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Activation of the Cholinergic Antiinflammatory Pathway Ameliorates Obesity-Induced Inflammation and Insulin Resistance XianFeng Wang,* ZhengGang Yang,* Bingzhong Xue, and Hang Shi Section on Endocrinology and Metabolism (X.W., B.X.), and Section on Gerontology and Geriatric Medicine (Z.Y., H.S.), Department of Internal Medicine, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157

Obesity is associated with a chronic inflammatory state characterized by adipose tissue macrophage infiltration and inflammation, which contributes to insulin resistance. The cholinergic antiinflammatory pathway, which acts through the macrophage ␣7-nicotinic acetylcholine receptor (␣7nAChR), is important in innate immunity. Here we show that adipose tissue possesses a functional cholinergic signaling pathway. Activating this pathway by nicotine in genetically obese (db/db) and diet-induced obese mice significantly improves glucose homeostasis and insulin sensitivity without changes of body weight. This is associated with suppressed adipose tissue inflammation. In addition, macrophages from ␣7nAChR⫺/⫺ [␣7 knockout (␣7KO)] mice have elevated proinflammatory cytokine production in response to free fatty acids and TNF␣, known agents causing inflammation and insulin resistance. Nicotine significantly suppressed free fatty acid- and TNF␣-induced cytokine production in wild type (WT), but not ␣7KO macrophages. These data suggest that ␣7nAChR is important in mediating the antiinflammatory effect of nicotine. Indeed, inactivating this pathway in ␣7KO mice results in significantly increased adipose tissue infiltration of classically activated M1 macrophages and inflammation in ␣7KO mice than their WT littermates. As a result, ␣7KO mice exhibit more severely impaired insulin sensitivity than WT mice without changes of body weight. These data suggest that the cholinergic antiinflammatory pathway plays an important role in obesity-induced inflammation and insulin resistance. Targeting this pathway may provide novel therapeutic benefits in the prevention and treatment of obesity-induced inflammation and insulin resistance. (Endocrinology 152: 836 – 846, 2011)

besity is the most important identified factor contributing to insulin resistance. Inflammation is a key feature of obesity and causatively links obesity to insulin resistance. Characteristics of obesity-induced inflammation include elevated production of proinflammatory molecules by adipose tissue, and the activation of a network of inflammatory signaling pathways, including the following inflammatory kinases: the c-Jun NH2-terminal kinase (JNK), inhibitor of nuclear factor (NF)-␬B kinase ␤

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(IKK␤), and protein kinase C-␪, which result in insulin resistance by modifying serine phosphorylation of insulin receptor (IR) substrate proteins in the insulin-signaling cascade (1). An important feature of obesity-induced inflammation is the infiltration of macrophages into adipose tissue (2, 3). These bone marrow-derived adipose tissue macrophages (ATMs) are the predominant source of TNF␣ and other proinflammatory molecules in adipose tissue (2, 3) and

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/en.2010-0855 Received July 27, 2010. Accepted December 9, 2010. First Published Online January 14, 2011 * X.W. and Z.Y. contributed equally to this work.

Abbreviations: ATM, Adipose tissue macrophage; BchE, butyrylcholinesterase; DIO, dietinduced obese; EWAT, epididymal adipose tissue; FACS, fluorescence-activated cell sorting; FFA, free fatty acid; GTT, glucose tolerance test; HF, high fat; IKK␤, inhibitor of NF-␬B kinase ␤; iNOS, inducible NOS; IR, insulin receptor; IRS, insulin receptor substrate; ITT, insulin tolerance test; JAK2, Janus Kinase 2; JNK, c-Jun NH2-terminal kinase; ␣7KO, ␣7 knockout; LF, low fat; LPS, lipopolysaccharide; MCP1, monocyte chemoattractant protein 1; ␣7nAChR, ␣7-nicotinic acetylcholine receptor; NF, nuclear factor; PKB, protein kinase B; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; SVF, stromal vascular fraction; WT, wild type.

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have been implicated in the development and maintenance of obesity-induced adipose tissue inflammation (1). The importance of macrophage in modulating insulin sensitivity is supported by genetic studies. Macrophage-specific disruption of the NF-␬B pathway (e.g. IKK␤) results in improved insulin sensitivity (4). Ablation of JNK1 in hematopoietically derived cells including macrophages also protects mice from diet-induced inflammation and insulin resistance without affecting adiposity (5). These data collectively demonstrate that macrophage inflammation is an important mediator of obesity-induced insulin resistance. Studying antiinflammatory mechanisms in macrophages may provide novel therapeutic targets in the treatment of obesity, inflammation, and insulin resistance. Like other physiological processes, the immune process is also regulated by neural reflexes, which comprise an afferent arm that senses inflammation and an efferent arm, i.e. signals transmitted via the efferent vagus nerve that inhibits innate immune responses. This inflammatory reflex has been defined as the “cholinergic antiinflammatory pathway” (6). Electrical stimulation of the vagus nerve or agonists (e.g. acetylcholine or nicotine) mimicking this effect ameliorates proinflammatory cytokine release from macrophages and protects against endotoxemia and other inflammation-related tissue damage; whereas vagotomy substantially increases lipopolysaccharide (LPS)-induced cytokine release (7, 8). The antiinflammatory effect of the cholinergic signaling pathway is further supported by the observation that pharmacological acetylcholinesterase inhibition decreases NF-␬B activity, reduces cytokine production, and improves survival in experimental sepsis (9). The ␣7-nicotinic acetylcholine receptor (␣7nAChR) on cytokine-producing cells such as macrophages is necessary in mediating this effect. Activation of the cholinergic antiinflammatory pathway by the nAChR agonist nicotine inhibits cytokine production, which can be blocked by specific ␣7nAChR antagonists (10). A selective ␣7nAChR agonist improves survival in murine endotoxemia and severe sepsis (11). Most importantly, ␣7nAChR⫺/⫺ mice produce significantly more LPS-induced TNF␣, IL-1␤, and other cytokines systemically and from macrophages than wild-type (WT) mice. Electrical stimulation of the vagus nerve attenuated LPS-induced inflammation in WT mice but was ineffective in ␣7nAChR⫺/⫺ mice (8). These data demonstrate that the effect of the cholinergic antiinflammatory pathway may be mediated by ␣7nAChR on macrophages. The cholinergic antiinflammatory pathway has been extensively studied in terms of its immunomodulating function and protective effects against a wide range of inflammation-related disorders, including sepsis, hemorrhagic shock, myocardial ischemia, postoperative ileus,

