Macrophage metalloelastase (MMP12) regulates adipose tissue expansion, insulin sensitivity, and expression of inducible nitric oxide synthase.

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BALANCE-OBESITY

Macrophage Metalloelastase (MMP12) Regulates Adipose Tissue Expansion, Insulin Sensitivity, and Expression of Inducible Nitric Oxide Synthase Jung-Ting Lee,* Nathalie Pamir,* Ning-Chun Liu, Elizabeth A. Kirk, Michelle M. Averill, Lev Becker, Ilona Larson, Derek K. Hagman, Karen E. Foster-Schubert, Brian van Yserloo, Karin E. Bornfeldt, Renee C. LeBoeuf, Mario Kratz, and Jay W. Heinecke Departments of Medicine (J.-T.L., N.P., N.-C.L., M.M.A., L.B., K.E.F.-S., B.V.Y., K.E.B., R.C.L., M.K., J.W.H.), Pathology (K.E.B.), and Epidemiology (E.A.K., M.K.), University of Washington, Seattle, Washington 98105; and Fred Hutchinson Cancer Research Center (D.K.H., M.K.), Public Health Sciences, Seattle, Washington 98103

Macrophage metalloelastase, a matrix metallopeptidase (MMP12) predominantly expressed by mature tissue macrophages, is implicated in pathological processes. However, physiological functions for MMP12 have not been described. Because mRNA levels for the enzyme increase markedly in adipose tissue of obese mice, we investigated the role of MMP12 in adipose tissue expansion and insulin resistance. In humans, MMP12 expression correlated positively and significantly with insulin resistance, TNF-␣ expression, and the number of CD14⫹CD206⫹ macrophages in adipose tissue. MMP12 was the most abundant matrix metallopeptidase detected by proteomic analysis of conditioned medium of M2 macrophages and dendritic cells. In contrast, it was detected only at low levels in bone marrow derived macrophages and M1 macrophages. When mice received a high-fat diet, adipose tissue mass increased and CD11b⫹F4/80⫹CD11c⫺macrophages accumulated to a greater extent in MMP12-deficient (Mmp12⫺/⫺) mice than in wild-type mice (Mmp12⫹/⫹). Despite being markedly more obese, fat-fed Mmp12⫺/⫺ mice were more insulin sensitive than fat-fed Mmp12⫹/⫹ mice. Expression of inducible nitric oxide synthase (Nos2) by Mmp12⫺/⫺ macrophages was significantly impaired both in vivo and in vitro, suggesting that MMP12 might mediate nitric oxide production during inflammation. We propose that MMP12 acts as a double-edged sword by promoting insulin resistance while combatting adipose tissue expansion. (Endocrinology 155: 3409 –3420, 2014)

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besity, the most common metabolic disorder in the United States, is a risk factor for atherosclerosis and type 2 diabetes (1). These diseases are linked to a constellation of metabolic factors, including insulin resistance, hypertriglyceridemia, and low HDL levels (2). The cellular hallmark of obesity is lipid accumulation by adipocytes (3). Moreover, macrophages that ingest invading microorganisms and scavenge damaged cells accumulate in adipose tissue of obese mice and humans (4 – 8). It has there-

fore been proposed that adipose tissue expansion in obesity triggers and maintains a low-grade inflammatory state (1, 4 – 8). Chronic inflammation promotes insulin resistance and type 2 diabetes (1, 2, 4, 5), and recent studies also implicate macrophages in insulin resistance (1, 4 – 6, 8 –14). For example, deleting I-␬-B kinase-beta in myeloid cells that prevent the activation of transcription factors that trigger immune and inflammatory responses improved insulin

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received January 14, 2014. Accepted May 29, 2014. First Published Online June 10, 2014

* Both authors contributed equally to this work. Abbreviations: APC, allophycocyanin; BMI, body mass index; GM-CSF, granulocyte-macrophage colony-stimulating factor; HFD, high-fat diet; HOMA, homeostasis model assessment-insulin resistance index; iNOS, inducible nitric oxide synthase; IFN-␥, interferon-␥; LFD, low-fat diet; LPS, lipopolysacharide; M-CSF, macrophage colony-stimulating factor; MMP, matrix metallopeptidases; Mmp12⫺/⫺, macrophage metalloelastase-deficient mice; Mmp12⫹/⫹, wild-type mice; MS, mass spectrometry; SVF, stromal vascular fraction.

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Endocrinology, September 2014, 155(9):3409 –3420

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Macrophage Metalloelastase In Obesity and Insulin Resistance

action in adipose tissue and liver (9). Also, myeloid-specific deletion of peroxisome proliferator-activated receptor-␥, the molecular target of the insulin-sensitizing thiazolidinedione drugs, increased levels of inflammatory mediators and rendered fat-fed mice more insulin-resistant (10, 14). Increased adiposity triggered the accumulation of CD11c-expressing macrophages in mouse adipose tissue (11–13). Those cells have been proposed to be polarized toward an inflammatory M1-like phenotype that promotes insulin resistance (1, 11, 12). In contrast, adipose tissue macrophages in lean, insulin-sensitive mice have an anti-inflammatory M2-like phenotype that promotes insulin sensitivity (14). Collectively, these observations provide strong evidence that macrophages in obese mice promote insulin resistance. Tissue matrix metallopeptidases (MMPs) have been proposed to contribute to adipose tissue remodeling, and their levels are elevated in adipose tissue of obese mice (15, 16). Moreover, macrophage metalloelastase (MMP12), a metallopeptidase secreted by macrophages and trophoblasts (17, 18), is implicated in the pathogenesis of inflammatory disorders, including aortic aneurysms and smoking-induced emphysema (19, 20). However, it is unclear whether macrophage-derived MMPs are involved in adipose tissue expansion (21). Recent work suggests that adiposity is unaffected when fat-fed mice are deficient in Mmp12, but that study used only a few animals of mixed genetic backgrounds (22). Expression of Mmp12 mRNA in adipose tissue is dramatically enhanced in mice with diet-induced and genetic forms of obesity (15, 16), though much less is known about MMP12 in humans. We therefore investigated the role of MMP12 in adipose tissue expansion and insulin resistance in both humans and mice.

