Epididymal Fat-Derived Sympathoexcitatory Signals Exacerbate Neurogenic Hypertension in Obese Male Mice Exposed to Early Life Stress

Abstract

[Figure: see text].

Keywords: adiposity; blood pressure; capsaicin; leptin; serotonin.

Figure 1.

Acute eWAT stimulation with capsaicin (CAP) exacerbated mean arterial pressure (MAP) response in obese MSEW male mice. A, Blood pressure trace in mice fed a low fat diet (LF). B, Blood pressure trace in mice fed a high fat diet (HF) (C), 30-min area under the curve (AUC) in response to CAP. Data were analyzed by ANOVA repeated measures for continuous blood pressure trace and 2-way ANOVA followed by Tukey multiple comparisons post hoc test for AUC analysis. Data were reported as mean±SEM. n=8 per group. eWAT indicates epididymal white adipose tissue; MSEW, maternal separation and early weaning; and VEH, vehicle. *P<0.05 vs control.

Figure 2.

Acute eWAT stimulation with capsaicin (CAP) increases neuronal activation in organum vasculosum of the lamina terminalis (OVLT), paraventricular nucleus of the hypothalamus (PVN), and rostroventrolateral medulla (RVLM) in obese MSEW male mice. A, Pattern of Fos immunoreactivity in the OVLT (0.50 mm from bregma; top), posterior PVN (−1.06 mm from bregma; middle), and RVLM (−6.72 mm from bregma; bottom) of control and MSEW mice infused with vehicle (VEH) or CAP in eWAT. A dotted shape delimits the brain areas in each image in which the quantification was performed. White arrows indicate Fos positive cells. Microphotographs (×10) for OVLT, PVN, and RVLM were acquired using upright Zeiss LSM 880 multiphoton microscope and ZEISS ZEN Digital Imaging for Light Microscopy software (cFos: red filter). Scale bar, 100 μm. Representative schematic figures in stereotaxic coordinates for each brain area analyzed are included in the Data Supplement. B, Average number of Fos positive cells in OVLT, posterior PVN, RVLM, and nucleus of the solitary tract (NTS) control and MSEW mice infused with VEH or CAP in eWAT. Data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons post hoc test. Data were reported as mean±SEM. n=6-7 per group. For Fos positive cell quantification, ×10 magnification images were visualized with Nikon Super Resolution Inverted Microscope (cFos: red filter) and acquired with Nikon NIS-Elements (NIS-Elements Software, version 4.00.08; Nikon Instruments, Inc). 3 V indicates third ventricle; eWAT, epididymal white adipose tissue; MSEW, maternal separation early weaning; and NA, nucleus ambiguus. *P<0.05 vs MSEW-high fat (HF), VEH and control-HF CAP.

Figure 3.

Renal denervation abolishes the differences in blood pressure in obese MSEW male mice. A, Bilateral renal denervation (RNDX) abolished the acute changes in mean arterial pressure (MAP) in response to epididymal white adipose tissue stimulation with capsaicin (CAP). B, RNDX blunts the differences in chronic MAP. C, Renal cortex norepinephrine (NE) content was dramatically reduced in all mice subjected to RDNX. Data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons post hoc test. Data were reported as mean±SEM. n=5 to 6 per group. HF indicates high fat diet; MSEW, maternal separation early weaning; and VEH, vehicle. *P<0.05 vs control; #P<0.05 vs SHAM.

Figure 4.

Selective afferent denervation in eWAT lowers blood pressure and sympathetic index in obese MSEW male mice. A, Blood pressure trace before and after resiniferotoxin (RTX) injections in eWAT. B, Twenty-four–hour blood pressure after 2 d of sham or RTX surgery. C, Acute mecamylamine-induced blood pressure reduction. D, Representative images of Sham and RTX-treated eWAT using the CGRP (calcitonin gene-related peptide) reporter for sensory neuron Calca mouse. White arrows indicate the GFP (green fluorescence protein) labeled sensory neurons in eWAT. Intravital imaging of exposed fat pads was assessed on the stage of the multiphoton photon microscope (30 Hz full frame acquisition, Scientifica, Ltd, Uckfield, United Kingdom) using a 16X Nikon objective. Green pseudo coloring and contrast were applied after images were acquired using Image J processing software (Image J 1.53c, http://imagej.nih.gov/ij; National Institutes of Health). Scale bar, 50 µm. Data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons post hoc test. Data were reported as mean±SEM. n=8 per group. HF indicates high fat diet; eWAT, epididymal white adipose tissue; MAP, mean arterial pressure; and MSEW, maternal separation early weaning. *P<0.05 vs control.

Figure 5.

Obese MSEW male mice display increased serotonin content in epididymal eWAT. A, RT-qPCR profiler showing significant increases in trytophan hydroxylase 1. B, Serotonin content is eWAT determined by ELISA. Data were analyzed by the Student t test. Data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons post hoc test when considering low fat (LF)–high fat (HF; Table SIV). Data were reported as mean±SEM. n=5 to 8 per group. Bdkrb1 indicates bradykinin receptor, beta 1; Bdkrb2, bradykinin receptor, beta 2; Htr2a, hydroxytryptamine (serotonin) receptor 2A; IL1β, interleukin 1 beta; IL6, interleukin-6; Lepr, leptin receptor; Ngf, nerve growth factor; NOX4, NADPH oxidase 4; p47phox (Ncf1), neutrophil cytosolic factor 1; Tnf, tumor necrosis factor; Tph1, tryptophan hydroxylase 1; and TrpV1, transient receptor potential cation channel, subfamily V, member 1. eWAT indicates epididymal white adipose tissue; MSEW, maternal separation early weaning; and RT-qPCR, Real Time quantitative Reverse Transcription Polymerase Chain Reaction. *P<0.05 vs control.