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ulcerative colitis, arthritis, and pancreatitis (6, 12), which results in successful clinical applications. However, this pathway is not well characterized in the context of obesity and insulin resistance. Mabley et al. (13) reported that nicotine treatment significantly reduced incidence of diabetes in both streptozotocin-induced type 1 diabetes and spontaneous nonobese diabetic type 1 diabetes mouse models, which is associated with decreases in pancreatic inflammatory status. Oral nicotine administration for 8 wk also improved insulin sensitivity with the concomitant reduction of visceral fat TNF␣ content in obese and insulin-resistant Zucker fatty (fa/fa) rats without changes in body weight (14, 15). These data indicate that this pathway may also have protective effects against both type 1 and 2 diabetes. Therefore, the current study is designed to more thoroughly characterize the role of ␣7nAChR-mediated cholinergic antiinflammatory pathway in obesity, inflammation, and insulin resistance. We demonstrate that adipose tissue and macrophages express ␣7nAChR. In addition, activation of the cholinergic antiinflammatory pathway by nicotine significantly suppresses free fatty acids (FFAs)and TNF␣-induced inflammation in wild-type (WT), but not in ␣7nAChR⫺/⫺ [a7 knockout (␣7KO)] macrophages. Most importantly, activation of this pathway by nicotine significantly suppresses inflammation and improves insulin sensitivity in genetically obese (db/db) and diet-induced obese (DIO) animal models, whereas inactivating this pathway in ␣7KO mice results in abnormal inflammation and impaired insulin sensitivity.

Materials and Methods Animals For nicotine injection studies, 6-wk-old male C57BL/6J (B6) mice (Jackson Laboratories, Bar Harbor, ME) were put on chow (LabDiet 5P00, fat content 14% by calorie) or high-fat (HF) diet (Research Diets D12492; fat content 60% by calorie) for 11 wk. Male db/db and lean control (⫹/?) mice, 8 wk of age (Jackson Laboratories, Bar Harbor, ME). were on chow diet. Mice were randomly assigned to receive saline or nicotine injection ip twice daily for 3 wk [nicotine dose, 400 ␮g/kg, (-)-nicotine hydrogen tartrate salt (Sigma Aldrich, St. Louis, MO) calculated by free base (16)]. Male ␣7nAChR⫺/⫺ (␣7KO) mice on B6 background (Jackson Laboratories) and their WT littermates were put on low fat (LF) (Research Diets D12329, fat content 11% by calorie) or HF diet (Research Diets D12331, fat content 58% by calorie) for up to 24 wk starting at 6 wk of age. All animals were housed with a 12-h light, 12-h dark cycle in a temperature-controlled facility and had free access to water and food. All aspects of animal care were approved by the Wake Forest University Health Sciences Institutional Animal Care and Use Committee.

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Cell culture Peritoneal macrophages from mice were isolated as described elsewhere (17). Briefly, 12- to 14-wk-old WT and ␣7KO mice were ip injected with 2 ml 3% thioglycollate to elicit peritoneal macrophages. Mice were euthanized by CO2 inhalation 48 –72 h later, and cells were collected by lavaging the peritoneal cavity. Cells were plated at a density of 1 ⫻ 106 cells per well in 24-well plates in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum for 3 h, and medium was then changed. In some experiments, cells were pretreated with nicotine (1 ␮M) for 16 –18 h. On the second day, cells were changed to serum-free medium for 1 h with freshly added nicotine and then treated with stearic acid (C18:0) (500 ␮M) or TNF␣ (10 ng/ml) in the presence or absence of nicotine for an additional 4 h. Fatty acids were dissolved in 95% ethanol at 60 C and then were mixed with prewarmed BSA (10%) to yield a stock concentration of 7.5 mM.

Metabolic measurements Metabolic measurements were conducted as described elsewhere (18). For measuring blood glucose levels at fed or fasted state or during glucose and insulin tolerance tests, blood was collected via tail bleed. At the end of studies, mice were killed by CO2 inhalation at the fed state, except for the in vivo insulin signaling and the hyperinsulinemic-euglycemic clamp studies, where mice were killed at the fasted state. Blood was collected via trunk bleed at when animals were killed. Blood glucose was measured using an OneTouch Ultra glucose meter (LifeScan, Inc., Milpitas, CA). Serum insulin and TNF␣ levels and adipose tissue TNF␣ levels were measured by ELISA (Crystal Chem Inc., Chicago, IL).

Glucose tolerance, insulin tolerance, and insulin-signaling studies For glucose tolerance tests (GTTs), mice were fasted overnight, and blood glucose was measured immediately before and 15, 30, 60, 90, and 120 min after ip injection of glucose (1.2–1.5 g/kg of body weight). For insulin tolerance tests (ITTs), food was removed for 4 h, and blood glucose was measured immediately before and 15, 30, 60, 90, and 120 min after ip injection of human insulin (Humulin, 1.5–1.75 U/kg of body weight; Lilly). For insulin-signaling studies, mice were fasted overnight. Humulin (10 U/kg of body weight) was injected ip; 10 min later, mice were killed by CO2, and tissues were quickly collected and snap frozen in liquid nitrogen. Tissues were stored at ⫺80 C until processing.

