Quercetin reduces obesity-associated ATM infiltration and inflammation in mice: a mechanism including AMPKα1/SIRT1.

Adipose tissue macrophage (ATM) plays a central role in obesity-associated inflammation and insulin resistance. Quercetin, a dietary flavonoid, possesses anti-inflammation and anti-insulin resistance properties. However, it is unclear whether quercetin can alleviate high-fat diet (HFD)-induced ATM infiltration and inflammation in mice. In this study, 5-week-old C57BL/6 mice were fed low-fat diet, HFD, or HFD with 0.l% quercetin for 12 weeks, respectively. Dietary quercetin reduced HFD-induced body weight gain and improved insulin sensitivity and glucose intolerance in mice. Meanwhile, dietary quercetin enhanced glucose transporter 4 translocation and protein kinase B signal in epididymis adipose tissues (EATs), suggesting that it heightened glucose uptake in adipose tissues. Histological and real-time PCR analysis revealed that quercetin attenuated mast cell and macrophage infiltration into EATs in HFD-fed mice. Dietary quercetin also modified the phenotype ratio of M1/M2 macrophages, lowered the levels of proinflammatory cytokines, and enhanced adenosine monophosphate-activated protein kinase (AMPK) α1 phosphorylation and silent information regulator 1 (SIRT1) expression in EATs. Further, using AMPK activator 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside and inhibitor Compound C, we found that quercetin inhibited polarization and inflammation of mouse bone marrow-derived macrophages through an AMPKα1/SIRT1-mediated mechanism. In conclusion, dietary quercetin might suppress ATM infiltration and inflammation through the AMPKα1/SIRT1 pathway in HFD-fed mice

(BATs), were rapidly dissected and weighed on ice. Then, the tissues were snap-frozen in liquid nitrogen and stored at Ϫ 80°C.

Glucose tolerance tests and insulin tolerance tests
Glucose tolerance tests and insulin tolerance tests were performed before mice were euthanized at 17 weeks old as previously described ( 34 ). Generally, mice were fasted overnight and intraperitoneally injected with D-glucose (1 g/kg body weight, Sigma-Aldrich) for the glucose tolerance test and with insulin (1.5 IU/kg body weight; WanBang BioPharma, Xuzhou, China) for the insulin tolerance test. The blood glucose level was measured from tail veins using a blood glucose meter (Omnitest Plus; B. Braun, Melsungen, Germany) at 0, 15,30,45,60,90, and 120 min after injection.

Histology and immunohistochemistry
For histopathology studies, adipose tissues were rapidly fi xed in 4% formalin at room temperature for 24 h. Then, the tissues were embedded in paraffi n and serially sliced at 5 m thickness. The histological characterizations, including adipocyte sizes (hematoxylin-eosin staining, Sigma-Aldrich) and ATM (Mac-2 monoclonal antibody, 1:800; BioLegend, San Diego, CA) and mast cell (toluidine blue staining, Sigma-Aldrich) levels were carried out as described previously ( 34,35 ). Five random fi elds from each section were examined, and Image-Pro Plus Version 6.0 (Media Cybernetics) was used to measure adipocyte average diameters and Mac-2 positive staining areas. Mast cell numbers, which presented as cell numbers per square millimeter, were counted under a light microscope (OPTEC).

Total RNA isolation and quantitative real-time PCR
Total RNA was isolated from adipose tissues or cultured cells using RNAiso Plus reagent (TaKaRa, Dalian, China). cDNA was synthesized from total RNA using Oligo d(T) 18 (TaKaRa) and M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The cDNA was used as a template for real-time PCR (Bio-Rad MyiQ2 Real-time PCR System) in the presence of SYBR® Premix Ex Taq™ II (TaKaRa). All primer sequences are listed in supplementary Table I. Data were processed using the ⌬ ⌬ CT method. GAPDH was used as reference gene.

