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The C-C motif chemokine receptor–2 (CCR2) regulates monocyte and macrophage recruitment and is necessary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the development of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ameliorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improving hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adipose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue macrophages and insulin resistance once obesity and its metabolic consequences are established.
Obesity is associated with a complex systemic inflammatory state that has been implicated in the development of common, medically important complications, including atherosclerosis, hepatic steatosis, and insulin resistance (1–4). Characteristics of obesity-induced inflammation include elevated expression and production of proinflammatory molecules by adipose tissue, liver, and skeletal muscle (5–11); increased circulating concentrations of proinflammatory proteins, including acute-phase reactants, procoagulant factors, cytokines, and chemokines (3, 12); and the activation of pathways that regulate inflammation, including JNK and NF-κB pathways (10, 13–15). Obesity also induces the accumulation of macrophages in adipose tissue without altering the macrophage content of liver or muscle (8, 16). Macrophages produce some of the proinflammatory molecules released by adipose tissue and have been implicated in the development and maintenance of obesity-induced adipose tissue inflammation (8, 16, 17), but the mechanisms that recruit and retain macrophages in adipose tissue remain obscure.
Monocyte chemoattractant proteins (MCPs) and their receptors play pivotal roles in the development of inflammatory responses and are crucial for the recruitment of immune cells to sites of inflammation. In obese rodents and humans, the adipose tissue expression of at least 1 MCP, C-C motif chemokine ligand–2 (CCL2 or MCP1), is increased in proportion to adiposity. Both the adipose tissue expression and circulating concentrations of CCL2 increase in obesity and decrease following treatment with thiazolidinediones (18–20). Recent studies further implicate CCL2 and its receptor, CCR2, in the regulation of adipocyte function. These studies found that CCL2 inhibits insulin-stimulated glucose uptake as well as the adipocyte expression of metabolically important genes (e.g., Glut4, Pparg, and Fabp4) in a murine adipocyte cell line (21). CCR2 is a receptor for other MCPs, including CCL8 (MCP2) and CCL7 (MCP3) (22, 23), and is necessary for the recruitment of monocytes/macrophages in murine models of atherosclerosis, rheumatoid arthritis, and mycobacterial infections (24).
Because CCR2 regulates monocyte and macrophage chemotaxis and local inflammatory responses, and is implicated in regulating glucose uptake in an insulin-responsive cell line, we hypothesized that monocyte chemoattractant molecules acting through CCR2 might regulate obesity-induced inflammation in adipose tissue and associated perturbations in systemic glucose homeostasis. We studied the expression of ligands for CCR2 in lean and obese mice and in obese mice treated with a thiazolidinedione. We examined the effects of Ccr2 deficiency on development of diet-induced obesity, insulin sensitivity, adipose tissue histology, and gene expression and on the plasma concentrations of proteins implicated in the inflammatory and metabolic consequences of obesity. We also examined the metabolic phenotype of obese mice treated with a selective, CCR2 antagonist after the onset of obesity. Congenital absence of CCR2 had no measurable metabolic effect in lean animals. However, in a mouse model of diet-induced obesity, CCR2 deficiency attenuated the development of obesity, adipose tissue macrophage (ATM) accumulation, adipose tissue inflammation, and systemic insulin resistance. In mice with preexisting obesity, short-term pharmacologic antagonism of CCR2 reduced ATM content and improved in vivo insulin sensitivity. The mechanistic relationships among these diverse metabolic phenotypes is unclear, but these findings suggest that CCR2 modulates metabolic and inflammatory effects induced by a high-fat diet.
The macrophage content of adipose tissue is increased by obesity and reduced by administration of thiazolidinedione (8, 16, 25). To identify potential regulators of macrophage accumulation in adipose tissue, we studied the expression profiles of epididymal adipose tissue from lean male C57BL/6J mice maintained on a low-fat diet (5% of calories derived from fat) for 24 weeks (body mass, 28.9 ± 2.5 g; fasting insulin, 0.31 ± 0.6 ng/ml); C57BL/6J mice made obese and insulin resistant on a high-fat diet (45% of calories derived from fat) for 24 weeks (45.8 ± 4.8 g; 2.3 ± 0.5 ng/ml); and C57BL/6J mice made obese on a high-fat diet for 22 weeks and then fed the high-fat diet mixed with pioglitazone (60 mg/kg) for 2 additional weeks (47.2 ± 1.8 g; 0.3 ± 0.05 ng/ml). The expression of Ccl2 (also known as Mcp1) is upregulated in adipose tissue of obese rodents (18) and humans (20, 26), but its receptor, CCR2, and the other CCR2 ligands (CCL7/MCP3 and CCL8/MCP2) have not be studied. In the adipose tissue of obese compared with lean mice, the expression of Ccl2 was increased 7.6-fold (P < 0.01); Ccl7 was increased 8.4-fold (P < 0.01); and Ccl8 was increased 2.1-fold (P < 0.05). Treatment with pioglitazone significantly decreased the expression of Ccr2 (P < 0.001), Ccl2 (P < 0.01), and Ccl7 (P < 0.01) but not Ccl8 (Figure (Figure1).1). The influence of obesity and a thiazolidinedione on the expression of Ccr2 and its ligands is consistent with obesity-dependent alterations in CCR2 signaling within adipose tissue.