Hyperinsulinemic-euglycemic clamp study Hyperinsulinemic-euglycemic clamp study was performed as described elsewhere (17). Mice were implanted with the indwelling catheters and allowed to recover for 5 d. After an initial 5-␮Ci bolus, [3-3H]glucose was infused at 0.05 ␮Ci/min for 2 h to measure basal glucose turnover. A 2-h hyperinsulinemic-euglycemic clamp was conducted with a prime and continuous infusion of insulin at a rate of 2.5 mU/kg/min, coupled with a variable infusion of 40% glucose to maintain blood glucose at 6 mM. Blood glucose was measured via tail bleed every 5 min in the first hour to achieve stable blood glucose levels and every 10 min until the end of the 2-h clamp to maintain constant blood glucose levels. The rate of whole-body glucose turnover was estimated using a continuous infusion of [3-3H]glucose at 0.1 ␮Ci/min. Tissue-specific glucose uptake was estimated by a bolus admin-

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istration of 10 ␮Ci 2-deoxy-D-[1-14C]glucose 45 min before the end of clamp experiments.

Immunoblotting analysis Tissue homogenizing and immunoblotting to detect phospho- and total protein levels were performed as described (18). After incubating with primary antibodies, blots were incubated with Alexa Fluor 680-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) and developed with a Li-COR Odyssey Infrared Imager system (Li-COR Biosciences, Lincoln, NE). Rabbit anti-iR, butyrylcholinesterase (BchE), and goat anti-␤actin polyclonal antibodies were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Rabbit anti-insulin receptor substrate (IRS)-1 polyclonal antibodies were from Upstate Biotechnology (Lake Placid, NY). Rabbit antiphospho-IR Tyr1162/Tyr1163 and antiphospho-IRS-1 Tyr612 polyclonal antibodies were from BioSource International, Inc. (Camarillo, CA). Rabbit antiphosphoAkt/protein kinase B (PKB) Ser473 and antitotal Akt/PKB polyclonal antibodies were from Cell Signaling Technology, Inc. (Beverly, MA) (18).

RNA extraction and reverse transcription RT-PCR RNA extraction and gene expression analysis using quantitative real-time RT-PCR were performed as described using Stratagene Mx3000 (Stratagene, La Jolla, CA) (18). Primer and probe sequences for cyclophilin were: 5⬘-GGTGGAGAGCACCAAGACAGA-3⬘ (forward), 5⬘-GCCGGAGTCGACAATGATG-3⬘ (reverse), and 5⬘-TCCTTCAGTGGCTTGTCCCGGCT-3⬘ (probe) (18). TaqMan primer/probes for all other genes were purchased from Applied Biosystems (Foster City, CA). To determine tissue expression levels of ␣7nAChR, 2 ␮g of total RNA were reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA product (1 ␮l) was used to amplify ␣7nAChR and ␤-actin using the following primers: ␣7nAChR (forward), 5-GGCCAACGACTCGCAGCCGCTC-3; ␣7nAChR (reverse), 5-GCAGGTCCAAGGACCACCCTC-3; ␤-actin (forward), 5-CGTACCACGGGCATTGTGAT-3; ␤-actin (reverse), 5-GGCAGCTCATAGCTCTTCTC-3. The PCR primers for ␣7nAChR span across exons 2– 6 on the ␣7nAChR gene, thereby eliminating the possibility of false-positive results from genomic DNA contamination. The PCR product was resolved with gel electrophoresis.

Isolation of adipocytes and ATMs Isolation of primary adipocytes and adipose tissue stromal vascular fraction (SVF) were performed as described elsewhere (19). Briefly, 1–2 g of epididymal fat were placed in Krebs-Ringer HEPES buffer containing 10 mg/ml fatty acid-poor BSA (SigmaAldrich), minced and digested with 1 mg/ml collagenase type I (Worthington Biochemical Co., Lakewood, NJ) at 37 C for 1 h. Samples were filtered through a 300-␮m nylon mesh (Spectrum Laboratories, Rancho Dominguez, CA), and the resulting suspension was centrifuged at 500 ⫻ g for 5 min to separate SVF cells from adipocytes. Adipocytes were then harvested and used for RNA extraction and gene expression analysis. SVF cells were incubated with rat antimouse F4/80 polyclonal antibodies (AbD Serotec, Releigh, NC), followed by a pull down of F4/80-positive cells with sheep antirat microbeads using MACS cell separation system according to manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Isolated F4/80⫹ ATMs were used for gene expression analysis.

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Fluorescence-activated cell sorting (FACS) analysis In some experiments, SVF cells were incubated in erythrocyte lysis buffer (eBioScience, San Diego, CA) for 5 min, washed, and then resuspended in FACS buffer (eBioscience). A portion of the cells was counted with a hemacytometer. Based on trypan blue exclusion, the percentage of live cells per sample was usually greater than 95%. Cells were incubated in the dark on a shaker with FcBlock (eBioscience) for 30 min at 4 C and further incubated for 1 h with allophycocyanin-conjugated F4/80 and phycoerythrin-conjugated CD11c antibodies (eBioscience). After incubation, cells were washed, fixed in 4% paraformaldehyde, and analyzed with a FACS Calibur machine (BD Pharmingen, Franklin Lakes, NJ). FACS data were analyzed with CellQuest software (BD Pharmingen).

Statistical analysis Results are presented as mean ⫾ SE. Differences between groups were analyzed for statistical significance by Student’s t test, one-way or two-way ANOVA followed by Bonferroni or Fischer’s probable least-squares difference post hoc test or ANOVA with repeated measures as appropriate.

