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Endocrinology, doi:10.1210/en.2007-0699
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Endocrinology Vol. 148, No. 12 6186-6194
Copyright © 2007 by The Endocrine Society

Obesity-Induced Inflammation in White Adipose Tissue Is Attenuated by Loss of Melanocortin-3 Receptor Signaling

Kate L. J. Ellacott, Jonathan G. Murphy, Daniel L. Marks and Roger D. Cone

Center for the Study of Weight Regulation and Associated Disorders (K.L.J.E., J.G.M., D.L.M., R.D.C.), Department of Pediatrics (D.L.M.), and Vollum Institute (R.D.C.), Oregon Health and Science University, Portland, Oregon 97239-3098

Address all correspondence and requests for reprints to: Roger D. Cone, Center for the Study of Weight Regulation and Associated Disorders (L481), Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239-3098. E-mail: cone{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Metabolic syndrome, a complex of highly debilitating disorders that includes insulin resistance, hypertension, and dyslipidemia, is associated with the development of obesity in humans as well as rodent models. White adipose tissue (WAT) inflammation, caused in part by macrophage infiltration, and fat accumulation in the liver are both linked to development of the metabolic syndrome. Despite large increases in body fat, melanocortin 3-receptor (MC3-R)-deficient mice do not get fatty liver disease or severe insulin resistance. This is in contrast to obese melanocortin 4-receptor (MC4-R)-deficient mice and diet-induced obese (DIO) mice, which show increased adiposity, fatty liver disease, and insulin resistance. We hypothesized that defects in the inflammatory response to obesity may underlie the protection from metabolic syndrome seen in MC3-R null mice. MC4-R mice fed a chow diet show increased proinflammatory gene expression and macrophage infiltration in WAT, as do wild-type (WT) DIO mice. In contrast, MC3-R-deficient mice fed a normal chow diet show neither of these inflammatory changes, despite their elevated adiposity and a comparable degree of adipocyte hypertrophy to the MC4-R null and DIO mice. Furthermore, even when challenged with high-fat chow for 4 wk, a period of time shown to induce an inflammatory response in WAT of WT animals, MC3-R nulls showed an attenuated up-regulation in both monocyte chemoattractant protein-1 (MCP-1) and TNF{alpha} mRNA in WAT compared with WT high-fat-fed animals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY-INDUCED INFLAMMATION has increasingly been implicated in the pathogenesis of metabolic syndrome (1, 2, 3). These inflammatory changes are caused in part by the infiltration of macrophages and other immune cells into white adipose tissue (WAT) and have been seen in both rodents and humans (4, 5, 6, 7, 8). Adipocyte hypertrophy, adipocyte apoptosis/necrosis, and local increases in free-fatty acids (FFAs) have been implicated in potentiating WAT inflammation (9). Similarly, endoplasmic reticulum stress may play a role (10, 11). Additionally, elevated circulating FFAs have also been proposed to play a role in this process; however, recent studies suggest that inflammation in WAT is independent of changes in FFAs in the circulation (12). FFAs stimulate inflammatory pathways via their action on toll-like receptor 4 leading to proinflammatory cytokine release via nuclear factor-{kappa}B (NF-{kappa}B) signaling (13, 14). The initial stimulus for the influx of these immune cells into adipose tissue may be complex, but elevated levels of the chemokine monocyte chemoattractant protein (MCP)-1 in WAT are thought to play a role (6, 15, 16, 17, 18). Data showing that macrophages are still present in WAT of MCP-1 null mice fed a high-fat diet demonstrate that the signals for macrophage infiltration into WAT are also multifactorial (19, 20).