Materials and Methods Human subjects All studies were approved by the Fred Hutchinson Cancer Research Center Institutional Review Board. All subjects provided prior written informed consent, were healthy, did not use medications or drugs, and were weight stable. Subjects with fasting hyperglycemia were included in the study if they were not on glucose-lowering therapy. Freshly prepared EDTA plasma collected after an overnight fast was immediately frozen at ⫺70°C until analysis. Subcutaneous adipose tissue was biopsied as previously described (23). Insulin resistance was estimated by the homeostasis model assessment-insulin resistance index (HOMA) (24).

Mice. All studies were approved by the Animal Care and Use Committee of the University of Washington. Macrophage metalloelastase-deficient mice (Mmp12⫺/⫺) and wild-type mice


(Mmp12⫹/⫹) on the C57BL/6 background were obtained from the Jackson Laboratory. Male mice were fed a low-fat diet (LFD; pelleted rodent chow, Wayne Rodent BLOX 8604, Harlan Teklad) until 7 weeks of age, when they were randomly assigned to continue the LFD or switched to a high-fat diet (HFD). The HFD contained 60% kcal as fat, mainly lard (D12492, Research Diets). Mice were killed by cervical dislocation following anesthesia with ketamine/xylazine. Tissue and plasma were prepared and frozen at ⫺80°C until analysis. Before tissues were removed, the hepatic artery was severed and the mice were perfused with sterile PBS (pH 7.4) via the left ventricle. The tissue specimens were weighed, snap frozen, and stored at ⫺80°C. Dendritic cells and macrophages derived from bone marrow cells were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively, as described (25).

Body composition Human body composition was measured by dual-energy Xray absorptiometry (General Electric Lunar Prodigy DEXAscanner, GE Healthcare). Quantitative magnetic resonance (EchoMRI whole body composition analyzer; Echo MRI) was used to determine the body composition of conscious immobilized male mice (26, 27).

Adipose tissue fractionation Human abdominal adipose tissue was biopsied as described (23, 28). Tissue specimens were immediately flash-frozen for gene expression studies (23) or digested with collagenase and separated into stromal vascular cells and adipocytes (28). Mouse adipose tissue was separated into stromal vascular and adipocyte fractions as described (29).

Immunoblot analyses Adipose tissue was homogenized in radioimmunoprecipitation assay buffer containing protease inhibitor cocktail (10 ␮L per 100 mg tissue protein; Sigma-Aldrich) and clarified by centrifugation. Proteins (40 ␮g) in the supernatants were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot analysis, using rat anti-Mac-2 (1:10,000 dilution; Cedarlane Laboratories Ltd) or rabbit polyclonal antibody to macrophage elastase (1:5,000 dilution; Millipore). They were detected with goat antirat or antirabbit IgG-HRP (1:5,000 dilution; Sigma-Aldrich).

Glucose homeostasis All studies were performed on mice fasted for 4 hours. Blood glucose levels were measured with a portable glucose-measuring device. Plasma insulin levels were quantified by ELISA (Linco). For insulin and glucose tolerance testing, mice were injected ip with human insulin (1.0 U/kg body weight) or sterile glucose (2 mg/kg body weight in phosphate buffered saline) as described (27, 30).

Quantitative real-time PCR Total RNA was extracted from frozen adipose tissue, using acid-phenol reagent (TRIzol; Invitrogen Corp). For white adipose tissue, the oil layer was removed prior to chloroform extraction to ensure RNA quality. mRNA was quantified in triplicate, using the ␦-delta CT method (31) with 18S ribosomal


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RNA as the control. Primer sequences were: Mmp12, F-ttgaccacttcgccaaaag, R-aatcagcttggggtaagcagg; Nos2, F-gaagaaaaccccttgtgctg R-tccagggattctggaacatt; Ym1, F-agaagggagtttcaaacctggt, R-gtcttgctcatgtgtgtaagtga; Arg1, F-ctcaagccaagtccttagag, R-aggagctgtcattaggacatc.

Immunohistochemical and morphometric analyses Tissue sections were immunostained as described (27). Adipocyte area was determined from 4 high-power (200x) fields/ animal (5 mice per group), using a digital imaging system (ImagePro Plus, Media Cybernetics). All studies were performed by an observer blinded to animal genotype.

Flow cytometry Human stromal vascular fractions were stained with a combination of antibodies (Alexa fluorophore-700-conjugated CD45, allophycocyanin [APC]-conjugated CD206, and APCCy7-conjugated CD14) and characterized by flow cytometry (LSR2, Becton Dickinson). Mouse stromal vascular fractions were treated with red cell lysis buffer (Sigma-Aldrich) and Fc receptor blocker before being stained with propidium iodide or with fluorophore-conjugated antibodies (eBioscience) for CD11b APC, CD11c (PE) or F4/80 (FITC) (13). Flow cytometry (FACS Canto II) quantified mouse CD11b⫹ macrophage populations identified by side and forward scatter; subpopulations were identified by immunostaining for F4/80 and CD11c. Mouse monocyte subpopulations were identified and quantified as described (11).

Liquid chromatography-electrospray ionizationtandem mass spectrometry (MS) LC-ESI-MS/MS studies were performed on tryptic digests of macrophage-conditioned medium as described (25, 32).