Results Using RT-PCR, we found that mouse adipose tissue and macrophages, including bone marrow-derived and peritoneal macrophages, expressed the ␣7nAChR (Supplemental Fig. 1A published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). In addition, the expression of ␣7nAChR tended to be higher in ATMs than in isolated adipocytes (Supplemental Fig, 1B). Acetylcholine, the endogenous ligand for ␣7nAChR, is degraded by two enzymes, acetylcholinesterase (AChE) and BChE (20). We found that AchE and BChE were the major cholinesterases in brain and adipose tissue, respectively (Supplemental Fig. 1C). The expression of BChe in epididymal adipose tissue (EWAT) was up-regulated with chronic nicotine treatment (Supplemental Fig. 1D), but down-regulated by 30% and 60% in mice fed a high-fat diet (Supplemental Fig. 1E) and in db/db mice (Supplemental Fig. 1F), respectively. Consistent with BChE RNA expression, adipose tissue BChE protein levels were also up-regulated by chronic nicotine treatment (Supplemental Fig. 1G) and down-regulated in db/db mice (Supplemental Fig. 1H). The up-regulated BChE levels in adipose tissue of chronic nicotine-treated mice may serve as feedback inhibition of an activated cholinergic signaling pathway, whereas the down-regulated BChE levels in adipose tissue of HF and db/db mice may reflect a suppressed cholinergic signaling pathway. Therefore, our data suggest that adipose tissue possesses a functional cholinergic signaling pathway. To test whether activation of the cholinergic antiinflammatory pathway protects against obesity-induced in-

FIG. 1. Activation of the cholinergic antiinflammatory pathway by nicotine improves glucose homeostasis in db/db mice. db/db and lean control mice (8 wk of age) on chow diet were treated ip with 400 ␮g/ kg nicotine twice daily for 3 wk. Fed (panel A) and fasting (panel B) glucose were measured after 3 wk of treatment (n ⫽ 5). Data are expressed as mean ⫾ SE. Statistical significance is indicated by the presence of different superscripts. Groups labeled with the same superscripts are not statistically different from each other. Groups labeled with different superscripts are statistically different from each other.

sulin resistance and diabetes, we administered nAChR agonist nicotine to 8-wk-old male db/db mice. To avoid the confounding effect of nicotine on body weight and food intake, we used a lower dose of nicotine, 400 ␮g/kg, which is effective in preventing LPS-induced cytokine production and sepsis (21) and reducing diabetes incidence in a type 1 diabetic mice model (13) without affecting food intake and body weight (16, 22). As expected, 8-wk-old db/db mice were obese (Supplemental Fig, 2A) and diabetic (Fig. 1A) before nicotine treatment. Nicotine treatment (400 ␮g/kg twice daily) for 3 wk did not alter body weight in either db/db mice or lean controls, compared with saline-injected mice (Supplemental Fig. 2A). Nicotine also did not change food intake in lean control mice (Supplemental Fig. 2B). However, nicotine treatment lowered fed blood glucose levels in db/db mice (588 ⫾ 7 mg/dl in db/db saline group vs. 452 ⫾ 30 mg/dl in db/db nicotine group; P ⬍ 0.05), whereas it did not change blood glucose levels in lean controls (Fig. 1A). Nicotine treatment also reduced fasting blood glucose levels in db/db mice (561 ⫾ 23 mg/dl in db/db saline group vs. 319 ⫾ 60 mg/dl in db/db nicotine group; P ⬍ 0.05) (Fig. 1B). Nicotine treatment tended to lower plasma insulin levels (Supplemental Fig. 2C). These data demonstrate that nicotine improves glucose homeostasis in genetically obese and diabetic animal models without changes in body weight and food intake. We have also tested our hypothesis in a DIO animal model. Male B6 mice were put on chow or HF diet for 11 wk starting at 6 wk of age and then treated with either saline or 400 ␮g/kg nicotine ip twice daily for 3 wk, whereas mice on the chow diet received only saline, because we have shown that nicotine had no effect on blood glucose and insulin levels in chow-fed lean mice

