Gene silencing in adipose tissue macrophages regulates whole-body metabolism in obese mice

Abstract

Adipose tissue (AT) inflammation and infiltration by macrophages is associated with insulin resistance and type 2 diabetes in obese humans, offering a potential target for therapeutics. However, whether AT macrophages (ATMs) directly contribute to systemic glucose intolerance has not been determined. The reason is the lack of methods to ablate inflammatory genes expressed in macrophages specifically localized within AT depots, leaving macrophages in other tissues unaffected. Here we report that i.p. administration of siRNA encapsulated by glucan shells in obese mice selectively silences genes in epididymal ATMs, whereas macrophages within lung, spleen, kidney, heart, skeletal muscle, subcutaneous (SubQ) adipose, and liver are not targeted. Such administration of GeRPs to silence the inflammatory cytokines TNF-α or osteopontin in epididymal ATMs of obese mice caused significant improvement in glucose tolerance. These data are consistent with the hypothesis that cytokines produced by ATMs can exacerbate whole-body glucose intolerance.

Keywords: RNAi; immune cells; obesity.

Fig. 1.

GeRPs injected i.p. are localized in epididymal ATMs in obese mice. (A–J) Five-week-old ob/ob mice were i.p. injected once a day for 5 d with 5.6 mg/kg FITC-labeled GeRPs loaded with 2.1 mg/kg EP and 0.262 mg/kg SCR siRNA. On day 6, liver, lung, spleen, pancreas, heart, kidney, and (SubQ AT), mesenteric (Mes AT), perirenal (PeriR AT), and epididymal AT (EPI AT) were isolated. Tissues were fixed, sectioned, and then stained with H&E. Tissues were then analyzed by fluorescent microscopy. Images were obtained using a Zeiss Axiovert 200 inverted microscope. (Scale bar: 50 μm.) (K) Five-week-old ob/ob mice were i.p. injected with ¹²⁵I labeled GeRPs. Seven days later, liver, lung, spleen, pancreas, heart, kidney, and SubQ, mesenteric, and epididymal AT were isolated.

Fig. 2.

GeRPs undergo phagocytosis by epididymal ATMs in obese mice. (A) Five-week-old ob/ob mice were i.p. injected once a day for 5 d with 5.6 mg/kg FITC-labeled GeRPs loaded with 2.1 mg/kg EP and 0.262 mg/kg SCR siRNA. On day 6, epididymal AT was isolated, fixed, sectioned, and stained with F4/80 antibody. Tissues were also counterstained with hematoxylin. Tissues were then analyzed by fluorescent microscopy. Spinning disk confocal microscopy showing crown-like structures composed of macrophages (dark brown) containing FITC-GeRPs (green). (Scale bar: 20 μm.) (B) Confocal microscopy showing F4/80 (red) and GeRPs (green) present in SVF cells 24 h after treatment. Nuclei were stained with DAPI (blue). (Scale bar: Left, 50 μm; Right, 10 μm.) (C) FACS analysis showing SVF cells isolated from mice treated with FITC-labeled GeRPs (FITC-GeRPs) and stained with F4/80 antibody. APC, allophycocyanin. (D) FITC level stain in epididymal AT total SVF cells, macrophages (F4/80+/CD11b+/Siglec-f-), monocytes (Ly6-C high and low), neutrophils (Gr1+), eosinophils (F4/80+/CD11b+/Siglec-f+), and T cells (CD3+) determined by flow cytometry.

Fig. 3.

Silencing genes in primary macrophages in vitro. (A and B) Expression of TNF-α and OPN measured by RT-PCR in isolated macrophages from epididymal and SubQ AT of 7-wk-old genetically obese ob/ob mice. Macrophages were isolated using CD11b antibody bound to magnetic beads. n = 4. Statistical significance was determined by t test. ***P < 0.001; *P < 0.05. Results are means expressed in fold change (F.C.) ± SEM. (C and D) 1 × 10⁶ peritoneal macrophages were treated with particles made with a mixture of 160 pmoles siRNA and 3 nmol EP. Forty-eight hours after the treatment, mRNA levels were measured by RT-PCR. (E) TNF-α in media of peritoneal macrophages treated with 1 μg/mL LPS for 6 h measured by ELISA. (F) OPN protein basal levels in media measured by ELISA. n = 3 with three technical replicates for each experiment. Statistical significance was determined by ANOVA and Tukey post test. ***P < 0.001. Results are mean ± SEM.

Fig. 4.

Gene silencing in epididymal ATMs in obese mice without affecting macrophages in liver or other adipose depots. Expression of TNF-α in (A) epididymal AT and (C) liver from mice treated for 10 d with SCR- or TNF-α-GeRPs. n = 23–24. Expression of OPN in (B) epididymal AT and (D) liver from mice treated for 5 d with SCR- or OPN-GeRPs. n = 28. Statistical significance was determined by t test. ***P < 0.001; *P < 0.05. Results are means in F.C. ± SEM. TNF-α expression in (E) SubQ (F) perirenal, and (G) mesenteric AT from mice treated for 10 d with SCR- or TNF-α-GeRPs. n = 5. (H) TNF-α protein levels and bioactivity in LPS-treated epididymal SVF media of mice treated with SCR- or TNF-α-GeRPs. n = 10. (I) OPN protein levels in epididymal SVF media of mice treated with SCR- or OPN-GeRPs. n = 10. Statistical significance was determined by t test. **P < 0.01; *P < 0.05. (J) Representative dot-plot of F4/80-stained epididymal SVF from mice treated with PBS or FITC-labeled SCR-GeRPs for 5 or 10 d. F4/80+ cells containing GeRPs (FITC+) were sorted by FACS and mRNA levels were measured by RT-PCR in mice treated with SCR-, TNF-α-, or OPN-GeRPs. For TNF-α, n = 5 mice pooled together. For OPN, n = 3 groups of three mice pooled together. Statistical significance was determined by t test. ***P < 0.001.

Fig. 5.

Gene silencing in epididymal ATMs in obese mice regulates whole-body metabolism. Five-week-old ob/ob mice were treated as described in Fig. 4. Twenty-four hours after the last injection, glucose tolerance tests (GTT) were performed on mice that were fasted for 16 h. Mice were treated with SCR-GeRPs, (A) TNF-α-GeRPs, or (B) OPN-GeRPs. (C and D) Area under the curve (AUC) of corresponding GTT graphs. n = 10–18. Statistical significance was determined by ANOVA and Tukey posttest. *P < 0.05. Results are mean ± SEM. (E) Insulin tolerance tests (ITT) were performed on day 6 in ob/ob mice treated with PBS or SCR- or OPN-GeRPs by injecting 1 U/kg of insulin. (F) AUC of corresponding ITT graph. n = 15. Statistical significance was determined by ANOVA and Tukey posttest. *P < 0.05. Results are mean ± SEM. PBS or SCR- or OPN-GeRPs treated mice were also fasted for 4 h and then treated by i.p. injection with 1 U/kg of insulin (15 min). Western blotting and multiplexed ELISA were used to detect Akt and activated (pSer473) Akt in (G) epididymal AT (EPI AT), (H) gastrocnemius muscle, and (I) liver. n = 5–6. Statistical significance was determined by ANOVA and Tukey posttest. ***P < 0.001; *P < 0.05. Results are mean ± SEM.