Statistical analyses Unless otherwise stated, results are given as means ⫾ SEM. Because tissue levels of human MMP12 mRNA exhibited a nonnormal distribution, logarithmic transformations were used in these analyses. Linear regression analysis used Pearson’s correlation coefficients. Significant P values were ⬍ .05 on two-tailed analysis (32). Analyses were performed using SPSS (version 16.0, SPSS Inc) or Stata (version 112, StataCorp IC) software.

Table 1.

a

Results MMP12 expression in human adipose tissue correlates with macrophage infiltration, adipose tissue inflammation, and insulin resistance To determine if MMP12 is expressed in human adipose tissue and to assess its relationship to adiposity and inflammation, we studied 18 subjects ranging widely in body mass index (BMI) (Table 1). Across all individuals, MMP12 mRNA expression levels in subcutaneous adipose tissue varied over a 100-fold range. MMP12 levels correlated highly and significantly (Pearson’s r ⫽ 0.67, P ⫽ .002) with the number of CD14⫹ (a lipopolysaccharide [LPS] coreceptor expressed by monocyte/macrophages) and CD206⫹ (a macrophage mannose receptor) macrophages as assessed by immunostaining and flow cytometry (Figure 1A). In contrast, MMP12 expression did not correlate significantly with BMI (Figure 1B; r ⫽ 0.33, P ⫽ .17) or body fat mass (r ⫽ 0.17, P ⫽ .64 in men; r ⫽ 0.53, P ⫽ .18 in women). Adjusting for BMI in logistic modeling had little effect on the relationship between MMP12 expression and the number of adipose tissue macrophages (r ⫽ 0.62, P ⫽ .008). These observations strongly suggest that macrophages are the major source of MMP12 in human adipose tissue. However, variations in adiposity have little impact on MMP12 expression in subcutaneous adipose tissue. To determine whether MMP12 might promote adipose tissue inflammation, we examined the relationship between MMP12 expression in adipose tissue and mRNA levels for proteins implicated in macrophage inflammation. MMP12 expression correlated highly and significantly with the expression of TNFa (r ⫽ 0.59, P ⫽ .01, Figure 1C), CCL2 (MCP-1; r ⫽ 0.68, P ⫽ .002, Figure 1D), and ICAM1 (r ⫽ 0.47, P ⫽ .048) (not shown). It correlated inversely with adiponectin (ADIPOQ) mRNA levels (r ⫽ ⫺0.57, P ⫽ .013, Figure 1E).

Baseline Characteristics of Human Subjectsa

Gender Age (years) Weight (kg) Height (m) BMI ( kg/m2) Body fat mass (% of total) Male Female Fasting glucose (mmol/L) Fasting insulin (␮U/mL) HOMA

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Non-obese (n ⴝ 9)

Obese (n ⴝ 9)

Total (n ⴝ 18)

4 males 5 females 41.6 ⫾ 12.1 76.4 ⫾ 8.8 1.75 ⫾ 0.13 25.0 ⫾ 2.2

6 males 3 females 45.4 ⫾ 10.7 103.7 ⫾ 12.0 1.79 ⫾ 0.08 32.5 ⫾ 1.9

10 males 8 females 43.5 ⫾ 11.2 90.0 ⫾ 17.3 1.77 ⫾ 0.11 28.7 ⫾ 4.3

22.6 ⫾ 3.6 40.5 ⫾ 6.6 5.1 (4.8 – 6.6) 5.1 (4.2 – 8.9) 1.2 (0.9 – 2.0)

39.0 ⫾ 6.0 49.2 ⫾ 1.3 5.8 (5.0 – 7.9) 15.0 (5.4 – 25.4) 3.9 (1.4 – 7.0)

32.4 ⫾ 9.8 43.7 ⫾ 6.8 5.4 (4.8 – 7.9) 7.2 (4.2 – 25.4) 1.7 (0.9 – 7.0)

, Values are means ⫾ SDs or median (range). Obese, BMI ⬎ 30.


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Figure 1. Quantification of adipose tissue adipokines, CD14⫹CD206⫹ macrophages, and MMP12 mRNA in human adipose tissue. Stromal vascular cells were isolated from biopsies of human abdominal subcutaneous fat, stained for CD14 and CD206 cell-surface proteins, and analyzed by flow cytometry. Levels of mRNA in the same biopsies were quantified by qPCR. Gene expression data was normalized with 3 housekeeping genes: ␤-glucuronidase, phosphoglycerate kinase 1, and 18s rRNA. Macrophages in panel (A) are CD14⫹CD206⫹ cells as percentage of total live cells.

MMP12 expression also correlated significantly with insulin resistance as assessed by HOMA (r ⫽ 0.54, P ⫽ .020, Figure 1F). Collectively, these observations indicate that MMP12 expression in human subcutaneous adipose tissue is linked with accumulation of CD14⫹CD206⫹ macrophages, insulin resistance, and the expression of macrophage inflammatory mediators. Mouse dendritic cells and M2 macrophages secrete high levels of macrophage metalloelastase in vitro To determine whether macrophages or dendritic cells express and secrete MMP12, we cultured bone marrow cells from Mmp12⫹/⫹ mice for 7 days with M-CSF or

GM-CSF, respectively (33, 34). To generate inflammatory (M1) or antiinflammatory (M2) macrophages, we then stimulated the M-CSF cells for 48 hours with IFN-␥ plus LPS (M1) or with IL-4 (M2). Macrophage polarization (25, 35) was confirmed by quantifying mRNA levels (M1 macrophages, Nos2, Tnfa, Il12b; M2 macrophages, Ym1, Arg1, Mrc2; data not shown). Dendritic cells and M2 macrophages respectively expressed 60and 10-fold greater levels of Mmp12 mRNA than bone marrow-derived macrophages and M1 macrophages (Figure 2A). To determine if protein secretion patterns reflected the mRNA patterns, we used LC-ESIMS/MS to analyze conditioned medium from each cell type (25). Of the more than 700 proteins we detected in the media, only 3 were MMPs: MMP12, MMP13, and MMP19 (Figure 2B). Dendritic cells produced high levels of MMP12, one of the most abundant proteins in the medium, and M2 macrophages produced about half as much. In contrast, much lower levels of the metallopeptidase were found in conditioned medium of the bone marrow-derived macrophages and M1 macrophages. Collectively, these observations indicate that M2 macrophages and dendritic cells produce high levels of MMP12 in vitro.