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nase and subsequent translocation of glucose transporter 4 in response to insulin (24). In muscle of chow-fed mice, insulin rapidly stimulated IR phosphorylation at Tyr1162/Tyr1163 and IRS1 phosphorylation at Tyr612 (Fig. 2, E and F). HF feeding resulted in reduced insulin-stimulated IR and IRS1 tyrosyl phosphorylation in muscle (reduced by 45% and 65%, respectively), which was restored to 90% and 80%, respectively, by nicotine treatment (Fig. 2, E and F). Insulin stimulation also resulted in a profound increase in Akt/PKB Ser473 phosphorylation in muscle of chow-fed mice (Fig. 2G). HF feeding led to reduction of insulin-stimulated Akt/PKB phosphorylation by 65%, which was restored by nicotine treatment to 80% of that seen in chow-fed FIG. 2. Activation of the cholinergic antiinflammatory pathway by nicotine improves insulin sensitivity in DIO mice. Male C57BL/6 mice (6 wk of age) were put on chow or HF diet for 11 mice (Fig. 2G). wk followed by saline or nicotine (400 ␮g/kg) injection (ip) twice daily for 3 wk. Fed glucose Similarly, insulin stimulated signifi(panel A) fed insulin (panel B) levels, GTT (panel C), and ITT (panel D) were assessed. GTT was cant IR phosphorylation at Tyr1162/ performed in overnight fasted mice with ip injection of glucose at 1.2 g/kg of body weight. Tyr1163 and Akt/PKB phosphorylation ITT was performed 4 h after food removal. Mice were ip injected with insulin at 1.5 U/kg (n ⫽ 10) for chow-saline for HF-saline (n ⫽ 5) and for HF-nicotine (n ⫽ 7) in panels A–D. E–G, at Ser473 in liver and fat of chow-fed Insulin signaling study was conducted at the end of 3-wk saline or nicotine treatment by ip mice (Supplemental Fig. 3, C and D). injection of 10 U/kg insulin into overnight-fasted mice. Gastrocnemius muscle was collected 1162 1163 612 This was decreased in HF-fed mice and 10 min later, and phosphorylation of IR at Tyr Tyr (panel E), IRS1 Tyr (panel F), Akt/ PKB at ser473 (panel G), and total IR, IRS1, and Akt/PKB were measured by Western blotting restored by nicotine treatment (Suppleanalysis. Bar graphs in panels E–G show phosphorylation levels of IR, IRS1, and Akt/PKB mental Fig. 3, C and D). These data normalized to total IR, IRS1, and Akt/PKB levels (n ⫽ 5 each for chow-saline and HF-saline demonstrate that nicotine improved groups and n ⫽ 6 for HF-nicotine group). Data are expressed as mean ⫾ SE. In panels C and D: *, P ⬍ 0.05 vs. other two groups; in panels A, B, and E–G, statistical significance is glucose homeostasis and insulin sensiindicated by the presence of different superscripts. Groups labeled with the same superscripts tivity in DIO mice without modifying are not statistically different from each other. Groups labeled with different superscripts are body weight. statistically different from each other. Nic, Nicotine; Ins, insulin. We explored whether nicotine’s effect on improving insulin sensitivity (Fig. 1, A and B and Supplemental Fig. 2C). As in db/db was associated with suppressions of obesity-induced inmice, nicotine treatment did not change body weight in flammation. Serum TNF␣ level was significantly elevated HF-fed mice over 3 wk (Supplemental Fig. 3A). Howin db/db mice, which was normalized by nicotine (Fig. ever, nicotine treatment normalized the fed (Fig. 2A) 3A). Nicotine also normalized the dramatically increased and fasting (Supplemental Fig. 3B) glucose levels and mRNA and protein levels of TNF␣ in EWAT of db/db fed insulin levels (Fig. 2B) in HF-fed mice. Nicotine also mice (Fig. 3B). The expression of monocyte chemoattracsignificantly improved insulin sensitivity in HF-fed tant protein 1 (MCP1), which is important in attracting mice as assessed by GTT and ITT, respectively (Fig. 2, macrophages into inflamed adipose tissue (25), and the C and D). We evaluated early steps in insulin signaling by deter- specific macrophage marker F4/80 was significantly inmining the tyrosyl phosphorylation sites critical for acti- creased in EWAT of db/db mice (Fig. 3C). The expression vation of the insulin-signaling cascade, including auto- of F4/80 was normalized, whereas the expression of phosphorylation of IR at the tandem tyrosyl residues MCP1 was reduced by 40% by nicotine treatment (Fig. Tyr1162 and Tyr1163, located in the activation loop of the 3C), suggesting a reduction of macrophage infiltration in IR kinase domain and required for IR activation and sub- adipose tissue (2, 3). Nicotine treatment also normalized sequent phosphorylation of other IR tyrosines (23), and significantly increased proinflammatory cytokine levels in the tyrosyl phosphorylation of IRS1 at Tyr612, which is EWAT of db/db mice, including IL-6, IL-1␤, and inducible important for full activation of phosphatidylinositol 3-ki- NOS (iNOS) (Fig. 3C). Similarly, nicotine treatment in

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ically (db/db) and diet-induced obese mice, which likely contributes to their improved insulin sensitivity. The effect of cholinergic antiinflammatory pathway is mediated through the ␣7nAChR on macrophages (8). We therefore tested whether macrophage ␣7nAChR also mediates the antiinflammatory effects of nicotine against FFAs and TNF␣, important mediators of obesity-induced inflammation and insulin resistance (1). We found that saturated fatty acids [myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0)] stimulated significant TNF␣ expression in WT peritoneal macrophages, with stearic acid being the most potent (Supplemental Fig. 5A). Therefore, we used stearic acid in the following experiments. As shown in Fig. 4, A–D, stearic acid significantly induced the expression of proinflammatory cytokines, including TNF␣, IL-1␤ and IL-6, as well as TNF␣ secretion from peritoneal macrophages isolated from WT mice. This effect was further increased in macrophages isolated from ␣7nAChR⫺/⫺ FIG. 3. Activation of the cholinergic signaling pathway by nicotine suppresses adipose tissue inflammation in db/db mice. Serum TNF␣ (␣7KO) mice. Nicotine significantly suppressed stearic acid(panel A), epididymal white adipose tissue (WAT) TNF␣ mRNA, and induced cytokine production in WT, but not ␣7KO macroprotein levels (panel B) were measured by ELISA. The expression of phages. The adipocyte- and macrophage-derived cytokine MCP1, F4/80, IL-6, IL-1␤, and iNOS (panel C) was measured by realtime RT-PCR and normalized to cyclophilin (n ⫽ 5) in panels A–C. Data TNF␣ plays an important role in the development of insulin are expressed as mean ⫾ SE. Statistical significance is indicated by the resistance (1) and can further promote the expression of propresence of different superscripts. Groups labeled with the same inflammatory cytokines including itself, probably via actisuperscripts are not statistically different from each other. Groups vation of NF-␬B and activator protein 1 transcription factors labeled with different superscripts are statistically different from each other. Nic, Nicotine. (1). We found that TNF␣ also significantly induced the expression of proinflammatory cytokines, including IL-1␤, DIO mice also normalized the elevated expression of F4/80 IL-6, and TNF␣ itself, in WT macrophages, which was furand proinflammatory cytokines including TNF␣, IL-6, IL- ther increased in ␣7KO macrophages (Supplemental Fig. 5, 1␤, and iNOS, and reduced the expression of MCP1 by 60% B–D). Nicotine significantly suppressed TNF␣-induced cyin EWAT (Supplemental Fig. 4). These data suggest that ac- tokine expression in WT, but not ␣7KO macrophages (Suptivating the cholinergic antiinflammatory pathway by nico- plemental Fig. 5, B–D). These data demonstrate that the antine suppresses ATM infiltration and inflammation in genet- tiinflammatory effect of nicotine against FFAs and TNF␣ is dependent on the presence of ␣7nAChR in macrophages. To further explore the role of ␣7nAChR in obesity, inflammation, and insulin resistance, we put ␣7KO and their WT littermates on either LF or HF diet. There was no difference in body weight (Supplemental Fig. 6, A and B), epididymal fat mass (Supplemental Fig. 6C), or food intake (SupFIG. 4. ␣7nAChR mediates nicotine’s antiinflammatory effect in peritoneal macrophages. plemental Fig. 6D) between WT and TNF␣ (panel A), IL-1␤ (panel B), IL-6 expression (panel C), and TNF␣ secretion (panel D) in ␣7KO mice on either diet. However, peritoneal macrophages treated with stearic acid (C18:0) in the presence or absence of nicotine. Peritoneal macrophages were isolated from WT or ␣7KO mice as described in adipose tissue SVF from LF-fed ␣7KO Materials and Methods. Cells were pretreated with nicotine (1 ␮M) for 16 –18 h and then mice contained significantly more F4/ treated with stearic acid (500 ␮M) in the presence or absence of nicotine for additional 4 h. 80⫹ macrophages compared with LFThe expression of target genes was measured by real-time RT-PCR and normalized to cyclophilin. TNF␣ secretion into the medium was measured by ELISA for treatments in WT fed WT mice, as assessed by FACS analgroups (n ⫽ 3– 4) and for treatments in KO groups (n ⫽ 5– 6). Data are expressed as ysis (Fig. 5A), indicating increased mean⫾SE. Statistical significance is indicated by the presence of different superscripts. Groups macrophage infiltration into adipose labeled with the same superscripts are not statistically different from each other. Groups tissue of ␣7KO mice. labeled with different superscripts are statistically different from each other. Nic, Nicotine.