To assess the role of CCR2 in the development of obesity and obesity-associated adipose tissue inflammation and insulin resistance, we fed either low-fat (5% calories derived from fat) or high-fat (60% of calories derived from fat) diets to male Ccr2-deficient (C57BL/6J Ccr2–/–) or wild-type (C57BL/6J Ccr2+/+) mice. Previous reports of mice fed standard chow diets had not noted any Ccr genotype-dependent effects on body mass (27, 28). Consistent with these reports, lean body mass, fat mass, and total body mass were not affected by Ccr2 genotype in mice fed the low-fat diet for 24 weeks (Figure (Figure2).2). Unexpectedly, however, after 24 weeks on the high-fat diet, Ccr2–/– mice weighed 15% less than Ccr2+/+ mice (39.4 ± 6.9 vs. 46.3 ± 4.1 g [mean ± SD]; P < 0.05). Even though the mean body mass of the high-fat diet–fed Ccr2–/– mice was lower than that of comparably fed Ccr2+/+ mice, most Ccr2–/– mice did in fact become obese, weighing between 40 and 50 g, with adiposity between 40% and 50% (Figure (Figure2A).2A). Two-way ANOVA confirmed that both high-fat diet (P < 0.001) and Ccr2 genotype (P < 0.05) contributed to the variation in body mass, though the genotype-diet interaction did not reach significance (P = 0.14). Hence, Ccr2 deficiency attenuated but did not prevent the development of obesity in mice fed a high-fat diet.
To better understand why Ccr2–/– mice gained less weight while ingesting the high-fat diet, we examined food intake and body composition of individually caged Ccr2–/– and Ccr2+/+ mice fed a high-fat diet for 6 weeks. Despite having similar body weights (19.16 ± 1.05 g vs. 19.48 ± 0.63 g; P > 0.05) and body compositions at the study’s outset, Ccr2–/– mice consumed fewer kilocalories per day than Ccr2+/+ mice during the observation period (18.32 ± 4.08 kcal/d vs. 28.27 ± 14.25 kcal/d; P < 0.005; Figure Figure2B).2B). At the end of the 6-week period, Ccr2–/– mice weighed significantly less than the Ccr2+/+ mice (26.96 ± 2.20 g vs. 31.02 ± 3.40 g, P < 0.05; Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI24335DS1 http://dx.doi.org/10.1172/JCI24335DS1); had modestly but significantly lower lean body mass (20.86 ± 0.69 g vs. 22.66 ± 1.57 g; P < 0.05; Supplemental Figure 1) and modestly but not significantly lower fat mass (5.36 ± 1.94 g vs. 7.82 ± 3.36 g; Supplemental Figure 1) and adiposity (20.06% ± 5.10% vs. 25.02% ± 8.36% fat). These data suggest an unrecognized role for CCR2 in the regulation of feeding behavior in the context of a highly palatable diet.
Decreased fat mass in Ccr2–/– mice fed a high-fat diet would be expected to reduce their inflammatory response and to improve systemic metabolic parameters, including insulin sensitivity. Therefore, a comparison of all high-fat diet–fed Ccr2–/– mice with their Ccr2+/+ counterparts might overstate the true effect of Ccr2 genotype on ATM accumulation, adipose tissue inflammatory response, and insulin sensitivity in obese mice. To distinguish the direct effects of CCR2 deficiency on insulin sensitivity and ATM accumulation from the secondary effects of reduced weight and adiposity, we compared ATM accumulation and insulin sensitivity in mice from both high-fat diet–fed groups that weighed more than 40 g. Among these high-fat diet–fed mice, there was no significant difference in body mass, total fat mass, or percent body fat (Table (Table11).
We measured fasting blood glucose and plasma insulin concentrations and calculated homeostasis model assessment of insulin resistance (HOMA-IR) values in 4 groups of mice: lean Ccr2+/+, lean Ccr2–/–, obese Ccr2+/+, and adiposity-matched obese Ccr2–/– mice. No difference was observed in the fasting blood glucose or plasma insulin concentrations between lean Ccr2–/– and lean Ccr2+/+ mice. Likewise, there was no effect of Ccr2 genotype on insulin sensitivity (as measured by HOMA-IR) in lean animals (Figure (Figure3).3). However, obese Ccr2–/– mice had lower fasting blood glucose and insulin concentrations compared with adiposity-matched obese Ccr2+/+ mice (Figure (Figure3).3). Consistent with increased insulin sensitivity, the HOMA-IR values of obese Ccr2–/– mice were 50% lower than those of equally obese Ccr2+/+ animals (P < 10–4; Figure Figure3).3). Two-way ANOVA demonstrated a significant interaction between body weight and Ccr2 genotype in modulating fasting glucose (P < 0.0005), insulin (P < 0.05), and HOMA-IR (P < 0.001).
Obese Ccr2–/– mice were also more insulin sensitive than obese Ccr2+/+ animals as measured by an insulin tolerance test (Figure (Figure4).4). Similarly, Ccr2 deficiency improved glucose tolerance during an intraperitoneal glucose tolerance test. Obese Ccr2–/– mice were less hyperglycemic compared with obese Ccr2+/+ animals at 45, 60, and 90 minutes following intraperitoneal injection of a glucose bolus (Figure (Figure44).
Hepatic insulin resistance in obese mice and humans is strongly associated with hepatomegaly and hepatic steatosis (29). To determine whether improved insulin sensitivity associated with Ccr2 deficiency ameliorated hepatomegaly or reduced hepatic steatosis, we analyzed the livers from obese Ccr2+/+ and Ccr2–/– mice. As expected, obese Ccr2+/+ mice had a more than 10-fold higher concentration of hepatic triglycerides (TGs) than lean Ccr+/+ mice (82.1 ± 19.6 vs. 7.8 ± 2.6 mg TG/g tissue; P < 0.001). Adiposity-matched obese Ccr2–/– mice had 50% lower hepatic TG content than obese Ccr2+/+ animals (43.9 ± 23.6 vs. 82.1 ± 19.6 mg TGs/g tissue; P < 0.001). Similarly, livers of obese Ccr2+/+ mice weighed 50% more than those of comparably obese Ccr2–/– animals (2.4 ± 0.5 g vs. 1.6 ± 0.6 g; P < 0.001).
