SUMMARY Omega-3 fatty acids (ω-3 FAs), DHA and EPA, exert anti-inflammatory effects, but the mechanisms are poorly understood. Here we show that the G protein-coupled receptor 120 (GPR120) functions as an ω-3 FA receptor/sensor. Stimulation of GPR120 with ω-3 FAs or a chemical agonist causes broad anti-inflammatory effects in monocytic RAW 264.7 cells and in primary intraperitoneal macrophages. All of these effects are abrogated by GPR120 knockdown. Since chronic macrophage-mediated tissue inflammation is a key mechanism for insulin resistance in obesity, we fed obese WT and GPR120 knockout mice a high fat diet with or without ω-3 FA supplementation. The ω-3 FA treatment inhibited inflammation and enhanced systemic insulin sensitivity in WT mice, but was without effect in GPR120 knockout mice. In conclusion, GPR120 is a functional ω-3 FA receptor/sensor and mediates potent insulin sensitizing and anti-diabetic effects in vivo by repressing macrophage-induced tissue inflammation.

padhyay G, Olefsky JM. SIRT1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity. Am J Physiol Endocrinol Metab 298: E419 -E428, 2010. First published December 8, 2009; doi:10.1152/ajpendo.00417.2009.-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 TNF␣ 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 TNF␣, 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. macrophage; insulin resistance FOR MANY YEARS, IT HAS BEEN KNOWN that caloric restriction extends life span over a wide range of species, including mammals (27). Silent information regulator 2 (Sir2) is a NADdependent deacetylase that is one of the components connecting the metabolic effects of caloric restriction to longevity in yeast, worms, and flies (7). Mammals express 7 homologs of yeast Sir2, identified as the SIRTUIN family, SIRT1-7 (7). SIRT1 has the closest homology to Sir2, and recent data suggest that activation of SIRT1 may be, at least partially, responsible for the extension of life span in mammals (4, 5, 7).

Macrophage-mediated inflammation is a key component of insulin resistance; however, the initial events of monocyte migration to become tissue macrophages remain poorly understood. We report a new method to quantitate in vivo macrophage tracking (i.e., blood monocytes from donor mice) labeled ex vivo with fluorescent PKH26 dye and injected into recipient mice. Labeled monocytes appear as adipose, liver, and splenic macrophages, peaking in 1–2 days. When CCR2 KO monocytes are injected into wild-type (WT) recipients, or WT monocytes given to MCP-1 KO recipients, adipose tissue macrophage (ATM) accumulation is reduced by ~40%, whereas hepatic macrophage content is decreased by ~80%. Using WT donor cells, ATM accumulation is several-fold greater in obese recipient mice compared with lean mice, regardless of the source of donor monocytes. After their appearance in adipose tissue, ATMs progressively polarize from the M2- to the M1-like state in obesity. In summary, the CCR2/MCP-1 system is a contributory factor to monocyte migration into adipose tissue and is the dominant signal controlling the appearance of recruited macrophages in the liver. Monocytes from obese mice are not programmed to become inflammatory ATMs but rather the increased proinflammatory ATM accumulation in obesity is in response to tissue signals.

Obesity represents a state of chronic, low grade inflammation and is associated with infiltration of increased numbers of adipose tissue macrophages (ATMs). Diet-induced obesity leads to an increase in non-inflammatory M1-like ATMs displaying the CD11c surface marker. We assessed the function of CD11c-positive ATMs when insulin resistant high fat diet (HFD) mice become insulin-sensitive after switching from HFD to normal chow (NC). HFD mice rapidly become insulin-sensitive in all major insulin-target tissues, including muscle, liver, and adipose tissue, after the diet switch. In adipose tissue the CD11c-positive macrophages remain constant in number despite the presence of insulin sensitivity, but these macrophages now assume a new phenotype in which they no longer exhibit increased inflammatory pathway markers. Adipose tissue markers of apoptosis and necrosis were elevated on HFD and remain high after the HFD 3 NC diet switch. Furthermore, ATM accumulation preceded detectable adipocyte necrosis at the early phase of HFD. Together, these results indicate that 1) CD11c-positive M1-like ATMs can exhibit phenotypic plasticity and that the polarization of these cells between inflammatory and non-inflammatory states is well correlated to the presence of absence of insulin resistance, and 2) adipocyte necrosis and apoptosis can be dissociated from ATM accumulation.