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compared with WT mice (Fig. 5C). Similarly, HF feeding also resulted in a significant increase of the percentage of F4/80⫹CD11c⫹ cells in SVF cells of ␣7KO mice compared with WT mice (Fig. 5D). The number of F4/ 80⫹CD11c⫹ cells per fat pad (Fig. 5E) or per gram of fat tissue (Fig. 5F) was also increased in ␣7KO mice on HF diet compared with WT mice. HF feeding also resulted in 2- to 2.5-fold increase in the expression of F4/80, TNF␣, and MCP1 in adipose tissue from ␣7KO mice compared with WT mice (Fig. 5G). These data suggest that ␣7KO mice have increased ATM infiltration and inflammation on both LF and HF diet. MCP1 plays an important role in atFIG. 5. ␣7KO mice have increased adipose tissue inflammation. A, Analysis of SVF cells for F4/80 tracting macrophage infiltration into and CD11c. Epididymal fat pads from male WT and ␣7KO mice on chow diet were separated into adipose tissue during the development adipocyte and SVF populations. SVF cells were stained with antibodies against F4/80, CD11c, and of obesity and insulin resistance (25). isotype controls and analyzed by flow cytometry. Samples were gated for F4/80⫹ cells (upper panels) and examined for coexpression of CD11c (lower panels). Data from a representative To identify the source of MCP1, we experiment are shown. The percentage of CD11c⫹ cells within the F4/80⫹ ATM is indicated for measured MCP1 expression in adieach genotype. B and C, Quantitation of F4/80⫹CD11c⫹ ATM subpopulations in epididymal fat pocytes and ATMs isolated from WT pads. Data were presented as total number of cells per mouse (panel B) and as cell numbers normalized to fat pad weight (panel C). In panels A–C, n ⫽ 8 for WT-LF and n ⫽ 6 for ␣7KO-LF. and ␣7KO mice. There was no differD–F, F4/80⫹CD11c⫹ cells in epididymal fat pads of WT and ␣7KO mice on HF diet. Data were ence in MCP1 expression in adipocytes presented as percentage of cells in SVF populations (panel D), number of cells per mouse (panel from LF-fed WT and ␣7KO mice, E), and number of cells per gram of fat pad (panel F). G, Gene expression profiles in epididymal whereas MCP1 was significantly infat pads from 6-month-old male WT and ␣7KO mice on HF diet. The expression of target genes was measured by real-time RT-PCR and normalized to cyclophilin (n ⫽ 4 in panels D–F and n ⫽ creased in ATMs from ␣7KO mice 3– 4 in panel G). Data are expressed as mean ⫾ SE; *, P ⬍ 0.05. compared with WT mice on the same diet (Supplemental Fig. 7A). On HF Recent reports suggest that obesity promotes the rediet, MCP1 expression was increased in both adipocytes cruitment of a subpopulation of macrophages into adipose and ATMs from ␣7KO mice compared with WT mice tissue, which is referred to as classically activated or M1 (Supplemental Fig. 7B). These data indicate that delemacrophages (26, 27). These macrophages express tion of ␣7nAChR results in increased MCP1 expression CD11c and produce more proinflammatory cytokines and primarily in ATMs, which may then contribute to inchemokines, such as TNF␣, IL-6, and MCP1, than resicreased macrophage infiltration and inflammation in dent macrophages in adipose tissue of lean mice, thus pro␣7KO mice. The increased MCP1 expression in adimoting a proinflammatory state in adipose tissue and thereby contributing to the induction of insulin resistance pocytes of ␣7KO mice on HF diet may be a result of (26, 27). Therefore, we also characterized the amount of macrophage-adipocyte interaction, which has been widely reported in the literature (28). CD11c⫹ macrophages within the F4/80⫹ population. ⫹ To investigate the physiological consequences of abOur data showed that within F4/80 macrophages, a small proportion of cells coexpressed CD11c in WT mice, normal ATM infiltration and inflammation in ␣7KO whereas the percentage of CD11c⫹ cells in F4/80⫹ cells mice, we characterized glucose homeostasis and was significantly higher in ␣7KO mice (Fig. 5A). The total insulin sensitivity in ␣7KO and their WT littermates on number of F4/80⫹CD11c⫹ cells isolated from epididymal both LF and HF diet. Plasma glucose level was not diffat pads increased in ␣7KO mice on LF diet compared with ferent between WT and ␣7KO mice on LF diet under WT mice (0.42 ⫾ 0.09 ⫻ 106/mouse and 3.01 ⫾ 1.64 ⫻ either fed or fasted states (data not shown). However, 106/mouse in WT and ␣7KO mice, respectively; P ⬍ 0.05) chow-fed ␣7KO mice had increased plasma insulin lev(Fig. 5B). Normalizing this data to fat pad weight indi- els (0.59 ⫾ 0.07 ng/ml in WT vs. 1.05 ⫾ 0.28 ng/ml in cated that there was a 5-fold increase in the content of ␣7KO; P ⬍ 0.05) (Supplemental Fig. 8A), indicating F4/80⫹CD11c⫹ cells in adipose tissue from ␣7KO mice impaired insulin sensitivity. Indeed, although their glu-