In earlier mouse studies, ATM content was strongly correlated with adiposity (8, 16). To determine whether the accumulation of ATMs during the development of obesity is dependent upon CCR2, we compared adipocyte size and ATM content in diet-induced obese Ccr2–/– and Ccr2+/+ mice that were matched for adiposity. We measured macrophage content and adipocyte morphology by immunohistochemistry in epididymal and subcutaneous adipose tissue depots obtained from obese Ccr2–/– and Ccr2+/+ mice. In epididymal adipose tissue, adipocyte morphology was similar in both genotypes. However, adipocyte size (median cross-sectional area) was greater in the Ccr2–/– mice (median, 4,457 ± 650 μm2; mean, 5,005 ± 621 μm2) compared with the obese Ccr2+/+ controls (median, 3,588 ± 561 μm2; mean, 4,345 ± 460 μm2; P < 0.05 for median). As a percentage of cells, adipose tissue from obese Ccr2+/+ mice contained more ATMs (25% ± 5.6%) than that from lean Ccr2+/+ animals (7.2% ± 4.9%). However, despite having adipocytes that were larger and total adiposity that was equal, the ATM content of epididymal adipose tissue in obese Ccr2–/– (16.3% ± 3%) mice was significantly less than the ATM content of adipose tissue in obese Ccr2+/+ mice (25% ± 5.6%; P < 0.005) (Figure (Figure5).5). A similar reduction in macrophage content was also detected in subcutaneous adipose tissue depots (Figure (Figure55)
Flow cytometry can also be used to isolate and quantify the fraction of ATMs that comprises stromal vascular cells (SVCs) of adipose tissue. However, a portion of ATMs is not isolated in the SVCs in standard protocols because of increased buoyancy of lipid-laden ATMs from obese animals and adherence of ATMs to adipocytes (8, 16). Nevertheless, characterization of SVC populations by flow cytometry has also shown a consistent increase in the fraction of ATMs in adipose tissue from obese compared with lean mice (8, 16). Flow cytometry, therefore, provides an alternative means of determining the relative abundance of ATMs. Using flow cytometry, we quantified the macrophage populations (F4/80+, Cd11b+) in SVCs of peri-renal adipose tissue from lean Ccr2+/+ mice and obese Ccr2–/– and Ccr2+/+ mice. Consistent with our immunohistochemical analysis, we found that the proportion of macrophages in SVCs isolated from obese wild-type mice (17.6% ± 5% of SVCs) was significantly greater than the proportion in the SVCs isolated from lean Ccr2+/+ mice (8.1% ± 2.9%) and obese Ccr2–/– mice (9.4% ± 1.7%; P < 0.001 vs. obese Ccr2+/+). Thus, in obese animals, intraabdominal and subcutaneous adipose tissue depots have fewer macrophages in Ccr2-deficient mice than in adiposity-matched wild-type animals. However, Ccr2 deficiency does not normalize ATM content to that observed in lean animals, indicating that macrophage accumulation is modulated by CCR2-independent factors as well.
The development of obesity and insulin resistance is associated with stereotypical changes in adipose tissue expression of inflammatory and metabolic genes. Increased expression of genes that encode proinflammatory and macrophage-associated proteins is a hallmark of adipose tissue from obese animals and humans (30). To assess the role of Ccr2 in mediating the transcriptional inflammatory response to obesity, we compared the expression of genes encoding proinflammatory and macrophage marker proteins in epididymal adipose tissue from lean and obese Ccr2+/+ and Ccr2–/– mice. As expected, obese Ccr2+/+ mice compared with lean Ccr2+/+ mice showed increased expression of genes encoding proinflammatory molecules including Tnfa (TNF-α) and Serpine1 (plasminogen activator inhibitor–1 [PAI-1]) and the macrophage markers Emr1 (F4/80) and Cd68 (CD68). Consistent with the histological data, expression of the macrophage markers Emr1 and Cd68 was reduced in obese Ccr2–/– mice compared with obese Ccr2+/+ mice. By 2-way ANOVA, the variation in the expression of these genes was attributable to a significant interaction between diet and Ccr2 genotype (Figure (Figure6;6; ANOVA interaction P < 0.05). Paralleling the decrease in macrophage-specific gene expression, Tnfa expression was reduced in obese Ccr2–/– mice (Figure (Figure6;6; ANOVA interaction P < 0.005). No significant differences in the expression of these genes were observed between lean Ccr2–/– and Ccr2+/+ mice (Figure (Figure6),6), nor were differences observed in Serpine1, Ccl2, or Ccl7 expression between obese Ccr2–/– and Ccr2+/+ mice (data not shown). In contrast to the alterations in Tnfa expression in adipose tissue, we were consistently unable to detect circulating TNF-α protein (<3.2 pg/ml) in mice of either genotype or level of adiposity (data not shown). These data suggest that the local proinflammatory changes that occur in adipose tissue during the development of obesity are, in part, CCR2 dependent.
Concomitant with an increase in inflammatory gene expression, obesity leads to decreased expression of genes required for mitochondrial function, TG metabolism, and adipocyte differentiation (8, 31–33). Recent studies suggested that CCL2 directly effects similar changes in adipocyte gene expression (21). We measured the expression of 4 genes downregulated in obesity and by Ccl2 treatment of adipocytes: a nuclear encoded mitochondrial gene (Gpam) (8, 32), a lipase (Lipe) (8, 31), a fatty acid binding protein (Fabp4) (32), and a transcription factor required for adipocyte differentiation (Pparg) (32). In lean animals there was no effect of Ccr2 genotype on the expression of these genes, but in obese mice the expression of these genes was increased in Ccr2–/– compared with Ccr2+/+ mice (Figure (Figure77).
