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. 2016 Jul;34(7):1883-95.
doi: 10.1002/stem.2358. Epub 2016 Mar 28.

Targeting IκB Kinase β in Adipocyte Lineage Cells for Treatment of Obesity and Metabolic Dysfunctions

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Free PMC article

Targeting IκB Kinase β in Adipocyte Lineage Cells for Treatment of Obesity and Metabolic Dysfunctions

Robert N Helsley et al. Stem Cells. .
Free PMC article

Abstract

IκB kinase β (IKKβ), a central coordinator of inflammation through activation of nuclear factor-κB, has been identified as a potential therapeutic target for the treatment of obesity-associated metabolic dysfunctions. In this study, we evaluated an antisense oligonucleotide (ASO) inhibitor of IKKβ and found that IKKβ ASO ameliorated diet-induced metabolic dysfunctions in mice. Interestingly, IKKβ ASO also inhibited adipocyte differentiation and reduced adiposity in high-fat (HF)-fed mice, indicating an important role of IKKβ signaling in the regulation of adipocyte differentiation. Indeed, CRISPR/Cas9-mediated genomic deletion of IKKβ in 3T3-L1 preadipocytes blocked these cells differentiating into adipocytes. To further elucidate the role of adipose progenitor IKKβ signaling in diet-induced obesity, we generated mice that selectively lack IKKβ in the white adipose lineage and confirmed the essential role of IKKβ in mediating adipocyte differentiation in vivo. Deficiency of IKKβ decreased HF-elicited adipogenesis in addition to reducing inflammation and protected mice from diet-induced obesity and insulin resistance. Further, pharmacological inhibition of IKKβ also blocked human adipose stem cell differentiation. Our findings establish IKKβ as a pivotal regulator of adipogenesis and suggest that overnutrition-mediated IKKβ activation serves as an initial signal that triggers adipose progenitor cell differentiation in response to HF feeding. Inhibition of IKKβ with antisense therapy may represent as a novel therapeutic approach to combat obesity and metabolic dysfunctions. Stem Cells 2016;34:1883-1895.

Keywords: Adipogenesis; Adipose stem cells; Antisense oligonucleotide; IκB kinase β; Obesity.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

