Deficiency for costimulatory receptor 4-1BB protects against obesity-induced inflammation and metabolic disorders

Abstract

Objective: Inflammation is an important factor in the development of insulin resistance, type 2 diabetes, and fatty liver disease. As a member of the tumor necrosis factor receptor superfamily (TNFRSF9) expressed on immune cells, 4-1BB/CD137 provides a bidirectional inflammatory signal through binding to its ligand 4-1BBL. Both 4-1BB and 4-1BBL have been shown to play an important role in the pathogenesis of various inflammatory diseases.

Research design and methods: Eight-week-old male 4-1BB-deficient and wild-type (WT) mice were fed a high-fat diet (HFD) or a regular diet for 9 weeks.

Results: We demonstrate that 4-1BB deficiency protects against HFD-induced obesity, glucose intolerance, and fatty liver disease. The 4-1BB-deficient mice fed an HFD showed less body weight gain, adiposity, adipose infiltration of macrophages/T cells, and tissue levels of inflammatory cytokines (e.g., TNF-α, interleukin-6, and monocyte chemoattractant protein-1 [MCP-1]) compared with HFD-fed control mice. HFD-induced glucose intolerance/insulin resistance and fatty liver were also markedly attenuated in the 4-1BB-deficient mice.

Conclusions: These findings suggest that 4-1BB and 4-1BBL may be useful therapeutic targets for combating obesity-induced inflammation and metabolic disorders.

FIG. 1.

HFD upregulates 4-1BB and 4-1BBL gene expression in adipose tissue and liver. C57BL/6 mice were fed an HFD or RD for 9 weeks. Levels of the mRNAs of 4-1BB and 4-1BBL in the epididymal adipose tissue (upper) and liver (lower) of mice fed an RD or HFD. Levels of mRNA were estimated by quantitative PCR. Results are means ± SEM (n = 4 mice per group). *P < 0.05, **P < 0.01 compared with RD. WAT, white adipose tissue.

FIG. 2.

Body weight change and adiposity in HFD-fed 4-1BB–deficient mice. WT and 4-1BB–deficient mice were fed an HFD for 9 weeks. A: Expression of 4-1BB mRNA in epididymal adipose tissue, liver, and skeletal muscle. Body weight changes and gross morphology of mice (B), energy intake (C), and adipose tissue weight (Ep, epididymal; Re, retroperitoneal; Me, mesenteric; and Sc, subcutaneous) of WT (n = 8) and 4-1BB–deficient mice (n = 8) fed an RD or HFD, and gross morphology of adipose tissues (D). Results are means ± SEM. *P < 0.05, **P < 0.01, #P < 0.005 compared with WT mice fed an HFD. E: Histological analysis of epididymal adipose tissue and size distribution of adipocytes from WT and 4-1BB–deficient mice fed an HFD. Sections were stained with hematoxylin-eosin. Hypertrophied adipocytes are indicated by asterisks. Original magnification is ×200 (scale bar = 50 μm). The sizes of adipocytes in randomly chosen fields were measured with a microscope (magnification ×200) and calculated using Axiovision AC software. #P < 0.005 compared with WT mice fed an HFD. WAT, white adipose tissue. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

Energy expenditure, locomotor activity, and body temperature in 4-1BB–deficient mice. A: Energy expenditure (EE) was measured in WT (n = 4) and 4-1BB–deficient mice (n = 4) fed an HFD. Locomotor activity (B) and body temperature (C) were measured in WT (n = 10–11) and 4-1BB–deficient mice (n = 7) fed an RD or HFD. Results are means ± SEM. *P < 0.05, ##P < 0.001 compared with WT mice fed an HFD. D: Levels of UCP-1 protein in BAT. Levels of protein were determined by Western blotting. The intensities of UCP-1 protein were normalized to those of β-actin and are expressed as means ± SEM of 4 mice per group. *P < 0.05 compared with WT mice fed an HFD. E: Histological analysis of BAT from WT and 4-1BB–deficient mice fed an HFD (hematoxylin-eosin). Original magnification is ×200 (scale bar = 50 μm). (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

Adipose tissue macrophages and T cells in HFD-fed 4-1BB–deficient mice. A: Histological analysis of epididymal adipose tissue from WT and 4-1BB–deficient mice fed an HFD. Sections were stained with hematoxylin-eosin (H&E) in epididymal adipose tissue from WT and 4-1BB–deficient mice fed an HFD. Stained cells are indicated by arrows. Original magnifications are ×200 (upper) and ×400 (lower) (scale bar = 50 μm). FACS quantification of immune cell population and numbers in visceral adipose SVF from WT and 4-1BB–deficient mice fed an HFD. SVFs were double stained with fluorescein isothiocyanate–conjugated phycoerythrin-conjugated anti-CD4 (helper T cell)/anti-CD8 (cytotoxic T cell), anti-F4/80/CD11b (macrophage), anti-CD4 (or CD8)/CD44^high (activated T cell), and anti-CD4 (or CD8)/CD62L^low (activated T cell). B: The values in the panels indicate the percentages of each cell population. C: The total immune cell numbers in adipose tissue. Results are mean ± SEM. *P < 0.05, **P < 0.01 compared with WT mice fed an HFD. (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

