3 Hz) bands. (D) The LF/HF ratio depicting cardiac sympathovagal balance. Values are means ± SEM. *P < 0.05 vs. “male control” values. ^&P < 0.05 vs. SO control values.

Fig. 2.

Representative images (A) and group data (B) of FluoroJade C (FJC) positive neurons, depicting neurodegeneration, in the hypothalamic PVN of the following diabetic (DM) and respective control groups (n=5 each): male, ovariectomized (OVX), sham operated (SO), and OVX with E₂ supplementation (OVX/E₂). Group data show the neurodegeneration level expressed as the number of FJC positive cells measured by NIH ImageJ analysis of confocal images. Values are expressed as means ± SEM. *P < 0.05 vs. male control values. ^#P < 0.05 vs. DM male values.

Fig. 3.

Representative immunohistochemical images (A) and group data (B) of c-Fos expression in the hypothalamic PVN of the following diabetic (DM) and respective control groups (n=5 rats each): male, ovariectomized (OVX), sham operated (SO), and OVX with E₂ supplementation (OVX/E₂). Group data showing the mean number of c-Fos expression measured by NIH ImageJ analysis of immunohistochemical images. Values are expressed as means ± SEM. *P < 0.05 vs. male control values. ^#P < 0.05 vs. DM male values.

Fig. 4.

Representative confocal images (A) and group data (B) showing superoxide level measured by dihydroethidium (DHE) staining (red) as well as group data (C) showing the ROS level, measured by 2′,7′-dichlorofluorescein, in the hypothalamic PVN of the following diabetic (DM) and respective control groups (n=5 rats each): male, ovariectomized (OVX), sham operated (SO), and OVX with E₂ supplementation (OVX/E₂). Values are expressed as means ± SEM. *P < 0.05 vs. male control values. ^#P < 0.05 vs. DM male values.

Fig. 5.

Western blots analysis showing ERα (A), ERβ (B) (both normalized to GAPDH) and phosphorylated death associated protein kinase-3, p-DAPK3 (C) (normalized to total DAPK3) expression in the hypothalamic PVN of the following diabetic (DM) and respective control groups (n=5 rats each): male, ovariectomized (OVX), sham operated (SO), and OVX with E₂ supplementation (OVX/E₂). Values are expressed as means ± SEM. *P < 0.05 vs. “male control” values. ^#P < 0.05 vs. DM male values. ^&P < 0.05 vs. SO control values.

Fig. 6.

Representative confocal immunohistochemical images (A) and group data (B) of TNF-α levels in the hypothalamic PVN of the following DM and respective control groups (n=5 rats each): male, ovariectomized (OVX), sham operated (SO), and OVX with E₂ supplementation (OVX/E₂). TNF-α images were analyzed by NIH ImageJ. Values are means ± SEM. *P < 0.05 vs. male control values. ^#P < 0.05 vs. DM male values.

Fig. 7.

Representative immunohistochemical images (A) and group data (B) showing adenosine A1 receptor (A1-R) expression in the hypothalamic PVN of the following diabetic (DM) and respective control groups (n=5 rats each): male, ovariectomized (OVX), sham operated (SO), and OVX with E₂ supplementation (OVX/E₂). A1-R expression was quantified by NIH ImageJ analysis. Values are expressed as means ± SEM. *P < 0.05 vs. male control values.

Fig. 8.

Representative immunohistochemical images (A) and group data (B) showing adenosine A2a receptor (A2a-R) expression in the hypothalamic PVN of the following diabetic (DM) and respective control groups (n=5 rats each): male, ovariectomized (OVX), sham operated (SO), and ovariectomized with E₂ supplementation (OVX/E₂). A2-R expression was quantified by NIH ImageJ analysis. Values are expressed as means ± SEM. *P < 0.05 vs. male control values.