R.G.L. is an employee at Ionis Pharmaceuticals. P.A.K. received research funding from Kindex Pharmaceuticals. The other authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Pharmacological inhibition of IKKβ with ASOs protects mice from diet-induced obesity, improves insulin sensitivity, and reverses hepatic steatosis in obese mice. (A): IKKβ mRNA expression in liver, kidney, spleen, skeletal muscle (Sk.M.), brown adipose tissue (BAT), subcutaneous white adipose tissue (subWAT), retroperitoneal WAT (retroWAT), and epididymal WAT (epiWAT) from mice treated with control ASO or IKKβ ASO for 8 weeks (n=6–10; *P<0.05, ***P<0.001, assessed by Student’s t-test). (B): Western blot analysis of IKKβ and IKKα expression in liver and epiWAT from mice treated with control or IKKβ ASO for 4 weeks. (C, D): Growth curves (C), and fat mass and lean mass (D) of ND and HFD-fed mice treated with control ASO or IKKβ ASO (n=10 for ND and 30 for HFD; *P<0.05, **P<0.01, and ***P<0.001, assessed by two-way ANOVA). (E): Representative images of adipose depots (top) and weight of adipose depots (bottom) from mice treated with control or IKKβ ASO for 8 weeks (n=20; ***P<0.001, assessed by Student’s t-test). Results are presented as means ± SEM. (F): Fasting plasma glucose and insulin levels of HFD-fed mice treated with control or IKKβ ASO (n=29–30; ***P<0.001, assessed by Student’s t-test). (G): Intraperitoneal glucose tolerance test (IPGTT), intraperitoneal insulin tolerance tests (IPITT), and area of curve (AUC) of IPGTT and IPITT of HFD-fed mice treated with control or IKKβ ASO (n=8–10; **P<0.01, ***P<0.001, assessed by Student’s t-test). (H): Western blot analysis of phosphorylated Akt and total Akt levels in liver, epiWAT, and skeletal muscle of control or IKKβ ASO-treated mice injected with saline or 0.35U/kg body weight. (I): Glucose uptake was measured in primary adipose tissues from mice treated with control or IKKβ ASO (n=9; **P<0.01, ***P<0.001, assessed by two-way ANOVA). (J, K): Representative appearance (J) and hematoxylin and eosin (top) and Oil-red-O (bottom) stained sections (K) of livers from mice treated with control or IKKβ ASO (scale bar =100 μm). (L): Hepatic cholesterol and triglyceride levels of mice treated with control or IKKβ ASO (n=10; ***P<0.001, assessed by Student’s t-test). Results are presented as means ± SEM.
Figure 2
Figure 2
IKKβ regulates murine adipocyte differentiation. (A): IKKβ mRNA expression in adipose SVF and mature adipocytes isolated from epiWAT of mice treated with control or IKKβ ASO (n=6; **P<0.01, assessed by Student’s t-test). (B): Oil-red-O staining of adipose SV cells isolated from epiWAT of mice treated with control and IKKβ ASO induced by differentiation media (scale bar=100 μm, bottom panels). (C): Expression of adipogenic genes in epiWAT of control and IKKβ ASO treated mice was measure by QPCR (n=6; **P<0.01, ***P<0.001, assessed by Student’s t-test). (D): Western blot analysis of Smurf2 protein levels in epiWAT. (E): Western blot analysis of nuclear β-catenin levels in epiWAT. (F): Western blot analysis of IKKβ and IKKα protein levels in control 3T3-L1 cells or CRISPR-mediated IKKβ-deficient 3T3-L1 cells. (G): Oil-red-O staining of control and IKKβ-deficient 3T3-L1 cells induced by differentiation media (scale bar=100μm, bottom panels). (H): Expression of adipogenic genes and adipocyte markers in control or IKKβ-deficient 3T3-L1 cells (n=6; **P<0.01, ***P<0.001, assessed by Student’s t-test). (I): Western blot analysis of Smurf2 protein levels in control or IKKβ-deficient 3T3-L1 cells. (J): Western blot analysis of nuclear β-catenin protein levels in control or IKKβ-deficient 3T3-L1 cells. (K): β-catenin reporter (TOP-flash reporter) activity in control or IKKβ-deficient 3T3-L1 cells (n=6; ***P<0.001, assessed by Student’s t-test). (L): Control or IKKβ-deficient 3T3-L1 cells were treated with vehicle control or 100 nM PS-341, as indicated, for 4 h. β-catenin was immunoprecipitated with anti-β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole cell lysates were probed with anti-β-catenin antibodies as an internal control. Results are presented as means ± SEM.
Figure 3
Figure 3
Generation of mice lacking IKKβ in the white adipose lineage. (A): Western blot analysis of IKKβ and IKKα protein levels in epiWAT, subWAT, liver, BAT, and skeletal muscle (Sk.M.) of IKKβF/F and IKKβΔPDGFRβ mice. (B): Western blot analysis of IKKβ and IKKα protein levels in adipose SV cells isolated from IKKβF/F and IKKβΔPDGFRβ mice. (C–G): Adipose SV cells isolated from IKKβF/F and IKKβΔPDGFRβ mice were treated with LPS for 3 h. The expression levels for proinflammatory cytokines including TNFα (C), MCP-1 (D), IL-1α (E), IL-1β (F), and IL-6 (G) were examined by QPCR (n=5; **P<0.01, ***P<0.001, assessed by two-way ANOVA). Results are presented as means ± SEM.