Adipose tissue inflammatory responses in HFD-fed 4-1BB–deficient mice. Concentrations of inflammatory proteins (TNF-α, IL-6, and MCP-1) (A) and an anti-inflammatory protein (adiponectin) (B) in adipose tissue from WT (n = 8) and 4-1BB–deficient mice (n = 8) fed an HFD. Adipose tissue (0.5 g) was homogenized with 1 mL of 100 mmol/L Tris-HCl and 250 mmol/L sucrose buffer (pH 7.4) supplemented with protease inhibitors. Lipids were removed by centrifugation at 10,000g for 10 min. Levels of cytokines/adipokines in homogenates were measured by enzyme-linked immunosorbent assay and normalized for protein content. Levels of HMW adiponectin were assessed in plasma samples from WT (n = 8) and 4-1BB–deficient mice (n = 8). Results are mean ± SEM. *P < 0.05, #P < 0.005 compared with WT mice fed an HFD. C: NF-κB activation in adipose tissue was determined using the p65 TransAM assay as described in research design and methods. Results are mean ± SEM (n = 6 mice per group). ##P < 0.001 compared with WT mice fed an HFD.

FIG. 6.

Deficiency of 4-1BB ameliorates insulin resistance and improves insulin signaling in mice fed an HFD. A: Fasting glucose and insulin levels. B: Plasma TG and total cholesterol levels in WT and 4-1BB–deficient mice fed an RD or HFD. Results are means ± SEM (n = 5–6 mice per group). *P < 0.05, **P < 0.01 compared with WT mice fed an HFD. C: Glucose tolerance tests. Mice fed an HFD for 7 weeks were fasted for 5 h before receiving an oral administration of 20% glucose solution at a dose of 2 g/kg, and blood samples were taken at the indicated times (n = 5). D: Insulin tolerance tests. Mice fed an HFD for 7 weeks were fasted for 5 h before receiving an intraperitoneal injection of 0.75 units/kg insulin, and blood samples were taken at the indicated times (n = 5). Results are means ± SEM. *P < 0.05, **P < 0.01, #P < 0.005 compared with WT mice fed an HFD. E: Western blots of phosphorylated Akt (p-Akt) and total Akt in adipose tissue, liver, and skeletal muscle from WT (n = 4) and 4-1BB–deficient mice (n = 4) fed an HFD. Mice were fasted for 5 h before receiving a 10 mU/g i.p. insulin injection and killed 4 min later, and tissues were collected for Western blotting. WAT, white adipose tissue. (A high-quality color representation of this figure is available in the online issue.)

FIG. 7.

Deficiency of 4-1BB ameliorates hepatic steatosis. A: Gross morphology and histological analysis (hematoxylin-eosin) of livers from WT and 4-1BB–deficient mice fed an HFD. Original magnification is ×200 (scale bar = 50 μm). B: Levels of TG content of livers from WT (n = 4) and 4-1BB–deficient mice (n = 4) fed an HFD. Results are means ± SEM. ##P < 0.001 compared with WT mice fed an HFD. C: Expression of lipogenic genes (SREBP-1, ACC1, and FAS) in livers from WT (n = 4) and 4-1BB–deficient mice (n = 4) fed an HFD. Levels of mRNA were estimated by quantitative PCR. Results are means ± SEM. *P < 0.05 compared with WT mice fed an HFD. D: Western blots of phosphorylated AMPK (p-AMPK), acetyl-CoA carboxylase (ACC), and PPAR-α in livers from WT and 4-1BB–deficient mice fed an HFD. The intensity of the bands was quantified by densitometry and is expressed as means ± SEM (n = 4 mice per group). **P < 0.05 compared with WT mice fed an HFD. E: Levels of inflammatory proteins (TNF-α, IL-6, and MCP-1) in livers from WT (n = 8) and 4-1BB–deficient mice (n = 8) fed an HFD. Levels of cytokines/chemokines in liver were measured by enzyme-linked immunosorbent assay. Results are means ± SEM. *P < 0.05, **P < 0.01 compared with WT mice fed an HFD. (A high-quality color representation of this figure is available in the online issue.)