We became interested in examining the inflammatory response to obesity in models of altered melanocortin signaling due to the finding that melanocortin-3 receptor (MC3-R) null mice are protected from the development of fatty liver disease and severe insulin resistance despite significantly elevated levels of body fat (21, 22). The central melanocortin system plays a pivotal role in the regulation of body weight and energy homeostasis as revealed by studies in animals and humans (for review, see Ref. 23). In animals, genetic alterations in melanocortin signaling lead to obesity (24, 25, 26, 27, 28, 29), highlighting the importance of this system in the regulation of energy homeostasis. However, genetic deletion of the melanocortin receptor MC3-R or MC4-R leads to two distinct models of obesity. Mice with genetic deletion of the MC3-R gene have a unique metabolic phenotype characterized by increased adiposity in the absence of hyperphagia, hypometabolism, or a notable increase in total body weight (24, 25). In contrast, MC4-R-deficient mice have a phenotype reminiscent of a diet-induced obese (DIO) mouse, with an increase in body weight due to increased adipose and lean mass caused by hyperphagia and hypometabolism (26, 30). Furthermore, the MC4-R null mouse shows elevated serum insulin, insulin resistance, and hepatic steatosis (21, 22, 26, 31).

We hypothesized that the insulin resistance and fatty liver disease in the MC4-R null mice would be associated with an increase in inflammation, whereas the apparent protection from hepatic steatosis and severe insulin resistance in the MC3-R null animals, despite increased adiposity, may be caused by a diminution in inflammation in WAT of these animals. In the present study, we tested this hypothesis by examining the inflammatory profile of WAT in MC3-R and MC4-R null mice at 28–32 wk, when their obesity phenotypes were fully developed, and compared them with age-matched wild-type (WT) and WT DIO mice. We also challenged MC3-R null animals with a high-fat diet to determine whether MC3-R deletion actually protects animals from diet-induced WAT inflammation. These studies provide novel cellular and molecular data potentially explaining the phenomenon of adipocyte hypertrophy and obesity in the absence of metabolic syndrome in the MC3-R null mouse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and husbandry
All animals used in experiments were male MC4-R null (26), MC3-R null (24), or wild-type C57BL6/J mice maintained in colonies at Oregon Health and Science University. MC4-R null and MC3-R null mice were crossed more than nine generations onto the C57BL6/J background. Unless stated otherwise, all animals were housed in groups, three to five per cage, at 21 ± 2 C with ad libitum access to standard chow (Purina rodent diet 5001; Purina Mills, St. Louis, MO) and water. Experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Oregon Health and Science University.

DIO
DIO mice were generated by placing WT C57BL6/J mice on a high-fat diet (60% calories from fat; D12492; Research Diets Inc., New Brunswick, NJ) for 22 wk (Table 1Go and Figs. 1–3GoGoGo) or 4 wk (Table 2Go and Fig. 4Go). MC4-R null and MC3-R null mice were also placed on the same high-fat chow in some experiments for 4 wk, as indicated.


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  		TABLE 1. Comparative body composition phenotypes of obesity models

 

Figure 1
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  		FIG. 1. Adipocyte hypertrophy and altered gene expression in MC3-R null, MC4-R null, and DIO mice. H & E staining shows an increase in adipocyte size in MC3-R null (B), MC4-R null (C), and DIO (D) mice, compared with WT animals fed a standard low-fat chow diet (A). Percent frequency distribution of adipocyte sizes indicates a shift in the size of the adipocyte population toward larger hypertrophied cells (E), reflected in a significant increase in the mean adipocyte size in MC3-R, MC4-R null, and DIO animals, compared with WT controls. MC3-R and MC4-R null mice were maintained on standard low-fat chow. ***, P < 0.001, compared with WT standard low-fat chow-fed animals; ##, P < 0.01, compared with MC3-R nulls; $$$, P < 0.001, compared with MC4-R nulls, one-way ANOVA. Adipocyte area was measured using Image J software from three different animals per genotype/diet (minimum of 60 cells measured per group). Taqman quantitative real-time PCR revealed an increase in WAT FAS gene expression in MC4-R null (G; n = 7) and DIO mice (H; n = 8), compared with low-fat-fed WT (G; n = 13, H; n = 8) and MC3-R null (G; n = 10) animals; *, P < 0.05, compared with WT, #, P < 0.05, compared with MC3-R null, one-way ANOVA (G). ***, P < 0.001, compared with control animals (CON), two-tailed t test. Adipocyte hypertrophy was associated with a significant reduction in WAT PPAR-{gamma} gene expression. ***, P < 0.001, **, P < 0.01, both compared with WT animals, one-way ANOVA (G); **, P < 0.01, compared with control group, two-tailed t test (H). RQ, Relative quotient. Scale bars, 100 µm.