Figure 4
Figure 4
Deficiency of IKKβ in adipocyte lineage cells renders mice resistant to diet-induced obesity. (A): Growth curves of ND or HFD-fed IKKβF/F and IKKβΔPDGFRβ mice (n=9–18, ***P <0.001 when comparing HFD-fed IKKβF/F mice to HFD-fed IKKβΔPDGFRβ mice, assessed by Student’s t-test). (B): Body weight, fat mass, percentage of fat, and lean mass of 10-week-old IKKβF/F and IKKβΔPDGFRβ mice fed a ND or HFD for 16 weeks (n=7–19; **P<0.01, ***P<0.001, assessed by two-way ANOVA). (C): Representative photographs of epiWAT, retroWAT, subWAT, and BAT from ND or HFD-fed IKKβF/F and IKKβΔPDGFRβ mice. (D, E): Representative coronal section MRI images (D) and visceral and subcutaneous adipose tissue volume (E) of HFD-fed IKKβF/F and IKKβΔPDGFRβ mice (n=3; ***P<0.001, assessed by Student’s t-test). Results are presented as means ± SEM.
Figure 5
Figure 5
IKKβ-deficient mice are protected from obesity-associated metabolic disorders. (A): Fasting plasma glucose and insulin levels in ND or HFD-fed IKKβF/F and IKKβΔPDGFRβ mice (n=5–11; **P<0.01, ***P<0.001, assessed by two-way ANOVA). (B): IPGTT, IPITT, and area under the curve (AUC) of IPGTT and IPITT of HFD-fed IKKβF/F and IKKβΔPDGFRβ mice (n=6–11; *P<0.05, **P<0.01, ***P<0.001, assessed by Student’s t-test). (C): Western blot analysis of phosphorylated Akt and total Akt levels in epiWAT, liver, and skeletal muscle of IKKβF/F and IKKβΔPDGFRβ mice injected with saline or 0.35U/kg body weight. (D): Glucose uptake was measured in primary adipose tissues from HFD-fed IKKβF/F and IKKβΔPDGFRβ mice (n=9; ***P<0.001, assessed by two-way ANOVA). (E): Western blot analysis of IKKβ protein levels in peritoneal macrophages of IKKβF/F and IKKβΔPDGFRβ mice. (F): Representative immunohistochemistry for the macrophage marker, F4/80 in epiWAT from HFD-fed IKKβF/F and IKKβΔPDGFRβ mice (scale bar = 100μm). (G): The expression levels of pro-inflammatory genes and macrophage markers in epiWAT of HFD-fed IKKβF/F and IKKβΔPDGFRβ mice were measured by QPCR (n=5; *P<0.05, **P<0.01, ***P<0.001, assessed by Student’s t-test). (H): Plasma cytokine levels of ND or HFD-fed IKKβF/F and IKKβΔPDGFRβ mice (n=5–7; **P<0.01, ***P<0.001, assessed by two-way ANOVA). Results are presented as means ± SEM.
Figure 6
Figure 6
Deficiency of IKKβ inhibits adipogenesis in mice. (A, B): Representative images (A) and quantification (B) of immunostaining for BrdU in epiWAT from BrdU-treated IKKβF/F and IKKβΔPDGFRβ mice fed a HFD for 7 days (n=4–5; **P<0.01, significance assessed by Student’s t-test). The nuclei were stained with DAPI (blue) and the BrdU-positive cells are indicated by arrows. (C): Oil-red-O staining of adipose SV cells isolated from epiWAT of IKKβF/F and IKKβΔPDGFRβ mice induced by differentiation media. (scale bar=100μm, bottom panels). (D): The expression levels of adipogenic genes and adipocyte markers in adipose SV cells of IKKβF/F and IKKβΔPDGFRβ mice were measured by QPCR (n=5–8; *P<0.05, **P<0.01, ***P<0.001, assessed by Student’s t-test). (E, F): Western blot analysis of Smurf2 protein levels (E) and nuclear β-catenin protein levels (F) in epiWAT of IKKβF/F and IKKβΔPDGFRβ mice. (G, H): Western blot analysis of Smurf2 protein levels (G) and nuclear β-catenin protein levels (H) of adipose SV cells isolated from IKKβF/F and IKKβΔPDGFRβ mice. (I): Adipose SV cells isolated from IKKβF/F and IKKβΔPDGFRβ mice were treated with vehicle control or 100 nM PS-341, as indicated, for 4 h. β-catenin was immunoprecipitated with anti-β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole cell lysates were probed with anti-β-catenin antibodies as an internal control. Results are presented as means ± SEM.
Figure 7
Figure 7
Inhibition of IKKβ decreases adipogenesis in human adipose stem cells. (A): Oil-redO staining of adult-derived human adipose stem cells induced by differentiation media or media containing 5 μM IKKβ inhibitor BMS-345541 (scale bar=100μm, bottom panels). (B): The expression levels of adipogenic genes and adipocyte markers of human adipose stem cells treated with vehicle control or 5μM BMS-345541 were measured by QPCR (n=4; *P<0.05, **P<0.01, ***P<0.001, assessed by Student’s t-test). (C, D): Western blot analysis of Smurf2 protein levels (C) and nuclear β-catenin protein levels (D) in control or BMS-345541-treated human adipose stem cells. (E): Control or BMS-345541-treated human adipose stem cells were treated with vehicle control or 100 nM PS-341, as indicated, for 4 h. β-catenin was immunoprecipitated with anti-β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole cell lysates were probed with anti-β-catenin antibodies as an internal control. **p<0.01, ***p<0.001. Results are presented as means ± SEM.

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