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FIG. 6. ␣7KO mice develop insulin resistance on HF diet. A–C, Fed insulin levels (panel A), GTT (panel B), and ITT (panel C) in 6-month-old male WT and ␣7KO mice fed HF diet. GTT was performed in overnight fasted mice with ip injection of glucose at 1.5 g/kg of body weight. ITT was performed 4 h after food removal in mice with ip injection of insulin at 1.75 U/kg of body weight (n ⫽ 10 in panel A, n ⫽ 7 for WT, and n ⫽ 5 for ␣7KO in panel B and n ⫽ 4 in panel C. D–F, Glucose infusion rate (panel D), glucose turnover (panel E), and muscle glucose uptake (panel F) in 7- to 8-month-old male WT and ␣7KO mice on HF diet during hyperinsulinemic-euglycemic clamp study. Hyperinsulinemic-euglycemic clamp was performed as described in Materials and Methods (n ⫽ 6 in panels D–F). G and H, Insulin signaling study was conducted in 6month-old male WT and ␣7KO mice on HF diet by ip injection of 10 U/kg insulin into overnight-fasted mice. Gastrocnemius muscle was collected 10 min later and phosphorylation of IR at Tyr1162Tyr1163 (panel G), IRS1 at Tyr612 (panel H), and total IR and IRS1 were measured by Western blotting analysis. Bar graphs show phosphorylation levels of IR and IRS1 normalized to total IR and IRS1 levels (n ⫽ 4). Data are expressed as mean ⫾ SE. *, P ⬍ 0.05 vs. WT mice.

cose tolerance was normal as determined by GTT (Supplemental Fig. 8B), ␣7KO mice exhibited mild insulin resistance as determined by ITT (Supplemental Fig. 8C). Although fed glucose level was not different between WT and ␣7KO mice on HF diet (data not shown), fasting plasma glucose level was significantly elevated in ␣7KO mice compared with WT mice (182 ⫾ 10 mg/dl in WT vs. 250 ⫾ 12 mg//dl in ␣7KO mice; P ⬍ 0.05). Fed insulin level was also elevated in ␣7KO mice compared with WT mice fed HF diet (2.02 ⫾ 0.43 ng/ml in WT vs. 4.02 ⫾ 0.73 ng/ml in ␣7KO on HF diet; P ⬍ 0.05) (Fig. 6A). HF-fed ␣7KO mice also exhibited impaired glucose tolerance and insulin resistance when compared with HF-fed WT mice, as determined by GTT and ITT, respectively (Fig. 6, B and C). We further performed hyperinsulinemic-euglycemic clamp studies to examine the insulin sensitivity and glucose metabolism in ␣7KO and WT mice fed HF diet. During a continuous insulin infusion at 2.5 mU/kg/min, the glucose infusion rate required to maintain euglycemia at 6 mM was reduced by 40% in ␣7KO mice compared with WT mice (Fig. 6D), indicating that ␣7nAChR deficiency induced insulin resistance. Similarly, insulin-stimulated glucose turnover was decreased by 40% in ␣7KO mice

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(Fig. 6E). Consistent with the measures of whole-body glucose turnover, insulin-stimulated glucose uptake into skeletal muscle was reduced by 40% in ␣7KO mice compared with WT mice (Fig. 6F). We also evaluated insulin-signaling events in insulin-targeting tissues (muscle and liver) in WT and ␣7KO mice on HF diet. Insulin stimulated IR tyrosyl phosphorylation at Tyr1162/Tyr1163 and IRS1 tyrosyl phosphorylation at Tyr612 in muscle of HF-fed WT mice, which was reduced by 30 – 40% in muscle of HFfed ␣7KO mice (Fig. 6, G and H). Insulin also stimulated IR phosphorylation at Tyr1162/Tyr1163 in liver of HF-fed WT mice, which was also reduced by 30% in HF-fed ␣7KO mice (Supplemental Fig. 9A). In addition, the expression of key enzymes in gluconeogenesis, including glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was increased by 50 –70% in liver of ␣7KO mice compared with WT littermates on HF diet (Supplemental Fig. 9B).