The effects of Ccr2 genotype on the expression of this select group of inflammatory and metabolic genes suggested that obesity-induced alterations in adipose tissue gene expression are, in part, CCR2 dependent. To more fully define the alterations in gene expression associated with Ccr2 deficiency in adipose tissue of obese mice, we used oligonucleotide microarrays to compare the expression profiles of epididymal adipose tissue from Ccr2–/– and Ccr2+/+ mice with dietary obesity. Using an algorithm that identifies predefined functional classes (based on the Gene Ontology [GO] Group gene classification scheme; http://geneontology.org) of coregulated genes, we found that 71 classes of genes were significantly downregulated (P < 0.001, corrected for multiple testing) (34). Forty-one of the downregulated groups represent inflammatory functional classes and include classes of genes involved in the regulation of immune responses (GO:0050778), interleukin receptor activity (GO:0004907), and NF-κB signaling (GO:0043122) (Supplemental Table 1). These data suggest that the inflammatory profile of adipose tissue in obese mice is broadly attenuated by deficiency of CCR2. We also found that 52 classes of genes were significantly upregulated in Ccr2–/– compared with Ccr2+/+ obese mice. Consistent with the relative normalization in metabolic gene expression and with the increase in expression of Gpam, Lipe, and Glut4, 23 of the upregulated functional classes in obese Ccr2–/– adipose tissue are involved in intermediary metabolism, most notably mitochondria and peroxisome function, and lipid metabolism (Supplemental Table 2).
The microarray dataset also revealed that several individual genes with higher levels of expression in Ccr2–/– than in Ccr2+/+ obese mice encode hormones produced by adipocytes — including leptin (Lep) (P < 0.05), resistin (Retn) (P < 0.05), and adiponectin (Acdc) (P < 0.05). Perturbations of each of these hormones has been implicated in the development of insulin resistance. Leptin deficiency leads to a syndrome of severe obesity and insulin resistance; elevation of circulating resistin is implicated in obesity-induced insulin resistance; and, conversely, decreases in the antiinflammatory and insulin-sensitizing protein adiponectin (encoded by Acdc), lead to the development of obesity-induced insulin resistance and hepatic steatosis (35, 36). Despite alterations in adipose tissue gene expression, we did not detect any differences in circulating concentrations of leptin or resistin between obese Ccr2–/– and Ccr2+/+ mice (Supplemental Table 3). We confirmed adipose tissue gene expression of adiponectin by quantitative PCR and found, as expected, decreased expression of adiponectin in obese compared with lean Ccr2+/+ mice. No difference in adipose tissue Acdc expression was detected between lean Ccr2+/+ and Ccr2–/– animals. However, consistent with their increased systemic insulin sensitivity, adiponectin gene expression was higher in obese Ccr2–/– mice compared with obese Ccr2+/+ animals (Figure (Figure7).7). Furthermore, the amount of circulating adiponectin in plasma, while similar in lean mice of both genotypes, was significantly higher in obese mice deficient in Ccr2 than in wild-type obese animals (Figure (Figure88).
IL-6 and PAI-1 are circulating proteins that are produced by multiple organs and tissues including adipose tissue, liver, leukocytes, and endothelial cells. Both have been implicated in obesity-induced insulin resistance, and the adipose tissue expression of both is increased in obesity (18, 26, 37–40). We did not detect Ccr2-dependent modulation in adipose tissue expression of IL-6 or PAI-1. Consistent with the gene expression data, there were no CCR2-dependent alterations in the circulating concentration of IL-6. In contrast, we detected lower circulating concentrations of PAI-1 in lean and obese Ccr2–/– mice when compared with weight-matched Ccr2+/+ mice (Supplemental Table 3). These data reveal that CCR2 modulates circulating PAI-1 by a mechanism that is not primarily dependent upon adipose tissue expression or the development of obesity.
In rodents and humans, obesity is associated with a dyslipidemia characterized by elevated serum concentrations of cholesterol (41, 42). As expected, fasting total plasma cholesterol concentrations were higher in obese compared with lean Ccr2+/+ mice. In obese Ccr2–/– mice, the increase was similar, and there were no genotype-dependent differences between fasting cholesterols of adiposity-matched mice (Table (Table1).1). Fasting plasma concentrations of TGs and nonesterified fatty acids were not different between lean and obese animals. No genotype-dependent effects on plasma nonesterified fatty acid (NEFA) concentrations were detected. TG concentrations were modestly higher in obese Ccr2–/– mice compared with obese Ccr2+/+ mice (Table (Table1),1), though still lower than TG concentrations in lean mice. In contrast to other metabolic phenotypes examined, Ccr2 deficiency did not significantly reverse the lipid abnormalities associated with obesity.
CCR2 has been identified as a potential therapeutic target in several inflammatory disorders, including rheumatoid arthritis and multiple sclerosis. INCB3344 (Incyte Corp.) was developed as a selective small molecule antagonist of CCR2. Previous studies characterized the pharmacokinetic and pharmacodynamic profile of this molecule and demonstrated that with a single daily subcutaneous dose (100 mg/kg), a significant inhibition (>50%) of murine CCR2 receptor activity is achieved in vivo (11). To test whether short-term treatment with a CCR2 antagonist reverses any of the obesity-induced effects that are prevented in CCR2-deficient animals, we treated C57BL/6J mice made obese by feeding a high-fat diet with daily subcutaneous injections of INCB3344 (100 mg/kg) for 17 days. At the end of the treatment period, body mass and composition were not significantly different between INCB3344- and vehicle-treated animals (Table (Table11).