Protein extraction and Western blot analysis
For total protein extraction, adipose tissues or macrophages were lysed in RIPA lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with total protease inhibitor and phosphatase inhibitor (Sangon, Shanghai, China) and centrifuged at 4°C and 12,000 g for 15 min. Plasma membrane (PM) proteins were extracted from adipose tissues and 3T3-L1 adipocytes as described previously ( 36 ). The protein concentration was determined with a BCA assay kit (Sangon). Samples of equal amounts were separated on SDS-PAGE and transferred to polyvinylidene difl uoride membranes (Millipore, Bedford, MA). Immunoblot analysis was performed with the corresponding primary antibodies, which are listed in supplementary Table II. HRPlabeled goat anti-rabbit IgG (1:4,000; KPL, Gaithersburg, MD) was used as secondary antibody, and the reactive bands were detected using an ECL kit (Thermo, Rockford, IL) in the ImageQuant LAS 4000 mini (GE Healthcare). Immunoblots were quantifi ed using ImageQuant TL 7.0 software (GE Healthcare) and expressed as the ratio of specifi c protein phosphorylation to the corresponding protein or specifi c protein to ␤ -actin.
In this study, we investigated the effects of quercetin on HFD-induced fat accumulation, insulin resistance, infl ammatory macrophage and mast cell infi ltration, proinfl ammatory cytokines, and AMPK ␣ 1/SIRT1 signal in adipose tissues in mice. Furthermore, we affi rmed the role of the AMPK ␣ 1/SIRT1 pathway in quercetin suppressing macrophage polarization and infl ammation.
All mice were housed in pathogen-free and ventilated cages with a 12 h-12 h light-dark cycle and allowed free access to autoclaved water and irradiated food. All research protocols were approved by Hefei University of Technology Standing Committee on Animals. Body weight and food intake were measured every week. After 12 weeks, mice were fasted overnight and then euthanized with CO 2 , and their blood and related adipose tissues were harvested. To obtain serum, blood stood overnight at 4°C and was then centrifuged at 5,000 g for 15 min. The serum was collected from the supernatant and stored at Ϫ 80°C for serum analysis. The adipose tissues, including epididymis adipose tissues (EATs), subcutaneous adipose tissues (SATs), and brown adipose tissues 30 min. After treatment, cell membrane proteins and total proteins were extracted for Western blot analysis. All aforementioned cell assays were performed in quadruplicate.

Statistical analysis
All data were presented as the mean ± SEM. The statistical differences among various animal groups were assessed with the Mann-Whitney test owing to our small data size and nonnormal distribution data. The difference in signifi cance of various cell assay groups was tested using Student's t -test. P < 0.05 was considered as statistical signifi cance.

Dietary quercetin reduced HFD-induced body weight gain and fat deposition
To reconfi rm the effect of quercetin on diet-induced obesity, 5-week-old C57BL/6 mice were fed LFD, HFD, or HFD supplemented with 0.1% quercetin for 12 weeks, respectively. There were no differences in food intake among all three groups ( Fig. 1A ). During weeks 1 to 7, dietary quercetin did not markedly affect HFD-induced body weight gain ( Fig. 1B ). However, after 8 weeks, dietary quercetin led to signifi cant body weight difference in HFD-fed mice ( Fig. 1B ). At 17 weeks old, the mice fed quercetin-containing HFD also had reduced adipose tissue weight ( Fig. 1C , supplementary Table III) and adipocyte sizes ( Fig. 1D, E ) compared with the mice only fed HFD. The results indicate that quercetin gently inhibits diet-induced obesity. Additionally, we used HPLC to determine serum quercetin concentration after hydrolysis of quercetin metabolites. The mice fed quercetin-containing HFD possessed signifi cantly higher serum quercetin levels ( Table 1 ). 100 l of water and then incubated in 250 l of 0.1 M acetate buffer (pH 5.0) containing 10,000 U ␤ -glucuronidase (Sigma-Aldrich) solution and 10 l of 0.1 M ascorbic acid at 37°C for 6 h. Hydrolyzed metabolites were extracted with 2 ml of ethyl acetate three times, dried under a nitrogen fl ow, and reconstituted in 80 l of methanol. Finally, 20 l of sample was applied to a Zorbax SB-C18 reversed-phase column (Agilent).

