Glucocorticoid Receptor β Induces Hepatic Steatosis by Augmenting Inflammation and Inhibition of the Peroxisome Proliferator-activated Receptor (PPAR) α

Abstract

Glucocorticoids (GCs) regulate energy supply in response to stress by increasing hepatic gluconeogenesis during fasting. Long-term GC treatment induces hepatic steatosis and weight gain. GC signaling is coordinated via the GC receptor (GR) GRα, as the GRβ isoform lacks a ligand-binding domain. The roles of the GR isoforms in the regulation of lipid accumulation is unknown. The purpose of this study was to determine whether GRβ inhibits the actions of GCs in the liver, or enhances hepatic lipid accumulation. We show that GRβ expression is increased in adipose and liver tissues in obese high-fat fed mice. Adenovirus-mediated delivery of hepatic GRβ overexpression (GRβ-Ad) resulted in suppression of gluconeogenic genes and hyperglycemia in mice on a regular diet. Furthermore, GRβ-Ad mice had increased hepatic lipid accumulation and serum triglyceride levels possibly due to the activation of NF-κB signaling and increased tumor necrosis factor α (TNFα) and inducible nitric-oxide synthase expression, indicative of enhanced M1 macrophages and the development of steatosis. Consequently, GRβ-Ad mice had increased glycogen synthase kinase 3β (GSK3β) activity and reduced hepatic PPARα and fibroblast growth factor 21 (FGF21) expression and lower serum FGF21 levels, which are two proteins known to increase during fasting to enhance the burning of fat by activating the β-oxidation pathway. In conclusion, GRβ antagonizes the GC-induced signaling during fasting via GRα and the PPARα-FGF21 axis that reduces fat burning. Furthermore, hepatic GRβ increases inflammation, which leads to hepatic lipid accumulation.

Keywords: NAFLD; NASH; PPARα; fatty acid metabolism; fatty acid synthase (FAS); fatty liver; glucocorticoid receptor; hepatic steatosis; inflammation; lipid.

FIGURE 1.

GRα and GRβ expression in mice on a high fat diet. Real-time PCR of GRα, GRβ, and TNFα in adipose (A), liver (B), and muscle (C) tissues. ***, p < 0.001 (versus regular diet) (±S.E.; n = 4).

FIGURE 2.

GRβ adenovirus overexpressed in liver. A, representative Western blot of GRβ, GRα, and heat shock protein 90 (HSP90) as well as merged Odyssey image from livers of GRβ-Ad and vec-Ad mice (note: the HSP90 blots for α and β are the same). The arrows are indicative of the 102- and 92-kDa GRβ bands that we have previously reported (9). The two arrows for GRα are indicative of the major GRα-A isoform at 97 kDa and the truncated 55-kDa GRα-D. Real-time PCR expression of GRβ and GRα in liver (B), adipose (C), and muscle (D) of GRβ-Ad and vec-Ad mice. ***, p < 0.001 (versus vec-Ad mice) (±S.E.; n = 7).

FIGURE 3.

GRβ increases hepatic lipid accumulation. A, Nile red lipid staining in livers of GRβ-Ad and vec-Ad mice. B, measurement of hepatic triglycerides (TGs). *, p < 0.05 (versus vec-Ad mice) (±S.E.; n = 7). C, measurement of serum triglycerides. ***, p < 0.001 (versus vec-Ad mice) (±S.E.; n = 7). D, Western blot and densitometry as well as real-time PCR of fatty acid synthase (FAS) expression in livers of GRβ-Ad and vec-Ad mice. *, p < 0.05 (versus vec-Ad mice) (±S.E.; n = 6). E, measurement of fasting glucose and serum insulin levels from GRβ-Ad and vec-Ad mice. **, p < 0.01 (versus vec-Ad mice) (±S.E.; n = 7).

FIGURE 4.

