SirT1 regulates adipose tissue inflammation

Abstract

Objective: Macrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation.

Research design and methods: We used antisense oligonucleotides to reduce SirT1 to levels similar to those seen during overnutrition and studied SirT1-overexpressing transgenic mice and fat-specific SirT1 knockout animals. Finally, we analyzed subcutaneous adipose tissue biopsies from two independent cohorts of human subjects.

Results: We found that inducible or genetic reduction of SirT1 in vivo causes macrophage recruitment to adipose tissue, whereas overexpression of SirT1 prevents adipose tissue macrophage accumulation caused by chronic high-fat feeding. We also found that SirT1 expression in human subcutaneous fat is inversely related to adipose tissue macrophage infiltration.

Conclusions: Reduction of adipose tissue SirT1 expression, which leads to histone hyperacetylation and ectopic inflammatory gene expression, is identified as a key regulatory component of macrophage influx into adipose tissue during overnutrition in rodents and humans. Our results suggest that SirT1 regulates adipose tissue inflammation by controlling the gain of proinflammatory transcription in response to inducers such as fatty acids, hypoxia, and endoplasmic reticulum stress.

Figures

FIG. 1.

Knockdown of SirT1 mimicking that seen in obesity causes anorexia-driven weight loss and TNF-α elevation. A–C: Body weight (***P < 0.001) (n = 8–12/group) (A), anorexia (n = 12/group) (B), and loss of epididymal fat mass (n = 3–4/group) (C) of ad libitum fed rats treated with ASO biweekly for 1 month. D: Results from parallel measurement of plasma appetite regulatory hormones in animals treated with control or SirT1 ASO showing elevated TNF-α and possibly IL-6 in the SirT1 group (n = 7–8/group, pooled). E: Plasma TNF-α levels (n = 5/group). F: Adipose tissue levels of TNF-α (n = 3–4/group). G: SirT1 protein expression in WAT from animals treated with control ASO and fed normal chow, animals treated with SirT1 ASO and fed normal chow, and mildly diabetic animals fed high-fat/high-fructose chow. H: Quantification of representative data presented in F (n = 6/group). I: Plasma TNF-α levels in normal chow–fed control ASO–treated rats, normal chow–fed SirT1 ASO–treated rats, and high-fat diet–fed, control ASO–treated rats by electrochemiluminescence (n = 4–5/group). HFD, high-fat diet.

FIG. 2.

SirT1 knockdown/deletion causes adipose tissue inflammation and macrophage infiltration. A: Cytokine array performed on plasma from normal chow–fed control ASO– and SirT1 ASO–treated rats (n = 4/group). *P < 0.05, **P < 0.01 (left). Cytokine array performed on plasma from chow-fed wild-type and fat-specific SirT1 knockout mice (n = 4/group). *P < 0.08, **P < 0.03 (right). B: Plasma IL-10 levels collected at the onset of hypophagia (1 week) (n = 10/group) and plasma IL-4 after 1 month of ASO treatment. C: Representative hematoxylin and eosin staining from epididymal WAT of individual rats fed normal chow and treated with control ASO or SirT1 ASO (representative of 5 rats/group). D: Reduced adipocyte diameter in SirT1 ASO–treated rats. E: Adipose tissue macrophage content, as assessed by FACS, and adipose tissue CD68 mRNA expression in mice (n = 5/group). F: Macrophage/monocyte (CD68, CD115) and macrophage/dendritic cell (CD11c) marker mRNA expression in rat adipose tissue (n = 4–7). G: Macrophage-related mRNA abundance in adipose tissue from SirT1 fat-specific knockout mice fed a chow diet (8–12 weeks of age, n = 4–6/group). H: Macrophage marker/cytokine mRNA abundance in adipose tissue from SirT1 fat-specific knockout mice fed a high-fat diet for 1 month (n = 3–7/group). GM-CSF, granulocyte-macrophage colony-stimulating factor; KO, knockout; N.S., nonsignificant. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

SirT1 knockdown in WAT results in NF-κB nuclear localization and gene expression through reduction of H3K9 deacetylation. A and B: Representative (n = 5/group) images showing that SirT1 knockdown stimulates nuclear translocation of NF-κB in rat WAT. Adipocytes are stained for caveolin (green) and NF-κB p65 nuclear localization sequence (red). Nuclei are stained with DAPI (blue). Inset box is expanded to individual channels in B.1-B.3 to demonstrate NF-κB and DAPI colocalization. Some NF-κB–positive nuclei (arrows) are in adipocytes. Because the nuclear localization sequence is masked in the cytosol, nonnuclear staining is nonspecific (i.e., erythrocyte or other autofluorescence). C: SirT1 knockdown increases abundance of phosphorylated (Ser536) and total p65 in rat WAT nuclear lysates. Line separates noncontiguous lanes from the same blot. D: Chow-fed fat-specific SirT1 knockout mice have increased phospho-p65 in WAT ∼12 h after stimulation with LPS (50 μg i.p.). E: SirT1 knockdown increases adipose tissue cytokine, complement, and TLR mRNA expression (n = 5–7/group). *P < 0.05, **P < 0.01. F: SirT1 knockdown increases H3K9 acetylation. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Overexpression of SirT1 reduces high-fat diet–induced increases in WAT macrophage content. A and B: Reduced F4/80 (A) and CD68 (B) expression in WAT of high-fat diet–fed SirT1-overexpressing mice by qPCR. C: Verification of SirT1 overexpression in WAT (n = 5/group). D: NEMO and CASP8 expression, also in WAT (n = 3/group).

FIG. 5.

SirT1 transcript level is inversely correlated with both BMI and adipose tissue macrophage content in humans. A: Control, obese, and severely obese subjects were stratified by BMI. Obese and severely obese subjects have decreased SirT1 expression (B) and increased macrophage content (C) in subcutaneous adipose tissue. Macrophages (CD68+ cells) within an entire section were counted by two independent observers using a light microscope. % macrophages = number of macrophages / number of adipocytes × 100. Obese and severely obese subjects have elevated HOMA-IR (D). **P < 0.01, *P < 0.05, #P < 0.10. Note: 18 of the 45 subjects examined are also being studied for the contributions of a gene polymorphism to SirT1 gene expression.

FIG. 6.

SirT1 transcript level is inversely correlated with macrophage markers in human adults. A: Correlation between SirT1 mRNA expression and CD14 (a macrophage/monocyte marker) in adipose tissue of a mixed-weight population of humans. B: Correlation between SirT1 and CD86 mRNA (another monocyte/macrophage marker) in WAT from the same group of subjects. C: Correlation between SirT1 and CX3CL1 (a monocyte chemoattractant) mRNA in WAT from the same group of subjects. D–F: SirT1 knockdown increases expression of CX3CL1, CX3CR1, and CD14 in rodent WAT. G: Schematic model of SirT1 regulation of inflammation. SirT1 deacetylation of inflammatory gene promoters causes decreased cytokine production in response to stimulation of inflammatory sensors by fatty acids, hypoxia, and ER stress. In turn, decreased SirT1 expression in obesity sensitizes these networks to activation by stressors. FFA, free fatty acid; NLR, NOD-like receptor.