Although the ubiquitin-editing enzyme A20 is a key player in inflammation and autoimmunity, its role in cancer metastasis remains unknown. Here we show that A20 monoubiquitylates Snail1 at three lysine residues and thereby promotes metastasis of aggressive basal-like breast cancers. A20 is significantly upregulated in human basal-like breast cancers and its expression level is inversely correlated with metastasis-free patient survival. A20 facilitates TGF-β1-induced epithelial-mesenchymal transition (EMT) of breast cancer cells through multi-monoubiquitylation of Snail1. Monoubiquitylated Snail1 has reduced affinity for glycogen synthase kinase 3β (GSK3β), and is thus stabilized in the nucleus through decreased phosphorylation. Knockdown of A20 or overexpression of Snail1 with mutation of the monoubiquitylated lysine residues into arginine abolishes lung metastasis in mouse xenograft and orthotopic breast cancer models, indicating that A20 and monoubiquitylated Snail1 are required for metastasis. Our findings uncover an essential role of the A20-Snail1 axis in TGF-β1-induced EMT and metastasis of basal-like breast cancers.

T he metabolic syndrome is defined as a constellation of impaired glucose metabolism, dyslipidemia, hypertension, and central obesity, and may lead to type 2 diabetes mellitus and cardiovascular diseases. Because nonalcoholic fatty liver disease is closely associated with metabolic syndrome and insulin resistance, this disease may represent the hepatic component of metabolic syndrome. Among the conditions grouped within metabolic syndrome, hyperinsulinemia results in the elevated lipogenesis of fatty acids and triglycerides (TGs) in the liver. In the regulation of lipogenesis, the nuclear hormone receptor liver X receptor-alpha (LXR␣)

Obesity-related insulin resistance is closely associated with macrophage accumulation and subsequent cytokine release in local tissues. Sirtuin 6 (Sirt6) is known to exert an anti-inflammatory function, but its role in macrophages in the context of obesity has not been investigated. We generated myeloid-specific Sirt6 knockout (mS6KO) mice and investigated the metabolic characteristics after high-fat diet (HFD) feeding for 16 weeks. Compared with their wild-type littermates, HFD-fed mS6KO mice exhibited greater increases in body weight, fasting blood glucose and insulin levels, hepatic steatosis, glucose intolerance, and insulin resistance. Gene expression, histology, and flow cytometric analyses demonstrated that liver and adipose tissue inflammation were elevated in HFD-fed mS6KO mice relative to wild type, with a greater accumulation of F4/80CD11bCD11c adipose tissue macrophages. Myeloid Sirt6 deletion facilitated proinflammatory M1 polarization of bone marrow macrophages and augmented the migration potential of macrophages toward adipose-derived chemoattractants. Mechanistically, Sirt6 deletion in macrophages promoted the activation of nuclear factor-κB (NF-κB) and endogenous production of interleukin-6, which led to STAT3 activation and the positive feedback circuits for NF-κB stimulation; this cross talk expedited an M1 polarization. We conclude that Sirt6 in macrophages is required for the prevention of obesity-associated tissue inflammation and insulin resistance.

Sirtuin (Sirt)6 has been implicated in negative regulation of inflammation and lipid metabolism, although its function in the progression from simple steatosis to nonalcoholic steatohepatitis (NASH) remains to be defined. To explore the role of hepatocyte Sirt6 in NASH development, we generated hepatocyte-specific Sirt6-knockout (KO) mice that were fed a high-fat and high-fructose (HFHF) diet for 16 wk. HFHF-fed KO mice had increased hepatic steatosis and inflammation and aggravated glucose intolerance and insulin resistance compared with wild-type mice. HFHF-induced liver fibrosis and oxidative stress and related gene expression were significantly elevated in KO mice. In the livers of KO mice, nuclear factor erythroid 2-related factor 2 (Nrf2) was down-regulated; conversely, BTB domain and CNC homolog 1 (Bach1), a nuclear repressor of Nrf2, were up-regulated. We discovered that Sirt6, which interacts with Bach1 under basal condition, induces its detachment from the antioxidant response element (ARE) region of heme oxygenase 1 promoter. Furthermore, we found that Sirt6 promotes Nrf2 binding to ARE in response to oxidative stimuli, which leads to the expression of phase II/antioxidant enzymes. Finally, we showed that HFHF-induced steatosis, inflammation, and fibrosis were ameliorated by adenoviral Sirt6 overexpression. Sirt6 may be a useful therapeutic target for amelioration of NASH by curbing inflammation and oxidative stress.-Ka, S.-O, Bang, I. H., Bae, E. J., Park, B.-H. Hepatocyte-specific sirtuin 6 deletion predisposes to nonalcoholic steatohepatitis by up-regulation of Bach1, an Nrf2 repressor.