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. 2010 Mar;298(3):E419-28.
doi: 10.1152/ajpendo.00417.2009. Epub 2009 Dec 8.

SIRT1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity

Takeshi Yoshizaki  1 , Simon Schenk, Takeshi Imamura, Jennie L Babendure, Noriyuki Sonoda, Eun Ju Bae, Da Young Oh, Min Lu, Jill C Milne, Christoph Westphal, Gautam Bandyopadhyay, Jerrold M Olefsky
Affiliations

SIRT1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity

Takeshi Yoshizaki et al. Am J Physiol Endocrinol Metab. 2010 Mar.

Abstract

Chronic inflammation is an important etiology underlying obesity-related disorders such as insulin resistance and type 2 diabetes, and recent findings indicate that the macrophage can be the initiating cell type responsible for this chronic inflammatory state. The mammalian silent information regulator 2 homolog SIRT1 modulates several physiological processes important for life span, and a potential role of SIRT1 in the regulation of insulin sensitivity has been shown. However, with respect to inflammation, the role of SIRT1 in regulating the proinflammatory pathway within macrophages is poorly understood. Here, we show that knockdown of SIRT1 in the mouse macrophage RAW264.7 cell line and in intraperitoneal macrophages broadly activates the JNK and IKK inflammatory pathways and increases LPS-stimulated TNFalpha secretion. Moreover, gene expression profiles reveal that SIRT1 knockdown leads to an increase in inflammatory gene expression. We also demonstrate that SIRT1 activators inhibit LPS-stimulated inflammatory pathways, as well as secretion of TNFalpha, in a SIRT1-dependent manner in RAW264.7 cells and in primary intraperitoneal macrophages. Treatment of Zucker fatty rats with a SIRT1 activator leads to greatly improved glucose tolerance, reduced hyperinsulinemia, and enhanced systemic insulin sensitivity during glucose clamp studies. These in vivo insulin-sensitizing effects were accompanied by a reduction in tissue inflammation markers and a decrease in the adipose tissue macrophage proinflammatory state, fully consistent with the in vitro effects of SIRT1 in macrophages. In conclusion, these results define a novel role for SIRT1 as an important regulator of macrophage inflammatory responses in the context of insulin resistance and raise the possibility that targeting of SIRT1 might be a useful strategy for treating the inflammatory component of metabolic diseases.