Cell culture and treatment
Bone marrow was isolated from the femurs and tibias of C57BL/6 mice. The cells were cultured in Dulbecco's modifi ed Eagle's medium (Gibco, Auckland, New Zealand) containing 20% fetal bovine serum (Gibco) and 30% L929 conditional medium for 6 days to differentiate into bone marrow-derived macrophages (BMDMs) ( 29,37 ). After differentiation, the cells were planted in a 24-well plate overnight. Then, the cells were stimulated with 100 ng/ml lipopolysaccharide (LPS) (Sigma-Aldrich) for 6 h. To assess the inhibitory effects of quercetin on cytokine expression and the infl ammatory signaling pathways in BMDMs, vehicle or 20 M quercetin was added during BMDM differentiation and LPS treatment; 2 mM 5-aminoimidazole-4carboxamide-1-␤ 4-ribofuranoside (AICAR) (Sigma-Aldrich) as positive control and 1 M Compound C as AMPK inhibitor (Santa Cruz) were added 2 h before LPS treatment. Adenine nucleotide levels of BMDMs were detected by HPLC with a Zorbax SB-C18 reversed-phase column as previously described ( 38 ).
The 3T3-L1 preadipocytes were purchased from Cell Culture Center of Peking Union Medical College, and cells were differentiated into adipocytes as previously described ( 39 ). For the glucose transporter 4 (GLUT4) translocation assay, differentiated 3T3-L1 adipocytes were starved for 2 h in serum-free medium before starting the experiment. Thereafter, 2 mM AICAR or 20 M quercetin was added into medium with or without 1 M Compound C for AKT is a central adaptor connecting insulin signaling with GLUT4 downstream traffi cking, and its activation regulates the translocation, orientation, and fusion of GLUT4 storage vesicles ( 41 ). Therefore, we further examined the expression and phosphorylation levels of AKT in EAT in mice after fasting overnight. Dietary quercetin reversed HFD-induced decline of the AKT phosphorylation level in EAT ( Fig. 2E ). Taken together, these results demonstrate that quercetin enhances glucose uptake in adipose tissues and may thereby protect HFD-fed mice against insulin resistance.

Dietary quercetin ameliorated HFD-induced insulin resistance and enhanced glucose uptake in adipose tissues
We next detected the glucose metabolism and insulin sensitivity in these mice. Along with their lower body weight gain, the mice fed quercetin-containing HFD had improved glucose tolerance ( Fig. 2A ) and insulin sensitivity ( Fig. 2B ) compared with the mice only fed HFD. Moreover, dietary quercetin also lowered the levels of insulin and leptin and enhanced the level of adiponectin in sera in HFD-fed mice ( Table 1 ).
The expression and translocation of insulin-dependent GLUT4, which is predominantly expressed in adipocytes and muscle cells, functionally regulates glucose uptake into adipose tissues in response to elevated levels of insulin in  GAPDH was used as reference gene in real-time PCR analysis. Quantifi cation of GLUT4 and AKT was described as the ratio of PM GLUT4 to total GLUT4 and phosphorylated AKT to total AKT, respectively. Statistical difference between groups was shown using a nonparametric Mann-Whitney test; n = 8 per group in (A)-(E). * P < 0.05, ** P < 0.01, *** P < 0.001. All data in (A)-(E) are mean ± SEM. investigated in EAT. Immunostaining with a macrophagespecifi c Mac-2 monoclonal antibody revealed that the mice fed with quercetin-containing HFD were similar to the mice fed with LFD in the number of EAT macrophages, which was signifi cantly less than in the mice only fed with HFD ( Fig. 4A , B ). The real-time PCR results of macrophagespecifi c marker F4/80 supported the above-mentioned action of dietary quercetin against HFD-induced macrophage infi ltration into EAT ( Fig. 4C ). To explore the subset levels of ATMs, we further analyzed the mRNA expression of some macrophage subtype-specifi c markers in EAT. Strikingly, dietary quercetin suppressed the expression of Cd11c and Nos2 , which are markers of M1 macrophages, in EAT of HFD-fed mice ( Fig. 4D ). In contrast, the mRNA level of M2 macrophage markers Mgl2 and Chil3l was signifi cantly increased by quercetin in EAT of the mice ( Fig. 4E ). These results indicate that dietary quercetin reduces the M1 macrophage number and increases the M2 macrophage number and thereby modifi es the M1/M2 subtype ratio in EAT.

