CCR2 modulates inflammatory and metabolic effects of high-fat feeding (original) (raw)
Adipose tissue expression of Ccr2 and its ligands is regulated by obesity. 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 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.
Expression of Ccr2 and its ligands in lean mice, obese mice, and obese mice treated with pioglitazone. Expression of Ccr2 and genes that encode 3 of its ligands, Ccl2, Ccl7 and Ccl8, were measured in lean mice (white bars), obese mice (black bars), and obese mice treated with the insulin sensitizer pioglitazone (gray bars). *P < 0.05, lean vs. obese; †P < 0.05 obese vs. obese pioglitazone-treated (n = 4). Values are expressed as mean ± SD.
Ccr2–/– mice are partially protected from diet-induced obesity. 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 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 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.
Body mass of Ccr2–/– mice. (A) C57BL/6J Ccr2+/+ (black symbols) and Ccr2–/– (gray symbols) mice were fed a low-fat (triangles) or a high-fat diet (squares) for 24 weeks. There was no significant difference in body mass between mice of each genotype on the low-fat diet (n = 5; P > 0.05). The mean body mass of Ccr2–/– mice fed a high-fat diet was significantly lower than that of the Ccr2+/+ mice (39.3 ± 6.9 g vs. 46.3 ± 4.1 g; n = 10; P < 0.05). (B) Average daily food intake was measured over a 6-week period for Ccr2+/+ and Ccr2–/– mice fed a high-fat diet. *P < 0.05 vs. Ccr2+/+.
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 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); 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.
Ccr2 genotype modulates insulin sensitivity in obesity. 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 1).
Body compositions and plasma lipids of animals examined in this study
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 3). However, obese Ccr2–/– mice had lower fasting blood glucose and insulin concentrations compared with adiposity-matched obese Ccr2+/+ mice (Figure 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 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).
Insulin sensitivity in obese Ccr2–/– and obese Ccr2+/+ mice. Fasting plasma insulin (A) and blood glucose concentrations (B) were measured in lean Ccr2+/+ (black bars) and Ccr2–/– (gray bars) mice and mice of both genotypes made obese following 20 weeks of high-fat diet feeding. There were no significant genotype-dependent differences in fasting glucose or insulin concentrations in lean animals. However, fasting glucose and insulin concentrations were lower in obese Ccr2–/– compared with obese Ccr2+/+ mice despite similar degrees of adiposity (insulin: P < 0.005; glucose: P < 10–4). (C) HOMA-IR values (expressed as IU-mg/dl) were significantly lower (P < 10–4) among obese Ccr2–/– than obese Ccr2+/+ mice. (D) A plot of HOMA-IR values against body mass among all Ccr2–/– (gray squares) and Ccr2+/+ (black circles) mice reveals that the relationship between insulin sensitivity and body mass differs between mice dependent upon Ccr2 genotype. **P < 0.01 compared with wild type. Values are expressed as mean ± SD.
Obese Ccr2–/– mice were also more insulin sensitive than obese Ccr2+/+ animals as measured by an insulin tolerance test (Figure 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 4).
Glucose homeostasis in obese Ccr2–/– and Ccr2+/+ mice. (A) The response of fasted obese Ccr2+/+ (black circles) and Ccr2–/– (gray squares) mice following a single intraperitoneal injection of insulin (1.5 U/kg) was monitored by serially measuring blood glucose concentrations. The percent reduction in glucose concentration in obese Ccr2–/– mice was significantly greater than that in obese Ccr2+/+ mice at 75, 90, and 130 minutes (*P < 0.05). (B) Intraperitoneal injection of a bolus of glucose to fasted obese Ccr2+/+ (black circles) and Ccr2–/– (gray squares) mice lead to similar peak glucose concentrations but lower glucose concentrations at 45, 60, and 90 minutes. *P < 0.05 compared with wild type. Values are expressed as mean ± SD.
Ccr2 deficiency attenuates obesity-induced hepatic steatosis. 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).
Ccr2 deficiency decreases macrophage content of adipose tissue. 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 5). A similar reduction in macrophage content was also detected in subcutaneous adipose tissue depots (Figure 5)
CCR2 deficiency lowers ATM content in obese mice. The fraction of F4/80-expressing macrophages was determined by immunohistochemical analysis of epididymal adipose tissue from obese Ccr2+/+ (A) and Ccr2–/– (B) mice with the macrophage-specific marker F4/80 (EMR1). (C) The fraction of ATMs (F4/80-stained cells/total cells) in periepididymal adipose tissue of obese Ccr2+/+ mice (white bar) was significantly greater than the fraction of ATMs in lean Ccr2+/+ mice (black bar) (P < 10–4) and obese Ccr2–/– mice (gray bar) (P < 0.005). (D) The average fraction of ATMs was also greater in subcutaneous adipose tissue of obese Ccr2+/+ mice (black bar) compared with obese Ccr2–/– mice (gray bar). Values are expressed as mean ± SD. **P < 0.01 compared with obese Ccr2+/+ mice.