Discussion Obesity is characterized by a state of chronic inflammation featuring altered production of proinflammatory cytokines and activation of the inflammatory signaling networks, including IKK/NF-␬B and the JNK pathways in key metabolic tissues as well as macrophages. Accumulated evidence strongly supports the notion that inflammation is a key link between obesity and insulin resistance/type 2 diabetes. Moreover, macrophage inflammation is a key component of obesity-induced inflammation and plays a key role in obesity-induced insulin resistance (1). Targeting antiinflammatory mechanisms may provide novel therapeutic approaches in the treatment of obesity, inflammation, and insulin resistance. The cholinergic antiinflammatory pathway has been extensively studied in terms of its immunomodulating function against chronic inflammatory disorders (6, 12). Here we have used both pharmacological and genetic approaches to show that targeting this pathway has beneficial effect on obesity-induced insulin resistance, the fundamental cause of which is inflammation. We find that activation of the cholinergic antiinflam-

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matory pathway by low-dose nicotine significantly suppresses inflammation in adipose tissue, an important site in mediating obesity-induced inflammation, in db/db and DIO mice. This is associated with the protective effect of nicotine on obesity-induced insulin resistance in db/db and DIO mice. In contrast, inactivation of this pathway in ␣7KO mice results in abnormal ATM infiltration and inflammation, which is associated with significantly impaired insulin sensitivity in ␣7KO mice. Most importantly, these consequences occur independently of changes of body weight and adiposity. Therefore, our data demonstrate that changing cholinergic tone potently modulates inflammatory responses and insulin sensitivity in animal models without modulating body weight and adiposity. This indicates that the endogenous cholinergic antiinflammatory pathway plays an important role in obesity-induced inflammation and insulin resistance. Both the RNA and protein levels of the major acetylcholine esterase BChE in adipose tissue is substantially down-regulated in obese and diabetic mice, indicating that in obesity, the cholinergic signaling pathway may be already suppressed in adipose tissue, which may contribute to increased adipose tissue inflammation typically seen in obesity. Reduced cholinergic activity has also been reported under other chronic inflammatory conditions. Using heart rate variability as a measurement of vagus nerve activity, it has been reported that vagal activity is significantly depressed in patients with rheumatoid arthritis and systemic lupus erythematosus (29, 30) and predicts the progression of coronary atherosclerosis (31). These data indicate that reduced cholinergic activity may be a common feature in chronic inflammation, including obesity and insulin resistance. However, the mechanism underlying obesity-associated deregulation of cholinergic activity is not clear. It has been reported that nicotine treatment in macrophages results in the recruitment of Janus Kinase 2 (JAK2) to ␣7nAChR, followed by phosphorylating and activating signal transducer and activator of transcription 3 (STAT3), which is crucial to nicotine’s antiinflammatory effect (32). This effect can be blocked by a selective ␣7nAChR antagonist (32). This suggests that JAK2-STAT3 pathway may serve as downstream signaling pathways in the ␣7nAChR-mediated cholinergic antiinflammatory pathway. The suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of the JAK2STAT3 pathway, and SOCS3 expression is up-regulated in adipose tissue of obese and insulin-resistant animal models (33). It is possible that increased SOCS3 expression in adipose tissue during the development of obesity results in a down-regulated JAK2-STAT3 signaling

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pathway, which in turn suppresses the ␣7nAChR-mediated cholinergic antiinflammatory pathway. Our data suggest that macrophage ␣7nAChR plays an important role in modulating inflammation under the context of obesity and insulin resistance and is important in mediating the antiinflammatory effect of nicotine. First, peritoneal macrophages isolated from ␣7KO mice exhibit significantly more proinflammatory cytokine expression than macrophages isolated from WT mice when challenged with FFA and TNF␣, two important molecules mediating obesity-induced inflammation and insulin resistance. Second, nicotine significantly suppresses FFA- and TNF␣-induced proinflammatory cytokine expression in macrophages isolated from WT, but not ␣7KO, mice. Third, adipose tissue from ␣7KO mice contains significantly more F4/ 80⫹CD11c⫹ macrophages, indicating increased infiltration of CD11c⫹ M1 macrophages into adipose tissue of ␣7KO mice. However, further experimental approaches, e.g. adoptive transfer of bone marrow between WT and ␣7KO mice, or using genetically engineered mouse models (Cre-lox system) to delete ␣7nAChR specifically in macrophages, will be needed to confirm the role of macrophage ␣7nAChR in the regulation of obesity-induced inflammation and insulin resistance. The cholinergic antiinflammatory pathway responses to signals originate from the central nervous system, e.g. the central muscarinic and melanocortin systems, in the regulation of peripheral inflammation (34, 35). Intracerebroventricular administration of muscarinic receptor agonists significantly increased vagus nerve activity and decreases serum TNF␣ levels during endotoxemia (35). Activation of central melanocortin pathway via the melanocortin MC4 receptor exerts a potent antiinflammatory effect in hemorrhagic shock through activation of the cholinergic antiinflammatory pathway (34). It is possible that, in addition to other physiological processes in energy and glucose homeostasis, e.g. food intake and energy expenditure, the central nervous system may also be important in the regulation of peripheral inflammatory processes during the development of obesity, and the cholinergic antiinflammatory pathway may mediate this effect. In conclusion, our data demonstrate that the cholinergic antiinflammatory pathway, which plays an important role in regulating innate immunity and has potent immunoprotective effect against diverse inflammation-related disorders (6, 12), may also be protective against obesity-induced inflammation and insulin resistance. In addition, this effect may be mediated through ␣7nAChR.

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Acknowledgements Address all correspondence and requests for reprints to: Hang Shi, Section on Gerontology and Geriatric Medicine, Department of Internal Medicine, Medical Center Boulevard, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, E-mail: [email protected]; or Bingzhong Xue, Section on Endocrinology and Metabolism, Department of Internal Medicine, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, North Carolina 27157. E-mail: [email protected] This work was supported by National Institutes of Health Grants R01DK084172 and P30 AG21332, a National Institutes of Health center grant for Wake Forest School of Medicine Claude D. Pepper Older Americans Independence Center (to H.S.), and American Heart Association Scientist Development Grant 10SDG3900046 (to B.X.). Disclosure Summary: The authors have no conflict of interests to disclose.

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