Quercetin reduced infl ammatory cytokine levels in adipose tissues and sera
During the development and progression of obesity, various proinfl ammatory cytokines can be secreted by hypertrophic adipocytes and infi ltrated infl ammatory cells ( 2 ). Here, we examined the expression levels of several important proinfl ammatory cytokines involved in fat deposition and insulin resistance. Real-time PCR and ELISA results showed that dietary quercetin signifi cantly suppressed the levels of proinfl ammatory cytokines TNF-␣ , IL-6, and MCP-1 in EAT ( Fig. 4F ) and sera ( Table 1 ). These results suggest that dietary quercetin reduces the obesity-induced adipose tissue and systematic infl ammation.

Quercetin regulated BMDM polarization and infl ammation through the AMPK ␣ 1/ SIRT1 pathway
To explore the cellular mechanism of quercetin-antagonized infl ammation and quercetin-promoted anti-infl ammatory macrophage phenotype in adipose tissue, we investigated whether quercetin directly resisted the polarization and infl ammation of differentiated BMDMs. Quercetin signifi cantly reduced the expression of Nos2 , the marker of M1 macrophage, in both basal and LPS-stimulated conditions ( Fig. 6A ). Subsequently, the expression of proinfl ammatory cytokines, including IL-6 , IL-1 ␤ , and Mcp-1 , was also reduced by quercetin treatment ( Fig. 6B-D ). On the contrary, M2 macrophage marker Mgl2 ( Fig. 6E ) and

Quercetin inhibited infl ammatory cell recruitment into adipose tissues
A large number of infl ammatory cells, such as mast cells and macrophages, infi ltrate into adipose tissue during diet-induced obesity, aggravating adipose tissue infl ammation and insulin resistance ( 1,3 ). Our recent study revealed that Western diets induced mast cell recruitment in WAT and promoted diet-induced obesity and insulin resistance ( 34 ). As a mast cell stabilizer, quercetin can inhibit the mediator releases of mast cells in vitro ( 42,43 ). To test the effects of quercetin on adipose tissue mast cells in HFD-fed mice, we examined the number of mast cells in EAT ( Fig. 3A , B ) and the expression of the mast cell marker gene mouse mast cell protease-4 ( mMcp-4 ) ( Fig. 3C ). The results showed that dietary quercetin dramatically reduced mast cell recruitment in EAT in HFD-fed mice. In addition, our previous study also showed that defi ciency and stabilization of mast cells improved energy expenditure in HFD-fed mice ( 34 ). Here, along with lower mast cell levels in EAT, the mice receiving quercetin-containing HFD possessed higher BAT uncoupling protein 1 ( Ucp1 ) mRNA expression ( Fig. 3D ), which is an important thermogenetic factor and energy expenditure marker in BAT. This implies that quercetin may enhance energy expenditure through suppressing mast cell recruitment and degranulation, a hypothesis that merits further investigation.