Following 2 weeks of treatment with INCB3344, we assessed glucose homeostasis in the INCB3344-treated and vehicle-treated obese animals. After a 6-hour fast, animals receiving INCB3344 were significantly less hyperglycemic than animals receiving injections of vehicle (143 ± 29 mg/dl vs. 181 ± 31 mg/dl; P < 0.05; Figure Figure9).9). After an overnight fast (14 hours), blood glucose concentrations in the 2 groups of animals were similar. However, fasting plasma insulin concentrations were approximately 33% lower in INCB3344-treated compared with control animals (P < 0.05); hence, insulin sensitivity as measured by HOMA-IR was improved (8.3 ± 2.5 vs. 13.5 ± 4.4 IR units; P < 0.05; Figure Figure9).9). Consistent with improved insulin sensitivity, an intraperitoneal injection of insulin lowered blood glucose concentrations more in INCB3344-treated than in vehicle-injected control obese mice (Figure (Figure9).9). Similar to the effects of Ccr2 deficiency, INCB3344 treatment also improved glucose homeostasis following a glucose tolerance test (Figure (Figure9).9). Treatment of obese mice with INCB3344 increased adipose tissue expression of adiponectin by 29.8% (P < 0.05), although the increase in circulating adiponectin concentration did not reach statistical significance (P > 0.05).
We also examined the effect of CCR2 inhibition with INCB3344 on the ATM content of periepididymal adipose tissue in wild-type mice with established obesity. Immunohistochemical analysis showed a modest but significant decrease in the fraction of F4/80-expressing ATMs in obese mice that had been treated with INCB3344 (21.8% vs. 15.7%, vehicle- vs. INCB3344-treated; P < 0.05). These data implicate CCR2-dependent pathways in the maintenance of the macrophage population in obese adipose tissue and imply a turnover sufficiently rapid to permit detectable differences after 2–3 weeks of CCR2 antagonism. In contrast to the ability of short-term treatment with a CCR2 antagonist to improve insulin sensitivity and lower macrophage content in adipose tissue, the effects of a short-term treatment regimen with INCB3344 on proinflammatory adipose tissue gene expression were less consistent. Short-term INCB3344 treatment consistently and significantly lowered Ccr2 expression by 65% (P < 0.01; n = 12). However, in contrast, a more modest (20–30%) reduction in expression of other inflammatory genes, including Tnfa, Csf1r, and Emr1, was not significant in all cohorts tested (data not shown). Similarly, short-term antagonist treatment did not have a measurable effect on hepatomegaly (data not shown). Thus, the development of obesity and the obesity-induced expression of several key inflammatory genes were not reversed with 2-week antagonism of CCR2.
CCR2 signaling plays pivotal roles in immune responses and atherosclerosis. Its role in metabolic disorders has not been previously appreciated. However, findings presented here demonstrate a complex and important role for CCR2 in both the development and maintenance of obesity and obesity-associated phenotypes. In lean animals, no effect of Ccr2 genotype on metabolic traits could be detected. In contrast, when mice were fed a high-fat diet, Ccr2 genotype modulated feeding behavior, the development of obesity, and the development of obesity-associated adipose tissue inflammation, ATM accumulation, adiponectin expression, hepatic steatosis, and insulin sensitivity.
Once obesity is established, short-term inhibition of CCR2 signaling with a specific antagonist attenuates obesity-induced macrophage accumulation in adipose tissue and insulin resistance. However, adipose tissue proinflammatory gene expression was not as consistently reduced. The expression of some genes, e.g., Ccr2 and Acdc, were modulated similarly by genetic deficiency and pharmacological antagonism of CCR2, but others, including the pro-typical inflammatory cytokines, were not consistently decreased in antagonist-treated mice. Similarly, circulating adiponectin and hepatomegaly were not significantly altered by drug treatment. That the effects of the antagonist were more modest than the developmental effects seen with genetic deficiency may reflect a developmental requirement for CCR2 in certain obesity-induced phenotypes and/or relate to the short duration of impaired or absent CCR2 action.
The requirement for CCR2 in the hyperphagic response to a high-fat diet was unexpected. Feeding behavior is integrated and regulated by centers in the central nervous system, in particular, by nuclei within the hypothalamus and brain stem. Populations of neurons, astrocytes, and glial cells express CCR2, and CCL2 binding within the hypothalamus and brain stem have been reported, although possible noninflammatory roles of CCR2 within the central nervous system have not been characterized. Thus, CCR2 may play a role in the ability of feeding centers to respond fully to a highly fat-laden palatable diet. Alternatively, CCR2-dependent regulation of feeding behavior may occur through the action of CCR2 on cells in the periphery, which in turn release molecules that act upon neurons in the central nervous system. Identifying specific hypothalamic and brainstem neuronal and non-neuronal populations that express CCR2, and determining whether central or peripheral INCB3344 can attenuate the development of diet-induced obesity before its onset, will be important initial steps in identifying mechanisms by which CCR2 modulates feeding behavior.
We had hypothesized that ATM content is CCR2 dependent. Indeed, the fraction of ATMs in adipose tissue was 35% lower in obese Ccr2–/– compared with obese Ccr2+/+ mice. The ability of a CCR2 antagonist to lower ATM content by 25% in mice with established obesity after 2 weeks of treatment suggests that the effects of genetic deficiency in Ccr2 are not simply attributable to developmental defects in hematopoiesis or chemotaxis. The data also indicate that the turnover of ATMs in obesity is relatively high (half-life in adipose tissue of ~1 month). Identifying the relevant ligand(s) for CCR2-dependent accumulation of ATMs will require further study, although the expression patterns of CCL2 and CCL7 make them strong candidates. The similarity in adipose tissue expression of CCL2 and CCL7 suggests an additional level of complexity and potential redundancy that will complicate studies. Furthermore, neither pharmacologic modulation nor genetic deficiency of CCR2 normalized ATM content, indicating that factors other than CCR2 participate in macrophage recruitment to adipose tissue.