 

Figure 2
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  		FIG. 2. MC3-R null mice are resistant to WAT inflammation. MC4-R null mice on low-fat chow (A–D; n = 7) and DIO (E–H; n = 12) mice showed increased gene expression of inflammatory markers MCP-1, CD68, IL-13, and TNF{alpha} in WAT, compared with MC3-R null mice on low-fat chow (A–D; n = 10) and WT low-fat chow-fed animals (A–D; n = 13, E–H; n = 8) as measured by Taqman quantitative real-time PCR. ***, P < 0.001, compared with WT; ###, P < 0.001; #, P < 0.01, compared with MC3-R null, one-way ANOVA (A–D); ***, P < 0.001; **, P < 0.01, compared with control animals (CON), two-tailed t test (E–H). RQ, Relative quotient. Immunohistochemistry for the macrophage marker F4/80 confirms the real-time PCR for CD68 and indicates a low macrophage infiltration into WAT in MC3-R null (J and N) animals, compared with MC4-R null (K and O) and DIO (L and P) animals. Black arrows indicate examples of crown-like structures of macrophages engulfing dying cells; see high-magnification image (K, inset). Tissue from WT low-fat-fed animals is shown in I and M for comparison. Scale bar, 200 µm (main images); 50 µm (K, inset). Upper panels, I–K, F4/80 immunoreactivity using the Caltag BM8 clone antibody. Lower panels, M–P, Immunoreactivity using the Serotec CI:A3–1 clone antibody.

 

Figure 3
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  		FIG. 3. MC3-R null mice are protected from fatty liver disease despite increased adiposity. Liver triglycerides, measured by gas chromatography, are elevated in MC4-R null (A; n = 7) and DIO (B; n = 12) mice but not MC3-R null (A; n = 10) or WT low-fat chow-fed animals (A; n = 13, B; n = 8). **, P < 0.01, compared with WT; ###, P < 0.001, compared with MC3-R null, one-way ANOVA (A); ***, P < 0.001, compared with control animals, two-tailed t test (B). H & E staining indicating the presence of lipid vacuoles in MC4-R null (E) and DIO (F) mice, compared with the relatively normal tissue morphology in the WT (C) and MC3-R null (D) animals. Real-time quantitative PCR revealed that increased liver triglyceride levels were associated with an increase in PPAR-{gamma} (G and I) and SCD-1 (H and J) mRNA levels in MC4-R null (G and H; n = 7) and DIO (I and J; n = 12) mice, compared with MC3-R null (G and H; n = 10) and control animals (G and H; n = 13, I and J; n = 8). RQ, Relative quotient.

 

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  		TABLE 2. Comparative body composition of WT and MC3-R null animals exposed to a high-fat (HF) diet

 

Figure 4
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  		FIG. 4. Attenuated inflammatory response to by high-fat feeding in MC3-R null mice. After exposure to a high-fat diet for 4 wk, animals of all genotypes showed the same mean adipocyte size (A). Adipocyte area was measured from H & E stains of visceral WAT using Image J software from three different animals (minimum of 55 cells measured per group). Increased adiposity caused by consumption of a high-fat diet was associated with an increase in inflammatory markers in WAT from WT and MC3-R null animals, compared with low-fat-fed controls (B–D: WT low fat; n = 5, MC3R low fat; n = 5); however, there was a significant attenuation of MCP-1 and TNF-{alpha} mRNA in MC3-R null high-fat-fed (B and C: MC3R high fat; n = 5) compared with WT high-fat-fed animals (B and C:WT high fat; n = 4). There was no significant difference in CD68 mRNA detected between WT high-fat and MC3R high-fat groups (D); however, when plotted as a function of the percent adiposity, there appears to be an alteration in the slope of the correlation between these two factors (E). ***, P < 0.001, P < 0.01, P < 0.05, one-way ANOVA. RQ, Relative quotient.

 
Analysis of body composition
Whole-animal body composition was assessed using a dual-energy x-ray absorptiometry (DXA) scanner (Piximus; GE Healthcare, IL). Animals were fasted for 16 h before scanning to reduce the variability attributed to food in the digestive tract. Before scanning, animals were deeply anesthetized using ketamine/xylazine/acepromazine mouse cocktail 0.1 cc/g.