Expression of MMP12 protein in epididymal adipose tissue increases in fat-fed obese mice To investigate the mechanism by which MMP12 contributes to adipose tissue homeostasis, we studied Mmp12⫹/⫹ and Mmp12⫺/⫺ mice under physiological and obese conditions. Previous studies have demonstrated markedly elevated levels of MMP12 mRNA in the stromal vascular fraction (SVF) of adipose tissue isolated from ob/ob mice and fat-fed obese mice (15, 36). To see if protein levels are also elevated, we fed male Mmp12⫹/⫹ mice a LFD or HFD for 10 weeks, isolated adipocytes and SVFs from adipose tissue, subjected the fractions to SDS-PAGE, and immunoblotted with a polyclonal antibody specific


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Figure 2. Quantification of Mmp12 mRNA and other matrix metallopeptidases in mouse macrophages activated by IL-4 or LPS and IFN-␥. Bone marrow-derived macrophages (Mac) or dendritic cells (DC) were derived from bone marrow precursor cells of C57BL/6J mice cultured with M-CSF or GM-CSF, respectively. Classically activated macrophages (M1) and alternatively activated macrophages (M2) were derived from M-CSF macrophages by treatment with IFN-␥ and LPS or IL-4. A, qRT-PCR of Mmp12. Results (n ⫽ 6) were standardized to 18S and are representative of 3 independent analyses. B, LC-ESI-MS/MS analysis of cell conditioned medium. Macrophages and dendritic cells were incubated in serum-free medium for 6 hours. Proteins were quantified by spectral counting (the total number of unique peptides detected for a given protein) by tandem MS/MS analysis (n ⫽ 6) and are representative of 3 independent analyses.

for MMP12. On either diet, MMP12 was undetectable in the adipocyte fraction of Mmp12⫹/⫹ mice (Figure 3A). It was also undetectable in the SVF of mice fed the LFD. In contrast, MMP12 was readily detected in the SVF of mice fed the HFD (Figure 3A). Moreover, immunohistochemistry localized MMP12 in crown-like structures in epididymal tissue of fat-fed mice, but not of mice fed the LFD (Figure 3B). Immunoblotting demonstrated a two-fold increase in Mac2 proteins levels of obese Mmp12⫺/⫺ mice relative to Mmp12⫹/⫹ mice. Collectively, these observations indicate that MMP12 protein expression is markedly elevated in adipose tissue macrophages of Mmp12⫹/⫹ mice fed a HFD.


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MMP12 deficiency promotes adipose tissue expansion To determine whether MMP12 helps control adipose tissue expansion during diet-induced obesity, we monitored body weight, body composition, and adipose tissue depot size of male Mmp12⫹/⫹ and Mmp12⫺/⫺ mice fed the LFD or HFD for 10 weeks (Figures 3, C–F). The two strains had comparable baseline weights (Mmp12⫹/⫹, 22.6 ⫾ 0.5 g, n ⫽ 69; Mmp12⫺/⫺, 21.9 ⫾ 0.3 g, n ⫽ 67). On the HFD, the Mmp12⫺/⫺ mice gained body weight more rapidly than the Mmp12⫹/⫹ mice (Figure 3C). Indeed, Mmp12⫺/⫺ mice fed the HFD doubled their weight (44.2 ⫾ 2.1 g, n ⫽ 57) by 10 weeks, whereas the Mmp12⫹/⫹ mice showed a more moderate weight gain (33.8 ⫾ 0.8 g, n ⫽ 59). After only 6 weeks on the HFD, Mmp12⫺/⫺ mice had twice as much fat mass as Mmp12⫹/⫹ mice (Figure 3D; 16.0 ⫾ 1.3 g, n ⫽ 3 vs 7.8 ⫾ 1.3 g, n ⫽ 4, P ⬍ .001). They also had slightly lower lean mass (17.6 ⫾ 0.3 vs 19.9 ⫾ 0.5, n ⫽ 4, P ⬍ .05) (Figure 3D). After 10 weeks on the HFD, the weights of mesenteric, retro-peritoneal, and inguinal adipose tissues isolated from Mmp12⫺/⫺ mice were significantly greater than those from Mmp12⫹/⫹ mice (Figure 3E; n ⫽ 24 per group). In contrast, there was no difference in the weights of epididymal adipose tissue between the two strains. Inspection confirmed greatly increased abdominal adiposity in the fat-fed Mmp12⫺/⫺ mice, which also had significantly larger adipocytes as assessed by light microscopy (Figure 3F). Thus, adipocyte hypertrophy may have contributed to the increase in total adiposity. In contrast, there was no difference in the weights of brown adipose tissue. These observations indicate that MMP12 restrains the expansion of white adipose tissue during diet-induced obesity but does not affect brown adipose tissue. MMP12 deficiency promotes insulin sensitivity Obesity associates strongly with insulin resistance and macrophage activation in adipose tissue, raising the possibility that glucose homeostasis is impaired in mice deficient in MMP12. We therefore measured fasting plasma glucose and insulin concentrations for Mmp12⫺/⫺ and Mmp12⫹/⫹ mice fed the test diets for 10 weeks (Figure 4A, B). Although adiposity of the fat-fed Mmp12⫺/⫺ mice greatly exceeded that of Mmp12⫹/⫹ mice, no significant differences in insulin or glucose levels were observed. To assess glucose homeostasis in more detail, we performed glucose tolerance tests and insulin tolerance tests. On the LFD, Mmp12⫹/⫹ mice had significantly higher glucose levels than the Mmp12⫺/⫺ mice at all 3 time points following ip injection of glucose (Figure 4C). With fat feeding, glucose excursions were greater than for mice fed