Recent data suggest that the expression of proinflammatory molecules by adipose tissue is in part due to the expression of these molecules by non-adipocytes, including macrophages (8, 16, 25, 43–45). Ccr2 deficiency attenuates the development of a proinflammatory expression profile by adipose tissue of obese animals, consistent with ATMs being a source of obesity-induced inflammation. However, short-term treatment with a CCR2 antagonist decreased the fraction of macrophages in adipose tissue and improved insulin sensitivity without a similarly broad impact on proinflammatory expression profile in mice with established obesity. This suggests that if the increased production of proinflammatory molecules in obesity were only the result of a quantitative increase in ATM number, then their expression should be uniformly reduced in both the CCR2-deficient and antagonist-treated obese mice. Therefore, the data from obese Ccr2–/– mice suggest that expression of macrophage-derived proinflammatory molecules is also dependent on the maturity or activation state of the ATMs, and not solely on reduction in ATM content per se. Furthermore, differences in reduction in proinflammatory gene expression observed in obese Ccr2–/– and antagonist-treated mice are likely in part attributable to adipocyte-derived proinflammatory gene expression being differentially affected by complete genetic deficiency and pharmacological antagonism of CCR2.
Adiponectin is an adipocyte-derived hormone that positively regulates insulin sensitivity in liver and muscle. The enhanced insulin sensitivity of Ccr2–/– compared with Ccr2+/+ obese mice is consistent with higher expression of adiponectin and higher circulating concentrations of adiponectin observed in obese Ccr2–/– mice. These findings suggest the existence of a CCR2-dependent mechanism for obesity-induced downregulation of adiponectin. Modulation of adiponectin expression may occur indirectly through interactions of adipocytes with CCR2-expressing ATMs and the modulation of obesity-induced inflammatory gene expression. Inflammatory cytokines, including TNF-α, are known to downregulate adiponectin expression by adipocytes, and therefore, the lower inflammatory expression profile of adipose tissue from obese Ccr2–/– mice may contribute to the maintenance of adiponectin expression. Alternatively or in addition, the expression of CCR2 by adipocytes raises the possibility that Ccr2-dependent downregulation of adiponectin may occur via a direct effect of CCR2 on adipocyte gene expression.
The improved insulin sensitivity of obese Ccr2–/– mice is not entirely attributable to elevated adiponectin expression. Enhanced insulin sensitivity of obese mice treated with a CCR2 antagonist, in which adiponectin expression is similarly elevated but total circulating adiponectin is not significantly different, implicates an additional CCR2-dependent but adiponectin-independent mechanism of insulin resistance. The identity and location of the CCR2-expressing cell(s) required for this effect are not apparent. While the correlation of ATM content with systemic insulin resistance is intriguing, we cannot determine whether ATM content and insulin sensitivity are causally related. Defining the functional relationship between these 2 phenotypes requires development of tools that will permit specific manipulation of ATMs as opposed to other hematopoietic or macrophage populations (e.g., ATM-specific promoter for Cre-based genetic manipulations).
The adiponectin-independent mechanism(s) of CCR2 regulation of insulin sensitivity may occur through direct action on insulin-responsive cell types. The expression and function of CCR2 in nonhematopoietic cells has not been well characterized. It has been reported that CCR2 is expressed by adipocytes, neurons, endothelial cells, and myocyte precursors. To our knowledge, there is no evidence that mature myocytes or hepatocytes express CCR2. However, we detected increased CCR2 transcript expression in livers of obese mice in the absence of any increase in the number of Kupffer cells (hepatic macrophages) (ref. 8 and S.P. Weisberg and A.W. Ferrante Jr., unpublished observations). Thus, CCR2 in the liver, either in Kupffer cells or nonhematopoietic cells, may modulate the insulin responsiveness of hepatocytes. In contrast, we have not detected any obesity-associated changes in CCR2 expression in whole skeletal muscle.
Here we have shown that CCR2 plays important roles in the pathogenesis of obesity and obesity-associated phenotypes and identify CCR2 as a possible therapeutic target in efforts to treat obesity and its complications.
Ccr2–/– were previously generated and have been backcrossed 10 times to the C57BL/6J background (46). C57BL/6J mice used in the INCB3344 studies were obtained from the Jackson Laboratory at 6–8 weeks of age. All mice were housed in Plexiglas ventilated cages (1–3 animals/cage) within a pathogen-free barrier facility that maintained a 12-hour light/12-hour dark cycle. Mice had free access to autoclaved water and irradiated pellet food. In all studies, including studies of Ccr2 genotype effects, and INCB3344 treatment, obesity was induced by a high-fat diet that derived approximately 60% of calories from lipids (D12492; Research Diets Inc.) for 24 weeks beginning at age 6 weeks. Prior to age 6 weeks, mice were fed a standard pellet diet, in which 5% of calories were provided as fat (PicoLab Rodent 20; LabDiet). Mice on a low-fat diet received the standard chow pellet throughout life.
Mice were sacrificed by CO2 asphyxiation during the second and third hour of the light cycle. Animals were weighed, and adipose tissues (epididymal, subcutaneous, and perirenal) and liver were removed. Tissues analyzed by FACS were processed immediately. Other samples were frozen in liquid nitrogen and stored at –80°C prior to RNA extraction and immunohistochemical analysis.
Mice treated with INCB3344 were lightly anesthetized with inhaled 2% isoflurane and injected subcutaneously with INCB3344 (100 mg/ml) emulsified in 10% carboxymethylcellulose as vehicle. Control animals were similarly anesthetized but subcutaneously injected with vehicle alone. Body composition was determined by dual x-ray absorbance spectroscopy using a PIXImus DEXA scanner specifically designed for mice (Lunar Corp.). Calibration was performed before each set of measurements and data obtained according to manufacturer’s protocols. All procedures were approved by Columbia University’s Institutional Animal Care and Use Committee.
INCB3344 was synthesized by the medicinal chemistry department of Incyte Corp. The specificity of this compound for CCR2 and its pharmakinetics of INCB3344 have been described elsewhere (11).