Serum leptin ELISA
Blood was collected from anesthetized mice via cardiac puncture after DXA scanning. Serum was separated after spinning the blood for 10 min at 4 C and stored at –80 C until use. Fasting serum leptin was measured by commercially available ELISA (CrystalChem Inc., Downers Grove, IL) according to the manufacturer’s instructions.

Histology and immunohistochemistry
Hematoxylin and eosin staining (H & E) was performed using standard techniques on formalin-fixed, paraffin-embedded tissues by the Cancer Pathology Core of the Veterans Affairs Cancer Research Center at the Portland Veterans Affairs Hospital.

F4/80 immunohistochemistry was performed on 5-µm sections of WAT that had been formalin fixed and paraffin embedded before sectioning. This antibody, and its use in detecting macrophages, has been previously characterized (32, 33). After sectioning, the slides were deparaffinized with three changes of xylene followed by washes in 100, 95, and 85% ethanol. For the Serotec antibody, the slides were incubated in 0.01 M citrate buffer (pH 6) containing 0.1% Triton X-100 at 70 C for 40 min (antigen unmasking). The sections were then incubated in 3% hydrogen peroxide in 1x PBS (0.01 M PBS) for 10 min to deactivate endogenous peroxidases. After repeated washing in 1x PBS, the slides were then incubated with blocking reagent (2% normal goat serum, 1% BSA, 0.1% Triton X-100, 0.05% Tween 20, and 0.05% sodium azide) for 1 h at room temperature. The slides were incubated over-night at 4 C with primary antibody against F4/80 (rat antimouse F4/80 BM8 clone catalog no. MF48000; Caltag Laboratories Inc., Carlsbad, CA; or rat antimouse F4/80 CI:A3–1 clone catalog no. MCA497R; AbD Serotec Inc., Raleigh, NC) diluted 1:100 in blocking reagent. After repeated washing in 1x PBS, the slides were incubated with the antirat avidin biotin complex kit according to the manufacture’s instructions (Vector Laboratories Inc., Burlingame, CA), followed by diaminobenzadine (Vector Laboratories). Finally, the slides were counterstained briefly with hematoxylin and dehydrated with increasing concentrations of ethanol and xylene. The sections were coverslipped using a xylene-based mounting medium (Sigma-Aldrich, St. Louis, MO). The specificity of the primary antibodies was verified by repeating the procedure and replacing the primary antibody with normal serum. No positive staining was seen with either antibody (data not shown).

Adipocyte sizing
Adipocyte area was measured, using visceral adipose, from at least 60 cells per animal from three animals per genotype or diet/genotype combination. Adipocyte area (square micrometer) was measured by using Image J software (NIH, Bethesda, MD) by an investigator blinded to the genotypes and/or diets.

Taqman quantitative real-time PCR
Tissues were rapidly dissected from animals after DXA scanning, frozen on dry ice, and stored at –80 C until use. RNA was extracted from tissues using Trizol, followed by cDNA synthesis using random hexamer priming and Moloney murine leukemia virus reverse transcriptase, both according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Taqman gene expression assays (primer/probe sets) specific to mouse were purchased from Applied Biosystems (Foster City, CA) [peroxisome proliferator-activated receptor-{gamma} (PPAR)-{gamma}, assay ID Mm00440945_m1; fatty acid synthase (FAS), assay ID Mm00662319_m1; MCP-1 assay, ID Mm00441242_m1; CD68, assay ID Mm00839636_m1; IL-13, assay ID Mm00434204_m1; TNF-{alpha}, assay ID Mm00443258_m1; stearoyl coenzyme A desaturase (SCD-1), assay ID Mm00772290_m1]. Samples were run in duplicate on 96-well plates according to the manufacturer’s instructions on a Taqman 7300 instrument (Applied Biosystems). Gene expression for each target was normalized to18S RNA (assay ID Hs99999901_s1).

Liver triglyceride measurements
Liver triglycerides were measured by gas chromatography by the mouse metabolic phenotyping center of Vanderbilt University (Nashville, TN).