ATM plays a critical role in obesity-induced infl ammation and insulin resistance ( 3 ), and we previously found that genetic defi ciency and pharmacological stabilization of mast cells reduce ATM infi ltration into WAT in HFDfed mice ( 34 ). Therefore, the number of macrophages was In the basal condition, although quercetin reduced BMDM infl ammation ( Fig. 6 ) and activated the AMPK/SIRT1 signal ( Fig. 7A, B ), quercetin did not affect ACC phosphorylation and Cpt1a expression in the same condition (supplementary Fig. I), suggesting that ACC phosphorylation and associated FA oxidation may not be involved in the inhibition of macrophage basal infl ammation by quercetin. By contrast, in the LPS-stimulated condition, both quercetin and AICAR increased ACC phosphorylation and

Quercetin increased GLUT4 translocation through AMPK activation in 3T3-L1 adipocytes
Because quercetin increased GLUT4 mRNA expression ( Fig. 2C ) and protein translocation ( Fig. 2D ) in EAT in HFD-fed mice and AMPK activation can promote GLUT4 translocation ( 40 ), we used Western blotting to detect the role of AMPK in GLUT4 translocation by quercetin in 3T3-L1 adipocytes. The results showed that either AICAR or quercetin strikingly increased the GLUT4 level in the cell membrane fraction and that Compound C reversed the action of quercetin and AICAR (supplementary Fig. II).
anti-infl ammatory cytokine IL-10 ( Fig. 6F ) expression was enhanced by quercetin in BMDMs. Compound C, a selective AMPK inhibitor, blocked these inhibitory effi ciencies of quercetin on BMDM infl ammation ( Fig. 6A-F ). Further, the AMPK ␣ 1 and SIRT1 signal was detected in BMDMs. Similar to the AMPK activator AICAR, quercetin dramatically induced phosphorylation of AMPK ␣ 1 and expression of SIRT1 in both basal and LPS-stimulated conditions. Compound C could reverse the effi cacies of quercetin and AICAR ( Fig. 7A , B ).
It has been reported that AMPK could be activated by an AMP-dependent pathway and liver kinase B1 (LKB1) is involved in activation of AMPK in response to an elevated AMP/ATP ratio ( 45 ). Therefore, we detected effects of quercetin treatment on LKB1 and adenine nucleotide levels in basal and LPS-stimulated BMDMs. Quercetin treatment increased LKB1 phosphorylation ( Fig. 7C ) and elevated the cellular AMP/ATP ratio ( Fig. 7D ).
Galic et al. ( 46 ) recently reported that activation of AMPK attenuated the infl ammatory response induced by palmitate or LPS through activation of acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase 1a (Cpt1a) in macrophages. To examine the effects of quercetin on the AMPK/ACC pathway in BMDMs, we detected ACC expression and phosphorylation and Cpt1a mRNA expression. GAPDH was used as reference gene in real-time PCR analysis. Statistical difference between groups was shown using a nonparametric Mann-Whitney test; n = 8 per group. * P < 0.05, ** P < 0.01, *** P < 0.001. All data in (B)-(E) are mean ± SEM. ( 16 ). Moreover, 0.5% or 1% quercetin diets, but not 0.1% quercetin diets, remarkably decreased the expression of ubiquitin C in healthy control mice ( 7 ). Therefore, to avoid the negative effects of excessive quercetin intake, we used 0.1% as the dose of dietary quercetin intervention in this study. After 12 weeks of feeding, the serum concentration of quercetin was 9.19 ± 0.46 mol/l in mice fed quercetin-containing HFD. The concentration was at a comparative level with those in other related topic studies ( 7,17,20 ).
Until now, the effect of quercetin on HFD-induced body weight gain has been debated, and several different groups have reported that dietary quercetin could not impact the body weight of HFD-fed animals ( 6,(16)(17)(18)(19). Similar to two other reports ( 20,21 ), we found here that the mice fed dietary quercetin had signifi cantly lower body weight gain than the mice fed only HFD during 8-12 weeks after diet interventions. Moreover, although dietary quercetin reduced EAT and SAT weights ( Fig. 1C , supplementary Table  III), it did not affect liver and BAT weights (supplementary Table III). It seems that quercetin is a mild weight-reducing and antilipid accumulation component; thus the effi ciency of quercetin on HFD-induced body weight gain may greatly depend on the experimental animal strain, the fat and glucose ratio in HFD, and the dose, age at start of feeding, and duration of dietary quercetin intervention.