Insulin tolerance testing was carried out in animals that were fasted for 6 hours beginning at approximately 10 am. After an intraperitoneal bolus injection of recombinant human regular insulin (1.5 U/kg) (Novolin R; Novo Nordisk Inc.), blood glucose concentrations were measured using a Glucometer ELITE XL blood glucose meter (Bayer Corp.) before and 15, 30, 45, 75, 90, and 130 minutes after injection. Glucose tolerance tests were performed after an overnight (14-hour) fast. Blood glucose concentrations were measured before and 15, 30, 45, 60, 90, 120 minutes after an intraperitoneal injection of dextrose dissolved in water (1 g/kg).
Total RNA was extracted from the perigonadal (epididymal or parametrial) adipose tissue of individual mice using a commercially available acid-phenol reagent (TRIzol; Invitrogen Inc.). RNA concentration was assessed by absorbance spectroscopy and RNA integrity confirmed by nondenaturing agarose gel electrophoresis. Twenty micrograms of RNA from each sample were further purified from contaminating organics and non-RNA species using a silica resin (RNEasy; QIAGEN) protocol according to the manufacturer’s instructions. Total RNA from single animals was individually converted into biotinylated, fragmented cRNA using protocols recommended by the microarray manufacturer (Affymetrix). cRNA samples derived from single animals were hybridized in recommended buffer to microarrays (Affymetrix Murine Genome Array U430 2.0) at 45°C for 16 hours. The samples were stained and washed according to the manufacturer’s protocol on a Fluidics Station 400 (Affymetrix) and scanned on a GeneArray Scanner (Affymetrix). Primary data extraction was performed with Microarray Suite 5.0 (Affymetrix), and signal normalization across samples was carried out using all probe sets with a mean expression value of 500.
We examined gene expression only for those genes deemed “present” by the Microarray Suite software. To calculate the probability that genes were up- or downregulated in Ccr2–/– compared with wild-type animals, we used Microsoft Excel (Microsoft Corp.) to calculate Student’s t test scores for each “present” gene. To identify functionally related groups of genes with similar expression patterns, we analyzed the list of Student’s t test scores using Gene Ontology Class Scoring software (34, 47).
Adipose tissue samples were fixed for 12–16 hours at room temperature in zinc-formalin fixative (Anatech Ltd.) and embedded in paraffin. Five-micrometer sections, cut at 50-μm intervals, were mounted on charged glass slides, deparaffinized in xylene, and stained for expression of F4/80 as described by Cecchini et al. with an anti-F4/80 monoclonal antibody (CALTAG Laboratories) (48). For each individual mouse tissue block, 15–20 different ×20 fields from each of 4 different sections were analyzed. The total number of nuclei and the number of nuclei of F4/80-expressing cells were counted for each field using a software algorithm designed for the image analysis software Image-Pro Plus 1.0 (Media Cybernetics Inc.). The fraction of F4/80-expressing cells for each sample was calculated as the sum of the number of nuclei of F4/80 expressing cells divided by the total number of nuclei in sections of a sample. Average adipocyte cross-sectional area was determined for each adipocyte in each field analyzed using image analysis software (SPOT version 3.3; Diagnostic Instruments Inc.).
Adipose tissue was isolated from mice immediately after CO2 asphyxiation. Tissues were handled using sterile techniques and minced into fine (<10-mg) pieces. Minced samples were placed in HEPES-buffered DMEM (Invitrogen Corp.) supplemented with 10 mg/ml fatty acid–poor BSA (FAP-BSA; Sigma-Aldrich) and centrifuged at 1000 g for 10 minutes at room temperature to pellet erythrocytes and other blood cells. An LPS-depleted collagenase cocktail (Liberase 3; Roche Diagnostics Corp.) at a concentration of 0.03 mg/ml and 50 U/ml DNAse I (Sigma-Aldrich) were added to the tissue filtrate and the samples incubated at 37°C on an orbital shaker (215 Hz) for 45–60 minutes. Once digestion was complete, samples were passed through a sterile 250-μm nylon mesh (Sefar Inc.). The suspension was centrifuged at 500 g for 10 minutes. The pelleted cells were collected as the SVCs, and the floating cells were collected as the adipocyte-enriched fraction. The SVCs were resuspended in erythrocyte lysis buffer and incubated at room temperature for 5 minutes. The erythrocyte-depleted SVCs were centrifuged at 500 g for 5 minutes and the pellet resuspended in FACS buffer (PBS, 0.2%, FAP-BSA, 5 mM EDTA).
SVCs isolated from adipose tissue samples were cooled on ice and counted using a hemocytometer. Cell survival rates ranged from 70% to 90% as determined by trypan blue exclusion. After counting, cells were centrifuged at 500 g for 5 minutes and resuspended in FACS buffer at a concentration of 7 × 106 cells/ml. Cells were incubated in the dark at 4°C on a bidirectional shaker for 30 minutes in FcBlock (20 μg/ml; BD Biosciences — Pharmingen), then for an additional 50 minutes with fluorophore-conjugated primary antibodies or isotype control antibodies. Antibodies employed in these studies included: CD11b-PE (2 μg/ml) and F4/80-APC (5 μg/ml) from (CALTAG Laboratories). Following incubation with primary antibodies, 1 ml FACS buffer was added to the cells. Cells were centrifuged at 500 g for 5 minutes and resuspended in 1 ml of FACS buffer. The wash was repeated 2 times. Cells were analyzed on a FACSCalibur and analysis was performed using CellQuest software (BD).