Statistical analysis
Statistical analysis was performed using Prism software (GraphPad, San Diego, CA). Data are expressed as mean ± SEM. Unpaired two-tailed Student’s t test was used to compare two groups. One-way ANOVA was used to compare data in three or more groups and post hoc tests were used to obtain multiple comparisons within the analysis. Significance was taken as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of WAT in genetic and diet-induced models of obesity
It has been shown previously that genetic deletion of MC4-R and MC3-R in mice leads to the development of two distinct obesity syndromes (24, 25, 26). Using DXA scanning, we confirmed that the 28- to 32-wk-old animals used in our study of these strains fed normal chow showed body composition phenotypes comparable with those published. The MC4-R null mouse displayed a phenotype reminiscent of that seen in an age- and sex-matched DIO mouse (Table 1Go), characterized by an increase in body weight, body fat, fat-free mass, and fasting serum leptin. In contrast, the MC3-R null mouse showed a modest increase in body weight caused principally by a highly significant increase in body fat combined with no significant change in fat-free mass, although there was a trend toward a decrease in this parameter. Histological staining (H & E) revealed that the increase in body fat seen in all three models of obesity was caused, at least in part, by adipocyte hypertrophy in visceral adipose depots (Fig. 1Go, A–F; mean cell area: WT, 2160 ± 68 µm2; MC3-R null, 4720 ± 197 µm2; MC4-R null, 4527 ± 178 µm2; DIO, 5509 ± 209 µm2).

Next, adipocyte gene expression was characterized in each of the three obesity models. Using quantitative real-time PCR, it was found that both DIO mice and MC4-R null mice had an increase in FAS and a reduction in PPAR-{gamma} gene expression in visceral WAT (Fig. 1Go, G–J). In contrast, the MC3-R null mice showed no change in FAS mRNA, whereas PPAR-{gamma} gene expression was also reduced, as in the adipose tissue from other obesity models (Fig. 1Go, G–J).

Absence of chronic inflammation in WAT of the MC3-R null mouse
The MC3-R null mouse has a unique metabolic syndrome with increased adiposity in the absence of a significant increase in food intake or body weight (24, 25) and without hepatic steatosis or severe insulin resistance (22). The lack of fatty liver disease and severe insulin resistance in this model are unexpected, given the high percentage body fat seen in these animals. It is now well established that obesity is a chronic low-grade inflammatory state characterized in part by elevated expression of proinflammatory cytokines and chemokines, and macrophage infiltration into adipose tissue, so expression of genes involved in the inflammatory process were measured in visceral adipose tissue from the DIO, MC4-R null, and MC3-R null mice. In agreement with published results (6, 16), we saw increased gene expression of a number of cytokines/chemokines in WAT of the DIO mouse, compared with control, WT chow-fed animals (Fig. 2Go, E–H). Furthermore, the elevation in MCP-1, IL-13, and TNF-{alpha} gene expression was also seen in the MC4-R null mouse (Fig. 2Go, A–D), reinforcing the similarities between these two models. Despite the increase in adipocyte size seen in the MC3-R null (Fig. 1Go, A–F), there was no significant increase in gene expression in any of the inflammatory markers examined in visceral adipose tissue from the MC3-R null mouse (Fig. 2Go, A–D). Furthermore, using CD68 gene expression (Fig. 2Go, B and F) as a marker for adipose tissue macrophages, we found that there was a significant increase in macrophage infiltration into WAT from DIO and MC4-R null mice but not in MC3-R nulls or WT normal chow-fed controls. These data were confirmed using F4/80 immunohistochemistry as another means of detecting macrophages (Fig. 2Go, I–P).

Absence of fatty liver disease in MC3-R null mice
To confirm the previous studies of Sutton et al. (22), the livers of DIO, MC4-R null, and MC3-R null mice were examined for evidence of fatty liver disease. Using a combination of histology (H & E staining) and chromatographic measurement of liver triglyceride levels, we found that the MC3-R null mice did not show an increase in triglyceride storage in the liver, compared with WT controls, whereas the DIO and MC4-R null animals showed substantial increases in stored triglycerides (Fig. 3Go, A–F). This was further confirmed using quantitative real-time PCR, which indicated an increase in SCD-1 and PPAR-{gamma} mRNA in the livers of DIO and MC4-R null mice, compared with MC3-R null animals. The MC3-R null animals were not significantly different from WT controls (Fig. 3Go, G–J).