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Figures

Fig. 1.
Fig. 1.
SIRT1 activators inhibit LPS-stimulated inflammatory pathways. After pretreatment of 50 μM resveratrol (RES; A, B, and E) or 1 μM SRT1720 (C–G) for 1 h, RAW264.7 cells (A–D, F and G) or peritoneal macrophage (E) were stimulated with (+) or without (−) 100 ng/ml LPS for 10 min (A–E) or 1 h (F and G) and lysed, and immunoblotting (IB) was performed with indicated antibodies. Scanned bar graphs show means ± SE, and values are expressed as %maximum compared with those observed in LPS-stimulated control cells (n = 3). G, top: schematic diagram of the mouse MMP9 promoter region. The positions of κB sites are indicated and pairs of arrows indicate the PCR-amplified regions. Soluble chromatin was immunoprecipitated with anti-NF-κB antibody (NF-κB) or control IgG (IgG). Enrichment of κB containing DNA sequences in the immunoprecipitated DNA pool was visualized by PCR. G, bottom: PCR-amplified MMP9 promoter band from input control. Graph shows mean ± SE from 3 independent experiments, and values are expressed as fold amount of immunoprecipitated promoter DNA copy numbers relative to their corresponding input controls, compared with those observed DMSO-treated cells. *P < 0.05, Etoh or DMSO vs. resveratrol or SRT1720. Etoh or DMSO, vehicle ethanol or DMSO control; p/total, phosphorylation/total; ns, nonspecific; ChIP, chromatin immunoprecipitation; ace, acetylation; m, marker.
Fig. 2.
Fig. 2.
SIRT1 knockdown increases inflammatory responses. A: RAW264.7 macrophages were electroporated with control (ctrl) or SIRT1 (SIRT1) siRNA. Forty-eight hours after electroporation, cell lysates were immunoblotted with indicated antibodies. B–F: cells were electroporated with control or SIRT1 (SIRT) siRNA. Forty-eight hours after electroporation, cells were stimulated with or without 100 ng/ml LPS for 10 min (B, C, and F) or 1 h (D and E), and Western blotting was performed with indicated antibodies. Data are percentages compared with ctrl siRNA electroporated cells and are means ± SE (n = 3). E, top: soluble chromatin was immunoprecipitated with anti-NFκB antibody or control IgG . Enrichment of κB containing DNA sequences in the immunoprecipitated DNA pool was visualized by PCR. E, bottom: PCR-amplified MMP9 promoter band from input control. Graph shows mean ± SE from 3 independent experiments, and values are expressed as fold amount of immunoprecipitated promoter DNA copy numbers relative to their corresponding input controls, compared with those observed DMSO-treated cells. *P < 0.05, control siRNA vs. SIRT1 siRNA.
Fig. 3.
Fig. 3.
SIRT1 is the target for the resveratrol and SRT1720 anti-inflammatory effects. After pretreatment of 50 μM resveratrol (A) or 1 μM SRT1720 (B) for 1 h, RAW264.7 cells electroporated with control or SIRT1 siRNA were stimulated with or without 100 ng/ml LPS for 10 min and then lysed, and immunoblotting was performed with indicated antibodies. Scanned bar graphs show means ± SE, and values are expressed as %maximum in phsphorylation compared with those observed in LPS-stimulated control cells (n = 3). C: RAW264.7 cells, electroporated with control or SIRT1 siRNA, were stimulated with or without 100 ng/ml LPS for 1 h, and conditioned medium (CM) was harvested for ELISA analysis. Secreted TNFα was assayed for mouse TNFα using ELISA assay. Data are presented as percentages of secretion compared with LPS-stimulated control siRNA electroporated cells and represent means ± SE (n = 4). *P < 0.05, Etoh vs. RES or DMSO vs. SRT1720. †P < 0.05, control siRNA vs. SIRT1 siRNA.
Fig. 4.
Fig. 4.
Macrophage SIRT1 affects adipocyte insulin sensitivity. A, B: 3T3-L1 adipocytes were incubated with CM, control CM from vehicle (Etoh), or CM from resveratrol-treated RAW cells, diluted 1:250 in DMEM, for 3 h before assays. Cells were stimulated with insulin and then measured 2-deoxyglucose uptake (A) or Akt phosphorylation (B). Graphs show means ± SE. Values are expressed as %control or fold basal (un-stimulated) (n = 4). *P < 0.01, vehicle (Etoh) vs. resveratrol.
Fig. 5.
Fig. 5.
SIRT1 activator improves glucose intolerance and insulin sensitivity in obese Zucker rat. Body weight (A), average daily food intake (B), epididymal fat mass (C), and plasma glucose on ad libitum feeding (D) were measured after Zucker fa/fa rats were placed on normal chow diet (Zucker) or normal chow diet supplemented with SRT2379 at 100 mg per kg of body weight per day (Zucker + SRT) for 24 days (B–D) or indicated time periods (A; n = 6). E: glucose and insulin responses during an oral glucose tolerance test (OGTT) are shown after normal chow diet or normal chow diet with SRT2379 for 24 days (n = 6). G: Insulin sensitivity evaluated through the average glucose infusion rate at equilibrium in a hyperinsulinemic euglycemic clamp (25 mU insulin·kg·−1min−1) in Zucker fa/fa rat treated with or without SRT2379 for 4 wk (n = 4). *P < 0.05, Zucker vs. Zucker + SRT.
Fig. 6.
Fig. 6.
SIRT1 activation leads to a reduction of CD11c expression in epididymal fat. Zucker fa/fa rats were placed on normal chow diet with vehicle (Zucker) or SRT2379 (Zucker + SRT), epididymal fat pad were isolated as described in Materials and Methods. A: representative histologic picture (hematoxylin and eosin staining) of the adipose tissue. Data are presented as percentages of mean area size of adipocyte compared with tissue from Zucker fa/fa rats and represent means ± SE. B–D: total RNA was purified, and then quantitative real-time-PCR was performed. After the mRNA expression differences were normalized to a standard housekeeping gene (GAPDH) mRNA level, data are presented as the relative expression. Error bars represent means ± SE from 4 independent experiments. Adipose tissue macrophage content was measured as described in Materials and Methods. Data are presented as percentages of macrophage per total nuclei and represent means ± SE. E: imunoblotting was performed with indicated antibodies. *P < 0.05, Zucker vs. Zucker + SRT. C: lean vs. Zucker or Zucker + SRT.
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