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Figure 3. Characterization of adipose tissue MMP12 protein expression in lean and obese wildtype mice and evidence that MMP12 deficiency results in increased body weight and body fat composition. A, Immunoblot and (B) immunohistochemical analyses of mac2 and MMP12 in adipose tissue of lean and obese Mmp12⫹/⫹ mice. Male C57BL/6J mice were fed a high-fat diet (HFD; fat 60% of calories) or low-fat diet (LFD, 12%) for 10 weeks. For immunoblotting, adipose tissue was separated into adipocyte and stromal-vascular fractions (SVFs). Equal amounts of detergent solubilized tissue protein (40 ug) were separated under reducing conditions by SDSPAGE and immunoblotted with an antibody to MMP12. For histology, paraffin-embedded adipose tissue sections were immunostained with a mouse monoclonal and rabbit polyclonal antibody for mac2 and MMP12 respectively. Immunoreactive material was detected (red color) with peroxidase-coupled antibody (eg, yellow arrows). C–F, Body weight and composition of male mice fed a LFD or HFD for 10 weeks. C, Weights of Mmp12⫺/⫺ mice fed a HFD were significantly greater than Mmp12⫹/⫹ mice at each time point after 2 weeks (P ⬍ .01; n ⬎ 30 all groups). Note that the Mmp12⫺/⫺ mice doubled their weight by 9 weeks on the HFD. D, Body composition was determined by magnetic resonance spectroscopy. Mice were fed a LFD or HFD for 6 weeks. *, P ⬍ .05 compared with the Mmp12⫹/⫹ group. E, White and brown adipose tissues (BAT) were harvested from mice fed a HFD for 10 weeks (n ⫽ 24 mice per group). *, P ⬍ .05 compared with the Mmp12⫹/⫹ group. White adipose tissues were mesenteric (Mes), epididymal (Epi), retroperitoneal (Retro), and inguinal (Ing). F, The mean size of adipocytes in epididymal (Epi) tissue was determined by light microscopy (n ⫽ 5 mice per group) for mice fed a LFD or HFD for 10 weeks. *, P ⬍ .05 compared with the Mmp12⫹/⫹ group.

the LFD, but no differences were seen between genotypes. These results are reflected in the area-underthe-curve for the glucose tolerance test that was lower for Mmp12⫺/⫺ mice (P ⫽ .002) than for Mmp12⫹/⫹ mice (Figure 4E). Although the fatfed Mmp12⫺/⫺ mice were 60% heavier, with twice as much fat mass, their glucose levels were significantly lower in response to insulin challenges at 60 minutes and 90 minutes than those of the fat-fed wild-type control mice (Figure 4D). Moreover, the area-under-the curve of the insulin tolerance test was significantly lower (35%, P ⬍ .001) in the fat-fed Mmp12⫺/⫺ mice (Figure 4F). These observations demonstrate that increased obesity did not make the fatfed Mmp12⫺/⫺ mice more insulin-resistant than the fat-fed Mmp12⫹/⫹ mice (Figure 4E). Indeed, the Mmp12⫺/⫺ mice might have been more insulin-sensitive. Collectively, these observations strongly suggest that MMP12 deficiency promotes insulin sensitivity even though it enhances fat mass and weight gain on a HFD. Fat-fed transgenic mice expressing high levels of adiponectin are markedly more obese than fat-fed wild-type mice but are more insulinsensitive (38). However, the adiponectin levels of the Mmp12⫹/⫹ and Mmp12⫺/⫺ mice differed only modestly (3.6 ⫾ 0.2 ␮g/mL and 2.8 ⫾ 0.2 ␮g/mL, respectively; P ⬍ .05). The two strains also had similar plasma levels of free fatty acids that increased modestly on the HFD (data not shown). Immunohistochemical analyses demonstrated higher lipid deposition in liver of Mmp12-deficient mice fed the HFD than in wild-type mice (data not shown). Biochemical analysis confirmed that the levels of both triglycerides and cholesterol were significantly higher in the livers of fat-fed Mmp12⫺/⫺ than Mmp12⫹/⫹ mice.