Attenuation of WAT inflammation in MC3-R null mice after exposure to a high-fat diet
In contrast to the MC4-R null mouse, the increased adiposity in the MC3-R null is not caused by an increase in food intake (hyperphagia) or a significant reduction in energy expenditure (24, 25) but is hypothesized to be caused by a change in substrate use, promoting the storage of energy as fat (21, 24). Like the MC4-R null, MC3-R null mice are known to be susceptible to further increases in body fat on a high-fat diet (21, 24). We placed MC3-R null mice on a high-fat diet to further challenge them and ascertain whether they would still be protected from the development of inflammation in WAT when faced with an even greater degree of adiposity. Animals were placed on a high-fat diet for 4 wk because this period of time has previously been shown to be sufficient to stimulate an inflammatory response in WAT of WT animals (16). After 4 wk on a high-fat diet, WT and MC3-R null mice gained similar amounts of body weight; however, in the WT animal, the increase in body weight occurred as a result of an increase in both fat and fat-free mass, as assessed by DXA scanning, whereas the increase in the MC3-R null animals was exclusively due to increased fat mass (Table 2Go). Analysis of adipocyte size using histology (H & E staining) revealed that after a 4-wk exposure to a high-fat diet, adipocytes from WT, MC3-R null, and MC4-R null mice reached a comparable mean area (Fig. 4AGo; mean cell area: WT low fat, 1597 ± 68 µm2, WT high fat, 5574 ± 280 µm2, MC3-R null low fat, 2981 ± 120 µm2, MC3-R null high fat, 5952 ± 319 µm2, MC4-R null low fat, 3518 ± 162 µm2, MC4-R null high fat, 5791 ± 267 µm2), despite the fact that MC3-R and MC4-R null mice had larger adipocytes at baseline on a low-fat diet suggesting the adipocytes have a maximal size/capacity. It is important to note here that the animals were younger at the time the animals were killed (21 wk as opposed to 28–32 wk) than the animals in Fig. 1Go, which is likely to be the cause of the lower baseline adipocyte size in this group.

As shown in the literature (6, 16) and the DIO model described in this study, exposure of WT mice to a high-fat diet was associated with an increase in MCP-1, TNF-{alpha}, and CD68 gene expression in WAT as assessed by quantitative real-time PCR (Fig. 4Go, B–E). In agreement with our earlier data, there was no significant difference in the expression of these inflammatory markers between WT and MC3-R null mice fed a regular, low-fat, diet. Moreover, MC3-R mice fed a high-fat diet showed a significant increase in inflammatory markers, compared with low-fat-fed MC3-R control animals; however, the increase in MCP-1 and TNF-{alpha} gene expression was significantly less than that seen in WT animals fed a high-fat diet (Fig. 4Go, B and C). The up-regulation of CD68 mRNA was not significantly different between WT and MC3-R null mice fed a high-fat diet, suggesting that the infiltration of macrophages into WAT can be driven by high-fat feeding of the MC3-R null mouse (Fig. 4DGo). However, when the relative amount of CD68 mRNA is plotted as a function of percent body fat for WT and MC3-R null mice, there is a reduction in the slope of the correlation for the MC3-R null mice, suggesting a reduced amount of macrophage infiltration for the amount of body fat (Fig. 4EGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to address the hypothesis that the protection from the development of fatty liver disease and severe insulin resistance in the MC3-R null mouse (21, 22), despite the frank obesity in this model might involve an altered inflammatory response to obesity. Initially we chose to examine obesity-associated inflammation in 28- to 32-wk WT, MC3-R null, and MC4-R null mice fed standard mouse chow ad libitum because at this time point, the phenotypes of each mouse obesity model are well developed. Twenty-eight- to 32-wk DIO mice, prepared by feeding WT mice high-fat chow from 6 wk of age, were also examined. The obesity phenotype in the MC3-R and MC4-R null animals develops without the need for high-fat feeding and allowed us to examine the effects of the genetic alteration on obesity-associated inflammation and avoid any potential influence of increased circulating triglycerides that may occur as a result of dietary intake. At this time point, we demonstrated that MC3-R null mice exhibited a doubling in adipose mass and a 6-fold increase in serum leptin levels, relative to WT control animals (Table 1Go). Furthermore, the degree of visceral adipocyte hypertrophy seen in the MC3-R, MC4-R, and DIO obesity models was comparable (Fig. 1Go). Thus, along with no increase in lean mass, as observed in the MC4-R null and DIO models, the MC3-R null mice are likely to contain adipose depots with fewer adipocytes overall. Despite the obesity and adipocyte hypertrophy, the MC3-R visceral adipose depot did not exhibit the metabolic or inflammatory changes represented by increases in FAS, MCP-1, IL-13, TNF-{alpha}, and CD68 mRNA gene expression observed in the obese MC4-R null and DIO models.