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.
Ccr2 deficiency attenuates obesity-induced changes in adipose tissue gene expression. 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 6; ANOVA interaction P < 0.05). Paralleling the decrease in macrophage-specific gene expression, Tnfa expression was reduced in obese Ccr2–/– mice (Figure 6; ANOVA interaction P < 0.005). No significant differences in the expression of these genes were observed between lean Ccr2–/– and Ccr2+/+ mice (Figure 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.
Expression of genes involved in inflammation. Quantitative RT-PCR was used to measure the expression in periepididymal adipose tissue of genes involved in macrophage function and inflammation (Tnfa, Cd68, Emr1, and Serpine1). Obese Ccr_2_–/– (light gray bars) and obese Ccr_2+/+_ (dark gray bars) mice with body mass greater than 40 g were studied, as were lean Ccr_2+/+_ mice (black bars) and lean Ccr_2_–/– mice (white bars). *P < 0.05, obese mice on high-fat chow compared with mice of the same genotype on low-fat chow; Χ_P_ < 0.05, obese Ccr2+/+ compared with obese Ccr2–/– mice. Values are expressed as mean ± SEM.
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 7).
Expression of genes involved in adipocyte function. Quantitative RT-PCR was used to measure the expression in periepididymal adipose tissue of genes involved in adipocyte function (Fabp4, Gpam, Lipe, Pparg, and Acdc). Obese Ccr_2_–/– (light gray bars) and obese Ccr_2+/+_ (dark gray bars) mice with body mass greater than 40 g were studied, as were lean Ccr_2+/+_ mice (black bars) and lean Ccr_2_–/– mice (white bars). *P < 0.05, mice with dietary obesity compared with lean mice of the same genotype; Χ_P_ < 0.05, obese Ccr2+/+ compared with obese Ccr2–/– mice. #NS. Values are expressed as mean ± SEM.
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 _Ccr_2 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).
Ccr2-dependent regulation of expression and circulating concentrations of adipose tissue–derived molecules. 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 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 8).
Plasma adiponectin in lean and obese Ccr2–/– and Ccr2+/+ mice. Proteins in 1 μl of plasma drawn from lean (white bars) and adiposity-matched obese (black bars) Ccr2–/– and Ccr2+/+ mice were denatured, reduced, and separated using SDS-PAGE. We performed immunoblotting for adiponectin with an antibody specific for adiponectin. Values are expressed as mean ± SD. *P < 0.05.
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.
Effects of Ccr2 genotype on plasma lipid concentrations. 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 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 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.
Short-term treatment with a CCR2 antagonist reduces ATM content and improves insulin sensitivity in obese mice. 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 1).
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 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 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 9). Similar to the effects of _Ccr2_ deficiency, INCB3344 treatment also improved glucose homeostasis following a glucose tolerance test (Figure 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).
Insulin sensitivity in CCR2 antagonist–treated mice. (A) Hyperglycemia after a daytime fast (6 hours) was reduced in high-fat diet–fed obese mice that received the selective CCR2 antagonist INCB3344 for 14 days (white bars) compared with high-fat diet–fed obese mice that received vehicle injections (black bars; n = 7 per group; *P < 0.05). (B) Following an overnight fast (14 hours), blood glucose concentrations of INCB3344 and vehicle-treated obese animals were not significantly different. However, fasting insulin concentrations and HOMA-IR values (expressed as IU-mg/dl) were significantly lower in the INCB3344-treated animals (n = 7 per group; *P < 0.05). (C) Following an intraperitoneal injection of glucose, obese mice treated with INCB3344 (9 days of daily injections; open circles) were significantly less hyperglycemic (n = 8 per group; **P < 0.01 at 10, 20, 30, and 60 minutes) than those treated with vehicle (filled squares). (D) Following an intraperitoneal injection of insulin (1.5 U/kg) the percentage reduction in blood glucose concentration was greater in obese mice treated with INCB3344 (14 days of daily injections) than in vehicle-treated controls (n = 7; *P < 0.05 at 30, 45, 75, 90, and 130 minutes). Values are expressed as mean ± SD.
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.