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Cited by 252Regulation of Appetite and ObesityAdipokines, Inflammation, and Metabolic DiseasesNeuroinflammation and Neurodegeneration Mechanisms

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

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Douglass, J.D., Dorfman, M.D., Fasnacht, R., Shaffer, L.D., Thaler, J.P.·2017·Molecular Metabolism
DOI 10.1016/j.molmet.2017.01.010PMC PMC5369266
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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 CreER Ikbkb 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 CreER TdTomato 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.

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Graphical abstract Graphical abstract Overview of astrocyte IKKβ inactivation in chronic HFD-fed mice. Rodents exposed to HFD have increased hypothalamic inflammation and accumulation of reactive astrocytes in the mediobasal hypothalamus. Conditional deletion of IKKβ/NFκB in astrocytes of HFD-fed mice results in obesity resistance, improved glucose homeostasis, and decreased hypothalamic inflammation. 3V = third ventricle
Graphical abstract Graphical abstract Overview of astrocyte IKKβ inactivation in chronic HFD-fed mice. Rodents exposed to HFD have increased hypothalamic inflammation and accumulation of reactive astrocytes in the mediobasal hypothalamus. Conditional deletion of IKKβ/NFκB in astrocytes of HFD-fed mice results in obesity resistance, improved glucose homeostasis, and decreased hypothalamic inflammation. 3V = third ventricleCC BY-NC-ND · PubMed Central
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 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.CC BY-NC-ND · PubMed Central
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 GfapCreERROSA26-stopfl/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 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 GfapCreERROSA26-stopfl/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.CC BY-NC-ND · PubMed Central
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 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.CC BY-NC-ND · PubMed Central
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.
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.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Douglass, J. D., Dorfman, M. D., Fasnacht, R., Shaffer, L. D., & Thaler, J. P.
Affiliations
University of Washington (US)
Corresponding author
Thaler, J.P.
ORCID
Venue
Molecular Metabolism
Publisher
Elsevier BV
Published
2017-04
Volume
6
Issue
4
Pages
366-373
ISSN
2212-8778 (ISSN-L)
DOI
10.1016/j.molmet.2017.01.010
PMC
PMC5369266
OpenAlex
W2581154578
Cited by
248
References
37
Citations by year
2026: 12 · 2025: 23 · 2024: 28 · 2023: 34 · 2022: 32 · 2021: 31 · 2020: 31 · 2019: 26 · 2018: 20 · 2017: 11
FWCI
14.72
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Metabolism (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • American Diabetes Association (1-14-ACE-51)
  • NIDDK (K08 DK088872, T32 DK7247-37, F32 DK108473-01)
  • NIH (DK035816)
  • Diabetes Research Center (DK017047)
Topics
  • Life Sciences › Neuroscience › Endocrine and Autonomic Systems › Regulation of Appetite and Obesity
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adipokines, Inflammation, and Metabolic Diseases
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
diet-induced obesity, hypothalamic inflammation, astrocytes, IKKβ, NF-κB signaling, astrocyte-specific knockout, high-fat diet, impaired glucose tolerance, insulin resistance, hyperphagia, energy expenditure, reactive gliosis, astrocyte activation, mediobasal hypothalamus, obesity treatment
MeSH terms
Animals, Astrocytes, Cells, Cultured, Gliosis, Hypothalamus, Inflammation, Male, Mice, Inbred C57BL, Obesity, Signal Transduction, NF-kappa B, Mice, I-kappa B Kinase, Diet, High-Fat

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