Once we had established that, unlike DIO and MC4-R-deficient animals, the 28- to 32-wk MC3-R null mice did not show increased WAT inflammation or fatty liver disease despite an increase in adiposity, compared with WT standard chow-fed animals, we decided that it would be important to control for the degree of adiposity. To further increase the adiposity seen in the MC3-R null animals, we placed them on a high-fat diet for 4 wk, a period of time known to stimulate a large inflammatory response to obesity in WT animals (16). MC3-R null mice increase weight rapidly on a high-fat diet, and indeed the MC3-R null mice in this study increased their body fat to 46%, compared with 36% in WT high-fat-fed animals. Despite this massive amount of body fat, comparable with the MC4-R null and DIO WT mice in the 28- to 32-wk group, the absence of the MC3-R appeared to blunt the increase in MCP-1 and TNF{alpha} mRNA in WAT induced in WT animals by high-fat feeding. Despite the attenuated induction of MCP-1 mRNA by high-fat feeding in the MC3-R null, macrophage infiltration into WAT did not appear to be blunted at this one time point, based on comparable levels of gene expression of the macrophage marker, CD68. However, whereas expression of CD68 mRNA may not be different when all animals are examined independent of adiposity, when the amount of CD68 is plotted as a function of body fat in individual animals (Fig. 4EGo), MC3-R null mice appear to express less CD68.

Changes in CD68 mRNA were used as a marker of macrophage infiltration throughout this study. Although CD68 is not exclusively expressed on macrophages (it is also expressed on neutrophils, basophils, and large lymphocytes), CD68 mRNA has been shown to increase in adipose tissue of obese mice to a similar degree as F4/80, another commonly used marker of macrophages (12). F4/80 immunohistochemistry appeared to confirm the results from CD68 quantitative real-time PCR, demonstrating a lack of macrophage infiltration in obese MC3-R null WAT depots when mice are fed on normal chow (Fig. 2Go). Thus, although CD68 is not solely expressed on macrophages, in this context, it provides a good indication of the infiltration of immune cells into WAT in response to increased adiposity.

Changes in proinflammatory gene expression in both macrophages and adipocytes can be induced via FFA stimulation of Toll-like receptor-4, leading to stimulation of the NF{kappa}B pathway (13, 14). The source of these FFAs in vivo is not known but is likely related to local increases due to increased adipocyte lipolysis or even apoptosis or necrosis of adipocytes (9). The cause of the reduced inflammatory response to obesity in the MC3-R null is not known but may be linked to an alteration in the function of the adipocyte in these animals. MC3-R null mice fed a standard chow diet have a comparable degree of adipocyte hypertrophy to MC4-R null animals; however, in contrast to MC4-R null animals, we did not detect any significant increase in FAS mRNA, an enzyme involved in fat storage in adipocytes. This suggests that the fundamental mechanism of adipocyte hypertrophy in MC3-R null animals is distinct from that of MC4-R nulls or DIO mice. Indeed, the double MC4-R/MC3-R null mouse is more obese than either single mutant animal (25), suggesting that the increased adiposity in these two models occurs through separate pathways. It is possible that the increase in adipocyte size in the MC3-R null is not related to an increased rate of fat synthesis, as indicated by the lack of significant increase in FAS mRNA, but is caused by a decreased rate of lipolysis within the adipocyte. An alteration in lipolytic activity of MC3-R adipocytes may, in turn, reduce the local increases in FFAs, which stimulate inflammatory pathways via Toll-like receptor-4. When the WT and MC3-R animals were placed on a high-fat diet, their adipocytes showed comparable degrees of hypertrophy, suggesting that adipocytes may have a maximal capacity for expansion. Furthermore, despite their comparable size, the inflammatory response to the increased adiposity in these two models was different, indicating the reduced inflammatory response in the WAT of the MC3-R null does not occur due to the absence of adipocyte hypertrophy.