Besides reducing body weight, antiadipogenesis ( Fig. 1C, E ) and anti-insulin resistance ( Fig. 2A, B ) effi ciency of dietary quercetin were also identifi ed in our study. Correspondingly, dietary quercetin also increased GLUT4 mRNA expression ( Fig. 2C ) and protein translocation ( Fig. 2D ) and AKT phosphorylation (Ser473) ( Fig. 2E ) in EAT in HFDfed mice, suggesting that it improves glucose uptake in adipose tissues. Moreover, several recent studies have shown that dietary quercetin could improve insulin sensitivity in liver ( 6,20 ) and skeletal muscle ( 47 ), indicating that quercetin could improve systemic insulin resistance in HFD-fed mice.
To explore the molecular mechanisms of quercetin improving HFD-induced adipose tissue infl ammation and insulin resistance, we further focused on two important DISCUSSION In this study, we demonstrate that dietary quercetin ameliorates obesity-associated insulin resistance and adipose tissue infl ammation. Meanwhile, quercetin antagonizes ATM infi ltration and infl ammation and normalizes AMPK ␣ 1 activity and SIRT1 expression in adipose tissues. Further, in vitro studies show that quercetin suppresses BMDM polarization and infl ammation through activating AMPK ␣ 1/SIRT1 signaling. These fi ndings suggest that dietary quercetin can suppress infl ammatory cell recruitment and regulate ATM polarization and infl ammation through a mechanism including AMPK ␣ 1/SIRT1.
Quercetin, an important dietary fl avonoid, has been widely investigated because of its benefi cial effects on diabetes ( 7,22,24 ), hepatic fat accumulation ( 20 ), and metabolic syndromes ( 6,23 ). Adipose tissue infl ammation, especially ATM recruitment, has been regarded as an important central event in obesity-associated insulin resistance ( 3 ). Therefore, we designed this study to test whether quercetin antagonizes adipose tissue infl ammation in HFD-fed mice. Moreover, although both visceral and SATs are white adipose depots, visceral adipose tissues have greater infl ammatory cell recruitment and secrete more abundant proinfl ammatory mediators than SAT responding to HFD ( 3 ). In visceral adipose tissues, mast cell and macrophage infi ltration of EAT is most severe during dietinduced obesity ( 35 ). Therefore, we mainly focus on the infl ammatory cells and signaling in EAT in this study.
Previous studies showed that 0.05% or 0.4% quercetin signifi cantly reduced body weight gain and blood glucose levels in HFD-fed mice ( 20,21 ), but 0.8% or 1.2% quercetin did not affect diet-induced obesity and insulin resistance Quantifi cation of AMPK ␣ 1 activity and SIRT1 expression was described as the ratio of phosphorylated AMPK ␣ 1 to total AMPK ␣ 1 and SIRT1 to ␤ -actin, respectively. Statistical difference between groups was shown using a nonparametric Mann-Whitney test; n = 8 per group in (A) and (B). * P < 0.05, ** P < 0.01. All data in (A) and (B) are mean ± SEM. polarization and inhibit macrophage infl ammation through activating AMPK ␣ 1/SIRT1 signaling. To answer this question, we studied in vitro the effects of quercetin on BMDM infl ammation.