GRβ activates GSK3β and suppresses gluconeogenic genes. A, glycogen content and glycogen synthase kinase 2 (GYS2) expression in livers of GRβ-Ad and vec-Ad mice. *, p < 0.05 (versus vec-Ad mice) (±S.E.; n = 7). B, GRβ overexpression decreases serine 9 phosphorylation of GSK3β. **, p < 0.01 (versus vec-Ad mice) (±S.E.; n = 7). C, gluconeogeneic genes were decreased in livers of GRβ-Ad mice compared with vec-Ad. **, p < 0.01 (versus vec-Ad mice) (±S.E.; n = 7). D, real-time expression of PTEN, Akt1, and Akt2 in livers GRβ-Ad and vec-Ad mice. *, p < 0.05; **, p < 0.01 (versus vec-Ad mice) (±S.E.; n = 6).

FIGURE 5.

GRβ increases NF-κB activity and M1 proinflammatory macrophages. A, representative Western blot and densitometry of serine 536 phosphorylation of NF-κB and total NF-κB as well as IκBα mRNA in livers of GRβ-Ad and vec-Ad mice. *, p < 0.05; **, p < 0.01 (versus vec-Ad mice) (±S.E.; n = 6). B, real-time PCR expression of F480 in livers of GRβ-Ad and vec-Ad mice. C, real-time PCR expression of TNFα, iNOS, Arg1, and FIZZ1 in livers of GRβ-Ad and vec-Ad mice. *, p < 0.05; **, p < 0.01 (versus vec-Ad mice) (±S.E.; n = 7). D, transient transfection of GRβ and p65 (NF-κB) and NF-κB-luc and PTEN-luc constructs in COS7 cells for 24 h. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 (versus p65- and GRβ− control); #, p < 0.05 (versus p65+ and GRβ− control) (±S.E.; n = 4). E, transient transfection of GRα and p65 (NF-κB) and NF-κB-luc and PTEN-luc constructs in COS7 cells for 24 h. *, p < 0.05; **, p < 0.01 (versus p65− and GRα− control); ##, p < 0.01; (versus p65+ and GRα− control) (±S.E.; n = 4).

FIGURE 6.

GRβ suppresses PPARα expression resulting in reduced FGF21 hormonal signaling. A, real-time PCR of PPARα, Glut1, and FGF21 in livers of GRβ-Ad and vec-Ad mice, as well as B, FGF21 serum levels. *, p < 0.05; **, p < 0.01 (versus vec-Ad mice); *, p < 0.05; **, p < 0.01 (versus vec-Ad mice) (±S.E.; n = 7), respectively. C, real-time PCR expression of Glut1, AMPK, and FGF21 in adipose of GRβ-Ad and vec-Ad mice. **, p < 0.01, ***, p < 0.001 (versus vec-Ad mice) (±S.E.; n = 7).

FIGURE 7.

GRβ inhibits PPARα transcriptional activity. A, transient transfection of GRβ, PPARα, RXRα, and PPRE-3tk-luc constructs in COS7 cells for 24 h following a 24-h WY-14,643 or vehicle (ctrl) treatment. ##, p < 0.01 (versus vector control); ***, p < 0.001 (versus vector WY) (±S.E.; n = 3). B, transient transfection of GRα, PPARα, RXRα, and PPRE-3tk-luc constructs in COS7 cells for 24 h following a 24-h WY-14,643 or vehicle (ctrl) treatment. ##, p < 0.01 (versus vector control); **, p < 0.01 (versus vector WY) (±S.E.; n = 3). C, transient transfection of GRβ, GRα, vector, and FGF21-luc constructs in COS7 cells for 24 h. **, p < 0.001 (versus vector) (±S.E.; n = 3). D, transient transfection of GRα, GRβ, PPARα, RXRα, and FGF21-luc constructs in COS7 cells for 24 h following a 24-h WY-14,643 or vehicle (ctrl) treatment. **, p < 0.01 (versus vector WY) (±S.E.; n = 3).

FIGURE 8.

Diagram of hepatic GRβ signaling to increase fatty liver and inflammation.