Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet

Abstract

Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR(betageo/+) mice were generated from embryonic stem (ES) cells with a gene trap integration of a beta-galactosidase-neomycin phosphotransferase (betageo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GR(betageo/+) mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin-aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GR(betageo/+) mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GR(betageo/+) mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet.

Figure 1.

The GR-βgeo fusion protein is transcriptionally inactive. A) Schematic view of the functional domains of GR (top), exon structure of GR cDNA (middle), and predicted structure of the GR-βgeo chimeric mRNA (bottom). The N-terminal domain (NTD) of GR is encoded by exon 2 (exon 1 is noncoding), the DNA binding domain (DBD) by exons 3–4, and the ligand binding domain (LBD) by exons 5–9. The βgeo cassette replaces exons 4–9. B, C) HEK293 cells were transiently transfected with WT mouse GR, GR-βgeo, or “empty” vector together with MMTV LTR-luciferase (B) or PMNT-998/-466-luciferase (C) reporters and then incubated without (white bars) or with (black bars) 1 μM dexamethasone. Data are means ± se of 3 independent experiments, each performed in triplicate. Values are expressed relative to vector, arbitrarily set to 1. ***P < 0.001 vs. untreated mice. Note the log scale for promoter activity. A.U., arbitrary units.

Figure 2.

GR^βgeo/+ mice have reduced GR mRNA levels in the brain, with normal distribution of GR-βgeo expression. A) Representative X-gal stained GR^βgeo/+ brain section showing strong staining in a pattern identical to GR mRNA distribution in hippocampal subfields CA1/2, dentate gyrus (DG), and paraventricular nucleus of the hypothalamus (PVN), with weaker staining in cortex and thalamus. B, C) Representative autoradiographs showing in situ hybridization of GR mRNA in brain coronal sections of GR^+/+ mice (B) and GR^βgeo/+ mice (C). D) Quantification of GR mRNA levels [optical density (OD)] in the PVN of GR^+/+ (white bars) and GR^βgeo/+ mice (black bars) (n=6/group; *P<0.05).

Figure 3.

GR^βgeo/+ mice have reduced functional GR protein levels. Representative Western blot analysis showing decreased normal GR (95 kDa) in epididymal fat of GR^βgeo/+ (+/−) mice compared with GR^+/+ (+/+) mice (top). The GR-βgeo fusion protein (191 kDa) was apparent only in GR^βgeo/+ mice. Tubulin (middle) and ponceau red staining (bottom) demonstrate equivalent protein loading.

Figure 4.

HPA hyperactivity in GR^βgeo/+ mice. A, B) Plasma corticosterone (cort) levels in LF-fed GR^+/+ (white bars), HF-fed GR^+/+ (hatched bars), LF-fed GR^βgeo/+ (black bars) and HF-fed GR^βgeo/+ mice (gray bars) in the morning (A) and evening (B). C) Left adrenal weights in LF or HF-fed GR^+/+ and GR^βgeo/+ mice (n=8–9/group). *^,†P < 0.05; **P < 0.01; ^†††P < 0.001. ‡P < 0.01 vs. HF-fed GR^+/+ mice.

Figure 5.

GR^βgeo/+ mice have larger adrenal glands. Representative images of hematoxylin and eosin stained sections of adrenal glands from LF-fed GR^+/+ mice (A, C); LF-fed GR^βgeo/+ mice (B, D); HF-fed GR^+/+ mice (E); and HF-fed GR^βgeo/+ mice (F). ZF, zona fasciculate; ZG, zona glomerulosa; MED, medulla. n = 8–9/group. Scale bars = 25 μm.

Figure 6.

Unaltered body composition and glucose homeostasis in GR^βgeo/+ mice after HF diet. Body weight (A) and plasma glucose levels (B) after GTT in LF-fed GR^+/+ mice (open squares, dashed line); HF-fed GR^+/+ mice (open circles, dashed line); LF-fed GR^βgeo/+ mice (black squares, solid line) and HF-fed GR^βgeo/+ mice (black circles, solid line) (n=6/group). ***P < 0.001.

Figure 7.

GR^βgeo/+ mice have elevated blood pressure. Systolic blood pressure (SBP) in LF-fed GR^+/+ mice (white bars), HF-fed GR^+/+ mice (hatched bars), LF-fed GR^βgeo/+ mice (black bars), and HF-fed GR^βgeo/+ mice (gray bars). *P < 0.05; **^,††P < 0.01.

Figure 8.

Activation of the RAAS in GR^βgeo/+ mice. Plasma renin activity (A), plasma aldosterone concentration (B), plasma angiotensinogen concentration (C), epididymal fat angiotensinogen (AGT) mRNA levels (D), and hepatic AGT mRNA levels (E) in LF-fed GR^+/+ mice (white bars), HF-fed GR^+/+ mice (hatched bars), LF-fed GR^βgeo/+ mice (black bars), and HF-fed GR^βgeo/+ mice (gray bars). For plasma measurements, n = 5/group. For mRNA levels, n = 8–9/group. *^,†P < 0.05; **^,††P < 0.01; ***^,†††P < 0.001.