As important tissue infl ammatory cells, macrophages undergo several stages of differentiation, including bone marrow hematopoietic stem cell, blood monocyte, and tissue macrophage ( 48 ). In diet-induced obesity, excess nutrition promotes more ATM recruitment and M1 polarization than in the normal diet state ( 3 ). Our study shows that dietary quercetin inhibits at least two stages of macrophage differentiation and polarization: macrophage infi ltration nutrient sensors and infl ammatory regulators, AMPK ␣ 1 and SIRT1 ( 27,28 ), in EAT. Dietary quercetin restored HFD-suppressed AMPK ␣ 1 activity ( Fig. 5A ) and SIRT1 expression ( Fig. 5B ) in EAT. This suggests that quercetin may impact AMPK ␣ 1/SIRT1 signaling in adipose tissues. Yang et al. ( 29 ) recently reported that AMPK ␣ 1 could activate SIRT1 and inhibit infl ammation in macrophages. Moreover, quercetin inhibited adipogenesis through upregulating the phosphorylation of AMPK ␣ and AMPK ␤ 1 in 3T3-L1 preadipocytes ( 8 ), and quercetin could directly or indirectly activate SIRT1 in vitro ( 31,33 ). Therefore, we asked whether quercetin could regulate macrophage could initiate obesity and insulin resistance ( 50 ). Moreover, LPS can induce macrophage M1 activation and proinfl ammatory gene expression ( 51 ). Therefore, in this study, LPS was used to induce obesity-associated macrophage M1 polarization and infl ammation.
In basal and LPS-stimulated conditions, quercetin downregulated M1 macrophage marker and proinfl ammatory cytokine expression and upregulated M2 macrophage marker and anti-inflammatory cytokine expression in BMDMs ( Fig. 6 ). Further, quercetin signifi cantly increased (from monocyte to macrophage) ( Fig. 4A-C ) and macrophage subtype transformation (from M2 to M1 subtype) ( Fig. 4D, E ). To mimic comprehensively the possible role of quercetin in macrophage differentiation and polarization, vehicle or 20 M quercetin was added during BMDM differentiation and LPS treatment. In type 2 diabetic patients and humans after a high-fat meal, circulating LPS levels were increased ( 49,50 ). In experimental animals, HFD enhanced circulating LPS levels through altering intestinal permeability, and subcutaneous infusion of LPS Quantifi cation of AMPK ␣ 1 activity, SIRT1 expression, and LKB1 activity was described as the ratio of phosphorylated AMPK ␣ 1 to total AMPK ␣ 1, SIRT1 to ␤ -actin, and phosphorylated LKB1 to total LKB1, respectively. (D) Changes of cellular AMP/ATP ratio by quercetin in BMDMs. Data are represented as fold changes compared with vehicle. C.c represented Compound C. Statistical difference between groups was shown using a Student's t -test; n = 4 per group (A-D). * P < 0.05, ** P < 0.01, *** P < 0.001. All data in (A)-(D) are mean ± SEM. confi rmation, including studies in oxygen consumption and CO 2 production, changes in the hourly respiration exchange rate, core temperatures, physical activity, morphological analysis of BAT, expression of series energy expenditure marker genes in BAT, and so on. Third, considering the paradoxical effects of quercetin on HFDinduced body weight gain in previous reports ( 6,(16)(17)(18)(19)(20)(21), dietary quercetin intervention started at 5 weeks old, an age that may be more sensitive to HFD-induced effi cacy. Thus, the metabolic impact of dietary quercetin during development needs to be investigated in our future work. Moreover, we did not use classic freeze clamp methodology here, which may lead to hypoxia and enhance AMPK activity post mortem. However, all the tissues were similarly handled in our study, and the non-freeze clamp methodology should not affect the relative difference of AMPK activities among EATs from the tested mice. Indeed, in accordance with a previously published study ( 29 ), we showed here that HFD reduced AMPK activities in EAT in mice ( Fig. 5A ). Therefore, dietary quercetin-abolished HFDinduced AMPK inactivation ( Fig. 5A ) should not be compromised by our non-freeze clamping manipulation.