In rodents, MC3-Rs are expressed at a number of central and peripheral sites. In the brain, one site of MC3-R expression is on proopiomelanocortin neurons of the arcuate nucleus of the hypothalamus (34) on which they are believed to exert an autoinhibitory action on central melanocortinergic tone (35). However, the fact that the MC3-R null mouse shows an obese and not lean phenotype suggests that the receptor plays a more complex role in the regulation of energy homeostasis. In addition to the arcuate nucleus, MC3-Rs are expressed in more than 30 other brain nuclei (36). Furthermore, in the rodent, MC3-R expression has been described at a number of peripheral sites including the adipose tissue, kidney, heart, skeletal muscle (37), stomach, duodenum, placenta, pancreas (38), and macrophages (39). The expression of the MC3-R on macrophages indicates the potential for a direct regulatory role for this receptor in macrophage action. Indeed, there is large body of literature describing a role of the melanocortin system in the modulation of immune function (for review see Ref. 40). However, in contrast to the results reported here, in which the absence of the MC3-R reduces inflammation, the melanocortin peptides have antiinflammatory activity in vivo and in vitro, and {alpha}-MSH has been shown to inhibit NF{kappa}B activation by inflammatory stimuli (41, 42, 43, 44, 45). Furthermore, the MC3-R specific agonist D-Trp8-{gamma}-MSH appears to inhibit cytokine release from macrophages both in vitro and in vivo (39). Whereas this rather extensive literature argues against a direct proinflammatory role for the MC3-R in the periphery, it remains possible that the reduced inflammatory responses reported here in the MC3-R null mouse result from a defect in the development or maturation of the immune system specifically due to the absence of the MC3-R.

Alternatively, the results reported here may also be mediated by absence of MC3-R expression in the central nervous system. Whereas {alpha}-MSH has been demonstrated to be antiinflammatory when administered centrally (46, 47), this peptide acts as a potent agonist at both the MC3-Rs and MC4-Rs in the central nervous system. Thus, both the site, central vs. peripheral, and the mechanism, developmental vs. pharmacological, of MC3-R-mediated effects on the inhibition of obesity-induced metabolic syndrome remain to be determined. Regardless of the mechanism, future studies may nonetheless provide valuable information regarding the link between obesity and metabolic syndrome.


   Acknowledgments
 
The authors thank Emily Larson and Robert Klein for the use of the DXA scanner and Paul Kievit for help with dissections. We also thank Carolyn Gendron for her excellent H & E staining and Carla Harris and Larry Swift of the Mouse Metabolic Phenotyping Core (Vanderbilt University) for performing the liver triglyceride measurements.


   Footnotes
 
This work was supported by American Heart Association Grant 0760014Z (to K.L.J.E.) and the National Institutes of Health Grant RO1 DK070332 (to R.D.C.).

Disclosure Statement: All authors have nothing to disclose.

First Published Online September 27, 2007

Abbreviations: DIO, Diet-induced obesity; DXA, dual-energy x-ray absorptiometry; FAS, fatty acid synthase; FFA, free-fatty acid; H & E, hematoxylin and eosin staining; MCP, monocyte chemoattractant protein; MC-R, melanocortin receptor; NF-{kappa}B, nuclear factor-{kappa}B; PPAR, peroxisome proliferator-activated receptor; SCD-1, stearoyl coenzyme A desaturase; WAT, white adipose tissue; WT, wild type.

Received May 23, 2007.

Accepted for publication September 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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