Total RNA was extracted from frozen adipose tissue (100 mg) using a commercially available acid-phenol reagent (TRIzol; Invitrogen Corp.). First-strand cDNA was synthesized using Superscript III reverse transcriptase and random hexamer primers as described in the manufacturer’s protocol (Invitrogen Corp.). Samples of cDNA were diluted 1:25 in nuclease-free water (QIAGEN). Samples from each cDNA pool were diluted 1:10, 1:30, 1:90, and 1:270 in order to create a standard curve for calculation of relative gene expression levels. PCR amplification mixtures (20 μl) contained 10 μl ×2 PCR SYBR Green I Quantitect Master Mix (QIAGEN), 0.4 μl of 25-μM reverse and forward primer mix, and 11.6 μl diluted cDNA template. Real-time quantitative PCR was carried out using the DNA Engine Opticon (MJ Research) instruments with the following cycling parameters — polymerase activation: 15 minutes, 95°C; amplification for 40 cycles: 15 seconds, 94°C; 20 seconds, 58°C; 20 seconds, 72°C. After amplification, melting curve analysis was performed as described in the manufacturers’ protocol (QIAGEN). Relative expression values were calculated based on the standard curve.
The expression rates of 2 macrophage-specific genes (Emr1, Cd68) that correlated with body mass in our microarray studies, an adipocyte-specific gene (Acdc), lipid metabolism genes (Lipe, Gpam, Fabp4), and proinflammatory genes (Tnfa, Ccr2, Ccl2/Mcp1, Ccl7/Mcp3, Serpine1) were determined by quantitative RT-PCR.
To normalize expression data, we used multiple internal control genes as described by Vandesompele et al. (49). The internal control genes encoding HMG-14 (Hmg14) and ribosomal protein S3 (Rps3) were selected from our murine microarray data set for having high expression and little sample-to-sample variability. For each transcript assayed, intron-spanning primers were designed using publicly available genomic contig sequences obtained through Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene), the public domain primer design software Primer3 (http://frodo.wi.mit.edu/primer3/primer3_code.html), and the DNA analysis software Vector NTI Suite Version 7 (Informax Inc.). Primer sequences were as follows: Rps3 forward, 5′-ATCAGAGAGTTGACCGCAGTTG-3′; Rps3 reverse, 5′-AATGAACCGAAGCACACCATAG-3′; Emr1 forward, 5′-CTTTGGCTATGGGCTTCCAGTC-3′; Emr1 reverse, 5′-GCAAGGAGGACAGAGTTTATCGTG-3′; Cd68 forward, 5′-CTTCCCACAGGCAGCACAG-3′; Cd68 reverse, 5′-AATGATGAGAGGCAGCAAGAGG-3′; Tnfa forward, 5′-CCAGACCCTCACTAGATCA-3′; Tnfa reverse, 5′-CACTTGGTGGTTTGCTACGAC-3′; Acdc forward, 5′-GCTCCTGCTTTGGTCCCTCCAC-3′; Acdc reverse, 5′-GCCCTTCAGCTCCTGTCATTCC-3′; Hmg14 forward, 5′-GCAGAAAATGGAGAGACGGAAAACC-3′; Hmg14 reverse, 5′-AAGGGAGGCGGGACCACTGAC-3′; Ccl2 forward, 5′-AGGTCCCTGTCATGCTTCTGG-3′; Ccl2 reverse, 5′-CTGCTGCTGGTGATCCTCTTG-3′; Pparg forward, 5′-GCCCTTTGGTGACTTTATGGAG-3′; Pparg reverse, 5′-GCAGCAGGTTGTCTTGGATG-3′; Lipe forward, 5′-ACGAGCCCTACCTCAAGAACTG-3′; Lipe reverse, 5′-ATCTGGCACCCTCACTCCATAG-3′; Fabp4 forward, 5′-AAGAAGTGGGAGTGGGCTTTG-3′; Fabp4 reverse, 5′-CTGTCGTCTGCGGTGATTTC-3′; Serpine1 forward, 5′-TCCTCATCCTGCCTAAGTTCTC-3′; Serpine1 reverse, 5′-GTGCCGCTCTCGTTTACCTC-3′; Gpam forward, 5′-TCCAGAAGGTGAAAAGGAAAGC-3′; Gpam reverse, 5′-GGCAAAAGAGGATGAAGGTGAG-3′.
For the experiments involving INCB3344, insulin levels were determined using an ultrasensitive insulin ELISA (Mercodia AB). In experiments comparing Ccr2–/– and Ccr2+/+ mice, we performed a multiplexed protein assay for IL-6, MCP1, PAI-1, insulin, and leptin (LincoPlex Mouse Adipokine Panel; Linco Research Inc.). Western blots for adiponectin were performed with 1 μl plasma from lean and adiposity-matched obese Ccr2–/– and Ccr2+/+ mice. Plasma proteins were denatured, reduced, and separated on a 16% Tris-glycine gel (Invitrogen Corp.) using SDS-PAGE. Proteins were transferred to nitrocellulose (Invitrogen Corp.). We performed immunoblotting for adiponectin and detected the bands using ECL.
Pairwise comparisons of quantitative phenotypes between mice of different genotypes (e.g., obese Ccr2–/– vs. obese Ccr2+/+) or diets (e.g., lean low-fat diet–fed Ccr2+/+ vs. obese high-fat diet–fed Ccr2+/+) were assessed by 2-tailed Student’s t test assuming unequal variance. To assess the interaction between genotype and diet for quantitative phenotypes, 2-way ANOVA was performed. Analyses were performed using Prism 3.0 (GraphPad Software Inc.). P < 0.05 was considered statistically significant.
We thank Philip Scherer for his gift of the anti-adiponectin antibody used for Western analysis. This work was supported by grants from the NIH (DK59960 and DK066525 to A.W. Ferrante Jr.) and the Columbia Diabetes and Endocrinology Research Center (DK063608).
See the related commentary beginning on page 33.
Nonstandard abbreviations used: ATM, adipose tissue macrophage; CCL, chemokine, C-C motif, ligand; CCR, CCL receptor; HOMA-IR, homeostasis model assessment of insulin resistance; MCP, monocyte chemoattractant protein; PAI, plasminogen activator inhibitor; SVC, stromal vascular cell.
Conflict of interest: K. Vaddi, D. Hunter, and R. Huber are employed by Incyte Corp.