In summary, as shown in supplementary Fig. III, we demonstrate here that dietary quercetin ameliorates HFDinduced obesity and insulin resistance in mice. Meanwhile, quercetin attenuates infl ammatory mast cell and macrophage infi ltration into adipose tissue and reduces adipose tissue infl ammation. More importantly, quercetin antagonizes M1 polarization and infl ammation of ATMs, which play a critical role in obesity-associated adipose tissue infl ammation and insulin resistance ( 3 ). Finally, we confi rm that quercetin enhances LKB1 activity and the AMP/ATP ratio, activates AMPK ␣ 1/SIRT1 and other associated antiinflammation (e.g., AMPK/ACC) signal pathways, and thereby inhibits macrophage polarization and infl ammation. Considering that quercetin is an abundant dietary fl avonoid in plants and its inherent antiadipogenesis, antiinfl ammation, and anti-insulin resistance actions, it might become a benefi c dietary supplement to protect against obesity-associated insulin resistance and diabetes. the phosphorylation level of AMPK ␣ 1 ( Fig. 7A ) and SIRT1 expression ( Fig. 7B ) in basal and LPS-exposed BMDMs. Meanwhile, the effects of quercetin were similar to those of AMPK activator AICAR and could be blocked by AMPK inhibitor Compound C ( Fig. 6; Fig. 7A, B ). LKB1 is an upstream kinase, which directly phosphorylates and activates AMPK in response to an elevated AMP/ATP ratio ( 46 ). To determine how quercetin activates AMPK, we detected the effect of quercetin on an AMP-dependent pathway including the AMP/ATP ratio, LKB1 expression, and phosphorylation. Quercetin treatment increased LKB1 activity ( Fig. 7C ) and the cellular AMP/ATP ratio ( Fig. 7D ) in BMDMs. Because LPS treatment promotes M1 activation of macrophages ( 51 ), the results suggest that quercetin can upregulate the AMP/ATP ratio and LKB1 activity and further activate AMPK ␣ 1/SIRT1 signaling to inhibit M1 polarization and infl ammation of macrophages.
In diet-induced obesity, adipose tissues possess hypertrophic adipocytes and more infl ammatory cells, such as mast cells and macrophages. The cells secrete more proinfl ammatory mediators and produce adipose tissue infl ammation ( 1,2 ). AMPK has been considered a central inhibitor of adipose tissue infl ammation ( 28,52 ). Activating AMPK may suppress c-Jun activation ( 52,53 ) and stimulate SIRT1 ( 28,29 ) to inhibit nuclear factor-B (NF-B). In macrophages, AMPK ␤ activation, which can mainly determine AMPK ␣ 1 activity and expression, attenuates palmitate-or LPS-induced infl ammation through increasing FA oxidation associated with ACC and Cpt1a ( 47 ). It is noteworthy that increased macrophage lipid accumulation is related to M1 macrophage polarization in adipose tissue during obesity ( 54 ) and SIRT1 is involved in AMPK-dependent FA oxidation enhancement ( 55 ). Additionally, AMPK activation also regulates GLUT4 translocation ( 40 ). Here, we found that quercetin not only activated AMPK ␣ 1/SIRT1 but also promoted ACC phosphorylation and Cpt1a expression in the LPS-stimulated condition (supplementary Fig. I). Previous studies reported that quercetin reduced macrophage (10)(11)(12)(13) and adipocyte ( 10 ) infl ammation through suppressing phosphorylation of c-Jun N-terminal kinase and c-Jun and inhibiting activation of NF-B. Therefore, AMPK/SIRT1 activation by quercetin might increase FA oxidation, depress infl ammatory signals such as c-Jun and NF-B, and enhance glucose uptake in adipose tissues, thereby improving adipose tissue infl ammation and insulin resistance.
This study has several potential limitations because we only focused on the possible action of quercetin on HFDinduced adipose tissue and macrophage infl ammation in mice. First, we did not assess the effects of dietary quercetin on lean body mass, although EAT, SAT, BAT, and liver weights were detected ( Fig. 1C , supplementary Table III). However, the results cannot give us any information about the alteration in lean body mass by quercetin, so we should perform a lean body mass study in the future. Second, we found here that dietary quercetin reduced the mast cell number in EAT and increased energy expenditure marker Ucp1 expression in BAT ( Fig. 3 ), suggesting that energy expenditure levels are enhanced in mice fed quercetincontaining HFD. However, this hypothesis requires further