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protein present on myeloid derived macrophages, and CD11c is an integral membrane protein present on dendritic cells but also expressed by macrophages (39). After identifying Cd11b⫹ cells, we gated that population for CD11c and F4/80 (11). The total number of macrophages, defined as CD11b⫹ cells, was comparable for Mmp12⫹/⫹ mice fed either diet, but were 25% higher in adipose tissue of fat-fed Mmp12⫺/⫺ mice (Figure 5B). There was also a higher percentage of CD11b⫹F4/80⫹ cells in the obese Mmp12⫹/⫹ animals than in the lean Mmp12⫹/⫹ controls (35%, P ⫽ .013). The fraction of CD11b⫹F4/80⫹ cells was even greater in obese Mmp12⫺/⫺ mice (Figure 5 C). When we gated for CD11b, F4/80, and CD11c, the number of adipose tissue-derived stromal vascular cells that were positive for all 3 markers was 50% higher in the fat-fed Mmp12⫹/⫹ mice (P ⫽ .002) than in the Mmp12⫹/⫹ mice on the LFD (Figure 5A, lower panel; Figure 5, B and C). Surprisingly, the ⫺/⫺ ⫹/⫹ Figure 4. Glucose metabolism in Mmp12 and Mmp12 mice. Male mice were fed a LFD number of CD11b⫹F4/80⫹ CD11c⫹ or HFD diet for 10 weeks. Fasting plasma glucose (A) and insulin (B) levels. Mice were fasted for 4 hours. C, Glucose tolerance test. Mice were fasted for 4 hours and then injected ip with macrophages that are implicated in inglucose (2 g/kg). Plasma glucose levels were significantly lower (*, P ⬍ .05) in Mmp12⫺/⫺ mice sulin resistance (11, 13) was even fed the LFD. D, Insulin tolerance test. Mice fed the two diets were fasted for 4 hours; they then greater (P ⫽ .02) in adipose tissue of received an ip injection of insulin (1 U/kg). Plasma glucose levels were significantly lower (*, P ⬍ .05) in the Mmp12⫺/⫺ mice. D, Areas under the curves for the glucose tolerance test (E) the obese Mmp12⫺/⫺ mice than in or insulin tolerance test (F). n ⱖ 5 mice per group for all studies. obese Mmp12⫹/⫹ mice, even though these animals were more insulin sensitive These observations suggest that alterations in plasma levCD11b⫹F4/80⫹CD11c⫹ and CD11b⫹F4/80⫹CD11c⫺ els of adiponectin and free fatty acids or hepatic lipids are unlikely to contribute to the phenotype of increased insu- macrophages in mouse adipose tissue are thought to have proinflammatory M1-like and anti-inflammatory M2lin sensitivity of obese Mmp12⫺/⫺ mice. like phenotypes (11, 13). Our in vitro studies indicated MMP12 deficiency alters macrophage recruitment that M2 macrophages expressed much higher levels of and polarization MMP12 than M1 or bone marrow-derived macrophages To test the hypothesis that MMP12 deficiency affects (Figure 2). To determine if the in vivo pattern was simmacrophage polarization towards an M1-like or M2-like ilar, we quantified Mmp12 levels in M1-like phenotype, we isolated the SVF of abdominal adipose tis- CD11b⫹F4/80⫹CD11c⫹ macrophages and M2-like sue from mice fed a LFD or HFD for 10 weeks, and per- CD11b⫹F4/80⫹CD11c⫺ macrophages isolated by formed flow cytometry to quantify different macrophage FACS from epididymal fat (11, 12). Consistent with our subpopulations. Examples of results and gating details are model system studies, M2-like macrophages isolated shown in Figure 5A, top and bottom panels. CD11b is an from fat-fed mice expressed the highest levels of Mmp12 integrin present on leukocytes, F4/80 is an adhesion (Figure 5D).


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Figure 5. Quantification of adipose tissue macrophages in Mmp12⫺/⫺ and Mmp12⫹/⫹ mice. Abdominal adipose tissue was harvested from mice fed a LFD or HFD diet for 10 weeks. Stromal vascular cells were isolated, stained for CD11b, CD11c, and F4/80 cell surface proteins, and analyzed by flow cytometry using propidium-negative selected (live) cells. A, Effects of LFD and HFD on macrophage number. Macrophages were quantified as CD11b⫹ cells. B, Quantification of CD11b⫹ cells and (C) F4/80⫹ and CD11c⫹ cells in CD11b⫹ macrophages. The numbers of cells, F4/80⫹ cells, doubly positive (F4/80⫹CD11b⫹CD11c⫺) cells, and triply positive cells, were calculated from flow cytometry data and stromal vascular tissue cell counts. D, Mmp12 levels in different populations of CD11b⫹ macrophages.


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MMP12 regulates expression of Nos2 by dendritic cells and M1 macrophages Inducible nitric oxide synthase (iNOS, gene name Nos2) is expressed at high levels by inflammatory macrophages (33). Recent studies indicate that Mmp12 and Nos2 are coordinately regulated in chondrocytes in a mouse model of inflammation (40). To determine whether there might be similar regulation in macrophages, we quantified levels of Nos2 in dendritic cells and macrophages derived from bone marrow of Mmp12⫺/⫺ and Mmp12⫹/⫹ mice fed the HFD (Figure 6; note log scale in Figure 6A). Levels of Nos2 mRNA were 3-fold higher in dendritic cells than in bone marrow-derived macrophages of wildtype mice. Nos2 levels rose 9-fold in dendritic cells stimulated with IL-4 (Figure 6A), but this increase failed to occur in dendritic cells derived from Mmp12⫺/⫺ mice. When dendritic cells or macrophages were stimulated with

