Astrocyte IKKβ/NF-κB signaling is required for diet-induced obesity and hypothalamic inflammation

Abstract

Objective: Obesity and high fat diet (HFD) consumption in rodents is associated with hypothalamic inflammation and reactive gliosis. While neuronal inflammation promotes HFD-induced metabolic dysfunction, the role of astrocyte activation in susceptibility to hypothalamic inflammation and diet-induced obesity (DIO) remains uncertain.

Methods: Metabolic phenotyping, immunohistochemical analyses, and biochemical analyses were performed on HFD-fed mice with a tamoxifen-inducible astrocyte-specific knockout of IKKβ (Gfap^CreERIkbkb^fl/fl, IKKβ-AKO), an essential cofactor of NF-κB-mediated inflammation.

Results: IKKβ-AKO mice with tamoxifen-induced IKKβ deletion prior to HFD exposure showed equivalent HFD-induced weight gain and glucose intolerance as Ikbkb^fl/fl littermate controls. In Gfap^CreERTdTomato marker mice treated using the same protocol, minimal Cre-mediated recombination was observed in the mediobasal hypothalamus (MBH). By contrast, mice pretreated with 6 weeks of HFD exposure prior to tamoxifen administration showed substantially increased recombination throughout the MBH. Remarkably, this treatment approach protected IKKβ-AKO mice from further weight gain through an immediate reduction of food intake and increase of energy expenditure. Astrocyte IKKβ deletion after HFD exposure-but not before-also reduced glucose intolerance and insulin resistance, likely as a consequence of lower adiposity. Finally, both hypothalamic inflammation and astrocytosis were reduced in HFD-fed IKKβ-AKO mice.

Conclusions: These data support a requirement for astrocytic inflammatory signaling in HFD-induced hyperphagia and DIO susceptibility that may provide a novel target for obesity therapeutics.

Keywords: ARC, arcuate nucleus; Agrp, Agouti-related peptide; Astrocytes; Bdnf, brain-derived neurotrophic factor; Cart, cocaine- and amphetamine-regulated transcript; Ccl2, C–C motif chemokine ligand 2; DIO, diet-induced obesity; DMH, dorsomedial hypothalamus; Energy homeostasis; GFAP, glial fibrillary acidic protein; GSIS, glucose-stimulated insulin secretion; GTT, glucose tolerance test; HFD, high-fat diet; Hypothalamus; IHC, immunohistochemistry; IKKβ, inhibitor of kappa B kinase beta; ITT, insulin tolerance test; Iba1, ionized calcium binding adaptor molecule 1; Il, interleukin; Inflammation; LPS, lipopolysaccharide; MBH, mediobasal hypothalamus; Metabolism; NF-κB, nuclear factor kappa B; Npy, neuropeptide Y; Obesity; Pomc, proopiomelanocortin; RER, respiratory exchange ratio; TMX, tamoxifen; Tnfa, tumor necrosis factor α; VMN, ventromedial nucleus; ir, immunoreactivity.

Graphical abstract

Figure 1

Deletion of IKKβ in astrocytes prior to HFD introduction does not alter DIO sensitivity or hypothalamic astrogliosis. (A) Experimental design. (B) Weight gain after HFD exposure (n = 6 per group). (C) Food intake prior to and after introduction of HFD (n = 6 per group). (D) Glucose tolerance test (GTT, 2 mg/kg dextrose i.p.) (n = 6–9 per group). (E) Area-under-curve of GTT. (F) Representative images of GFAP immunostaining in the MBH (ARC and VMN regions) of chow and HFD-fed mice at 21 weeks old. Scale bar is 100 μm. (G) Relative quantification of GFAP immunoreactivity (ir) in the MBH (4 sections per mouse analyzed, n = 4 per group). Values are mean ± SEM, 2-way ANOVA with Bonferroni post-hoc test, ***p < 0.001.

Figure 2

Efficiency of Cre-mediated recombination in MBH astrocytes is increased by HFD feeding. (A) Representative images of tdTomato fluorescence in vehicle (corn oil) and TMX-treated Gfap^CreERROSA26-stop^fl/fl-tdTomato reporter mice. Scale bars are 200 μm. (B) Experimental design to investigate the effect of HFD on CreER-mediated recombination. (C) Representative images of tdTomato fluorescence in hypothalami from age-matched mice administered TMX before (top row, TMX-HFD) or after 6 weeks HFD (bottom row, HFD-TMX). Scale bars: 200 μm for hypothalamus, 100 μm for MBH and DMH. (D) Relative quantification of tdTomato fluorescence in hypothalamic regions (2–4 sections per mouse analyzed, n = 3–4 per group). Values are mean ± SEM, Student's t-test, **p < 0.01.

Figure 3

Astrocyte-specific IKKβ deletion following 6 weeks of HFD feeding results in DIO resistance and metabolic improvements. (A) Experimental design. (B) Weight gain after TMX treatment. (C) HFD food intake prior to and after TMX. (G–J) GTT assessed at 3 weeks (G and H) and 9 weeks (I and J) after TMX. (K–L) Insulin tolerance test (ITT, 1.5 U/kg i.p.) at 20 weeks after TMX. (M–N) Glucose-stimulated insulin secretion (GSIS, 2 mg/kg dextrose i.p.) at 20 weeks after TMX. All analyses n = 5–7 per group. Values are mean ± SEM, repeated measures ANOVA and Student's t-test, ^#p = 0.06, ^∗p < 0.05, **p < 0.01.

Figure 4

HFD-TMX astrocyte IKKβ KO mice have decreased hypothalamic astrogliosis and inflammation. (A) Representative images of GFAP-ir in the MBH (top) and ARC (bottom). (B) Relative quantification of GFAP-ir (6 sections per mouse analyzed, n = 3 per group). (C) Total GFAP⁺ cell counts per unilateral ARC. (D) Quantification of individual cell area based on GFAP-ir. (E) Representative images of Iba1-ir. (F) Relative quantification of Iba1-ir. Hypothalamic expression by qRT-PCR (n = 4–6 per group) of (G) glial markers, (H) cytokines, and (I) neuropeptides. All scale bars are 100 μm. Values are mean ± SEM, Student's t-test, *p < 0.05.