A

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C

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IFN-␥ and LPS, the level of Nos2 mRNA increased 10,000-fold and 1,000-fold, respectively, but the increase was 100-fold lower in Mmp12⫺/⫺ dendritic cells and macrophages. SDS-PAGE and immunoblot analysis with an antibody specific for NOS2 confirmed that protein expression was markedly reduced in Mmp12⫺/⫺ dendritic cells stimulated with IFN-␥ and LPS (Figure 6B). These observations indicate that MMP12 promotes the expression of Nos2 in dendritic cells and M1 macrophages. To determine if MMP12 might affect NOS2 expression in vivo, we quantified mRNA levels of Nos2 in the SVF and in CD11b⫹F4/80⫹ CD11c⫹ macrophages isolated from adipose tissue of Mmp12⫹/⫹ and Mmp12⫺/⫺ mice (Figure 6, C and D). Nos2 mRNA expression was 5-fold lower in the SVF of fat-fed Mmp12⫺/⫺ mice (P ⬍ .0001). Nos2 levels were markedly lower in the CD11b⫹F4/80⫹ CD11c⫹ macrophages of fat-fed Mmp12⫺/⫺ mice than in those from Mmp12⫹/⫹ mice. They were also lower in F4/ 80⫹CD11c⫺ macrophages isolated from fat-fed Mmp12⫺/⫺ animals. These observations indicate that expression of Nos2, a marker of M1 macrophages and insulin resistance, was elevated in F4/80⫹CD11c⫹ macrophages isolated from fat-fed obese Mmp12⫹/⫹ mice and that expression was impaired in both SVF and macrophage subpopulations isolated from adipose tissue of Mmp12⫺/⫺ mice. Together, these observations suggest that MMP12 regulates both the in vitro and in vivo expression of Nos2, an enzyme that plays a central role in host defense and insulin resistance (41, 42).

Discussion

Figure 6. Expression of Nos2 by bone marrow-derived macrophages and by adipose tissue macrophages. Macrophages and dendritic cells were derived from bone marrow of Mmp12⫹/⫹ or Mmp12⫺/⫺ cultured with GM-CSF or M-CSF for 7 days. M1 and M2 cells were derived from macrophages stimulated for 48 hours with IFN-␥ and LPS or with IL-4. A, Expression levels. Levels of mRNA for MMP12 and Nos2 were measured by quantitative RT-PCR. B, Immunoblot analysis. Dendritic cells were stimulated with IFN-␥ and LPS. Equal amounts of protein were separated under reducing conditions by SDS-PAGE and immunoblotted with an antibody to iNOS (n ⫽ 4 mice per group). *, P ⬍ .05. C, Expression of Nos2 in adipose tissue of Mmp12⫺/⫺ and Mmp12⫹/⫹ mice fed a HFD for 10 weeks. RT-PCR was used to quantify mRNA levels of Nos2 (n ⫽ 7 per group). *, P ⬍ .05 compared with the Mmp12⫹/⫹ group. D, Expression of Nos2 in CD11b⫹ macrophages isolated from adipose tissue. Stromal vascular cells were isolated from abdominal fat, stained for CD11b, CD11c, and F4/80 cell surface proteins, and collected by flow cytometry using propidium-negative selected (live) cells. RT-PCR was used to quantify mRNA levels (n ⫽ 7 per group). Results are normalized to CD68 gene expression. N.D. indicates not detected. *, P ⬍ .05 compared with the Mmp12⫹/⫹ group.

Accumulation of inflammatory macrophages in adipose tissue has been proposed to promote insulin resistance during obesity (1, 4 –14). However, the factors that activate macrophages in that tissue are poorly understood, and little is known regarding the roles of macrophages in adipose tissue expansion. Because activated macrophages express high levels of MMP12 and because MMPs are implicated in tissue remodeling, we hypothesized that


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the enzyme might have previously unsuspected effects on adipose tissue expansion and insulin resistance. We first showed that expression of MMP12 mRNA varies 100-fold in human subcutaneous adipose tissue. Importantly, levels of that mRNA strongly correlated with the accumulation of CD14⫹CD206⫹ macrophages and levels of mRNA for proteins linked to macrophage inflammation. These results are consistent with MMP12 having proinflammatory activities. MMP12 expression also correlated significantly with insulin resistance as monitored by HOMA. In contrast, it did not correlate with BMI or adiposity. Collectively, these observations indicate that MMP12 expression in human adipose tissue is linked to the accumulation of CD14⫹CD206⫹ macrophages, insulin resistance, and the expression of macrophage inflammatory mediators, but is largely independent of body weight and fat mass. It is important to note that our study was confined to abdominal subcutaneous adipose tissue. Because macrophage accumulation can vary widely in different human fat depots, in future studies it will be important to determine if variations in adiposity affect macrophage accumulation and Mmp12 expression levels differentially in different tissues. We next demonstrated that MMP12 is dramatically upregulated at the levels of message and protein in the macrophage-rich stromal-vascular fraction of fat-fed mice. Moreover, immunohistochemistry detected localization of MMP12 in crown-like structures in adipose tissue. Crown-like structures have been proposed to be syncytia of macrophages that sequester and ingest adipocyte debris (12, 37). Unexpectedly, Mmp12⫺/⫺ mice were twice as obese as wild-type mice fed the HFD, and they accumulated more CD11b[plus[F4/80⫹ CD11c⫹ macrophages in adipose tissue. However, they were more insulin-sensitive than Mmp12⫹/⫹ mice. We also noted small but significant differences in muscle mass and glucose tolerance between lean Mmp12⫺/⫺ and Mmp12⫹/⫹ mice. The dissociation of the relationships between adipose tissue expansion, macrophage accumulation, and insulin resistance in obese Mmp12⫺/⫺ mice suggests that the enzyme affects the polarization of adipose tissue macrophages. Indeed, we observed marked dysregulation of the expression of iNOS, a classic inflammatory mediator, in Mmp12⫺/⫺ macrophages. Macrophage polarization in muscle and liver may also be involved, because both tissues contain resident macrophages and play important roles in insulin sensitivity. Previous studies have shown that Mmp12 mRNA expression increases in bone marrow-derived dendritic cells and that LPS inhibits its expression (25, 43). To determine


if protein expression and secretion were similarly affected, we used LC-ESI-MS/MS to quantify levels of the protein in conditioned medium of bone marrow-derived dendritic cells and macrophages. This strategy reliably detected more than 1,000 proteins in the medium. Of the more than 20 MMPs known to be expressed in mice, we identified only 3: MMP12, MMP13, and MMP19. MMP12 was by far the most abundant metallopeptidase. It was also one of the most prevalent proteins in conditioned medium of dendritic cells, where levels were twice as high as those in medium of M2 macrophages. In contrast, only low levels of MMP12 were found in medium of bone marrow-derived macrophages and M1 macrophages. Our model system studies may be physiologically relevant because Mmp12 was also expressed at much higher levels by M2-like macrophages (CD11b⫹F480⫹CD11c⫺ cells) than by M1-like macrophages (CD11b⫹F480⫹CD11c⫹ cells) isolated from adipose tissue of fat-fed mice. However, recent studies have shown that CD11b⫹F480⫹CD11c⫹ cells isolated from obese adipose tissue do not have all of the features of M1 cells (44). Thus, the M1/M2 classification system, though useful for in vitro studies, may not adequately reflect the in vivo state of macrophage polarization. Nevertheless, our observations indicate that Mmp12 expression and secretion are markedly affected by the polarization of macrophages both in vitro and in vivo. In future studies, it will be important to refine the definitions of the M1 and M2 model systems and to use additional markers to define the activation state of macrophages and the markers’ association with MMP12 expression levels in tissue. Recent work has implicated Mmp12 and Nos2 in the catabolic destruction of cartilage by inflammatory chondrocytes (40). We therefore used adipose tissue and bone marrow-derived cells from Mmp12⫺/⫺ and Mmp12⫹/⫹ mice to determine whether Mmp12 affects the expression of Nos2 by dendritic cells and macrophages. Unexpectedly, expression of Nos2, a marker of inflammatory M1 macrophages, was highly impaired in CD11b⫹F480⫹CD11c⫹ macrophages isolated from the SVF of fat-fed Mmp12⫺/⫺ mice. Levels of immunoreactive NOS2 protein greatly increased in Mmp12⫹/⫹ dendritic cells stimulated with LPS and INF-␥, whereas protein expression was markedly suppressed in Mmp12⫺/⫺ dendritic cells. Moreover, dendritic cells stimulated by LPS or INF-␥ and M1 macrophages derived from the bone marrow cells of Mmp12⫺/⫺ mice expressed levels of Nos2 mRNA that were approximately 100-fold lower than those in macrophages from Mmp12⫹/⫹ mice These observations raise the possibility that MMP12 regulates nitric oxide generation by macrophages during inflammation. The observation that Nos2 expression is markedly dysregulated in Mmp12⫺/⫺ macrophages is consistent with the idea that MMPs act primarily to regulate various as-


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derived macrophages and dendritic cells in adipose tissue. Elevated levels of free fatty acids and perhaps other dietderived factors trigger macrophage activation (10 –14). Macrophages and dendritic cells express MMP12 that restrains adipose tissue expansion while promoting the expression of inflammatory mediators that increase insulin resistance to restrict further adipose tissue growth. MMP12 therefore is a double-edged sword, because it promotes insulin resistance while combatting adipose tissue expansion. Figure 7. Proposed roles of MMP12 in adipose tissue homeostasis.

pects of inflammation and immunity (45). Indeed, many lines of evidence indicate that MMPs primarily act on proinflammatory cytokines, chemokines, and other proteins to regulate various aspects of inflammation and immunity (44). Because these enzymes proteolytically degrade matrix molecules in vitro, their ability to remodel the extracellular matrix might be another mechanism. For example, preadipocytes must express MT1-MMP (a membrane-bound MMP) to become adipocytes (46). Model system studies suggest that when proteolytic degradation of extracellular matrix by MT1-MMP is impaired, preadipocytes are unable to differentiate because they become entrapped in a collagen meshwork (46). Adipocyte hypertrophy also occurs in mice deficient in Mmp3 or Mmp19 (46, 47), suggesting that MMPs can affect adiposity by multiple different mechanisms. A key question is whether impaired Nos2 expression promotes insulin sensitivity in obese Mmp12⫺/⫺ mice. Although whole-body deficiency of Nos2 implicates this enzyme in obesity and insulin resistance (40), obesity and insulin resistance were not ameliorated when mice were transplanted with bone marrow from Nos2⫺/⫺ mice rather than wild-type mice (42). It appears, therefore, that myeloid NOS2 does not promote obesity and systemic insulin resistance. However, production of certain inflammatory mediators was enhanced when macrophages from those mice were treated with LPS. Thus, the failure of myeloid Nos2 deficiency to protect obese mice from insulin resistance could be partly due to continued production of NO by liver and muscle as well as to enhanced production of inflammatory mediators by macrophages following high-fat diet stimulation. Our observations suggest the following model for the involvement of MMP12 in diet-induced obesity in mice (Figure 7). When highly palatable nutrients become continuously available, excess caloric intake promotes adipocyte hypertrophy that induces adipose tissue stress and malfunction (1, 4, 5). Attempting to restore tissue homeostasis, hypertrophic adipocytes produce inflammatory mediators that promote the accumulation of monocyte-

Acknowledgments Address all correspondence and requests for reprints to: Jay Heinecke, Division of Metabolism, Box 358055, University of Washington, Seattle Washington 98105; E-mail: [email protected]. This work was supported by awards from the American Diabetes Association (7– 09-CT-36), the National Center for Research Resources (RR025015– 03), and the National Institutes of Health (HL055362, DK17047, HL112625, HL108897, R01HL062887, P01HL092969, and R01HL097365. P30DK035816). Proteomics analyses and mouse strains were provided by the Quantitative and Functional Core and the Viral Vector and Transgenic Mouse Core of the Diabetes Research Center (University of Washington). Disclosure Summary: All authors have nothing to disclose.

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E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression.

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