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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Circ Res. Author manuscript; available in PMC Jul 2, 2010.
Published in final edited form as:
PMCID: PMC2717797
NIHMSID: NIHMS127241

MIF-deficiency reduces chronic inflammation in white adipose tissue and impairs the development of insulin resistance, glucose intolerance and associated atherosclerotic disease

Abstract

Chronic inflammation in white adipose tissue (WAT) is positively associated with obesity, insulin resistance (IR) and the development of type-2 diabetes (T2D). The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is an essential, upstream component of the inflammatory cascade. This study examines whether MIF is required for the development of obesity, IR, glucose intolerance and atherosclerosis in the LDL-receptor-deficient (Ldlr−/−) mouse model of disease.

Ldlr−/−-mice develop IR and glucose intolerance within 15-w while Mif−/−Ldlr−/− littermates are protected. MIF-deficiency does not affect obesity and lipid risk factors but specifically reduces inflammation in WAT and liver, as reflected by lower plasma SAA and fibrinogen levels at baseline and under inflammatory conditions. Conversely, MIF stimulates the in vivo expression of human-CRP, an inflammation marker and risk factor of IR and cardiovascular-disease. In WAT, MIF-deficiency reduces nuclear c-Jun levels and improves insulin sensitivity; MIF-deficiency also reduces macrophage accumulation in WAT and blunts the expression of two proteins that regulate macrophage infiltration (ICAM-1, CD44). Mechanistic parallels to WAT were observed in aorta, where the absence of MIF reduces monocyte adhesion, macrophage lesion content and atherosclerotic lesion size.

These data highlight the physiological importance of chronic inflammation in development of IR and atherosclerosis, and suggest that MIF is a potential therapeutic target for reducing the inflammatory component of metabolic and cardiovascular disorders.

Keywords: inflammation, cytokines, atherosclerosis, insulin resistance, c-jun

Introduction

The intertwined medical problems of obesity, glucose intolerance, type-2 diabetes (T2D), dyslipidemia and atherosclerosis form one of the most serious threats to public health, worldwide. Insulin resistance (IR) is an integral feature of the medical sequelae that are collectively referred to as the metabolic syndrome1. Decreased insulin sensitivity is the underlying defect in >90% of patients with T2D, and it is also considered to be a major pathologic mechanism for the associated development of cardiovascular disease (CVD)2. Recent human and animal studies have established both correlative and causative links between IR and chronic inflammation, in particular within adipose tissue 3;4. For example, C-reactive protein (CRP), which is a serum marker of systemic inflammation, is independently related to insulin insensitivity and highly predictive for progression to overt T2D5. Mechanistic studies that have evaluated the impact of blocking specific inflammatory control points, such as c-Jun N-terminal kinase 1 (JNK1)6, support the concept that the persistent activation of pro-inflammatory transcription factors (e.g. c-Jun) in critical metabolic sites (adipose and liver tissue) may underlie the development of IR. When chronically inflamed, these tissues release pro-inflammatory molecules, including cytokines, acute-phase reactants and pro-coagulant factors (e.g. IL-6, SAA, CRP, fibrinogen), which can participate in the pathogenesis of IR and atherosclerosis3;79. A primary event in the pathogenesis of IR is the infiltration of macrophages into white adipose tissue (WAT). This process appears to be of critical importance for the development of low-grade adipose tissue inflammation, and it may be a unifying mechanism for the development of IR and atherosclerosis3. Nevertheless, our understanding of the factors that contribute to WAT inflammation is incomplete, and from a therapeutic perspective it remains unclear if inflammatory pathways can be manipulated for clinical benefit.

Macrophage migration inhibitory factor (MIF) is a widely expressed pro-inflammatory cytokine that participates in the development of many inflammatory disorders, including those that contribute to cardiovascular disease 1014. MIF amplifies the pro-inflammatory cascade and it controls the ‘set point’ and the magnitude of inflammatory responses (e.g. JNK1 response)10;15. In a recent study, we showed that MIF can exert chemokine-like functions thereby enhancing the tissue infiltration of macrophages during atherogenesis16.

Here, we have investigated whether genetic deletion of mif would result in a lower systemic and/or lower WAT-specific inflammation, and whether reducing MIF-dependent inflammation would prevent the development of IR, glucose intolerance and associated CVD. The LDL receptor-deficient mouse (Ldlr−/−) was chosen as a model because IR and atherosclerosis develop sequentially17 and under the mild conditions of a chow diet thereby mimicking the slow progression of disease in humans. Glucose tolerance testing and hyperinsulinemic-euglycemic clamp analysis in combination with functional, genome-wide pathway analysis and immunohistochemistry enabled us to explore for the first time the role of MIF in a chronic setting of acquired IR and atherosclerosis.

Materials and Methods

Animals and diets

All mouse lines used had a C57BL/6 background. Atherosclerosis-prone Ldlr−/− mice were crossbred with MIF-deficient mice (Mif−/−) mice18 to generate Ldlr−/−MIF−/− mice. Male littermates derived from crossbreeding of Ldlr−/−Mif+/− mice were used for the metabolic cage experiments, glucose tolerance tests, insulin tolerance tests, the hyperinsulinemic euglycemic clamp analysis, and the atherosclerosis experiments. Mice of both gender were used for the cytokine stimulation experiments.

Human CRP (huCRP) transgenic mice were challenged with IL-1β as described 19. Animal experiments were ethically approved by an independent Animal Care and Use Committee and were in compliance with European Community specifications regarding the use of laboratory animals.

Extended Material and Methods are provided in the Supplementary Information.

Results

Effect of MIF-deficiency on lipids and metabolic markers

Lldr−/− mice and Mif−/−Ldlr−/− littermates were fed a chow diet for 35 weeks and risk factors of the metabolic syndrome were monitored over time. The plasma levels of cholesterol, triglycerides and free fatty acids were comparable in both groups of mice (Table 1 shows values at t=12 and t=35 weeks). Lldr−/− and Mif−/−Ldlr−/− mice also had similar lipoprotein profiles indicating that the presence or absence of MIF does not affect the level of VLDL, LDL and HDL (Figure 1A).

Figure 1
A, Lipoprotein profiles of Ldlr−/− (open circles) and Mif−/−Ldlr−/− (solid squares) mice fed a chow diet at 35 w. B, Average fasting plasma insulin levels, C, average fasting whole blood glucose levels and ...
Table 1
Effects of MIF-deficiency on markers of cardiovascular and metabolic disease

Fasting plasma insulin concentrations at 12 weeks of age tended to be lower in Mif−/−Ldlr−/− (0.77±0.09 mM versus 1.27±0.38 mM), and this difference became significant at week 35 because insulin levels increased strongly in Ldlr−/− (4.0±0.7 mM) but only moderately in Mif−/−Ldlr−/− mice (1.9±0.2 mM; Figure 1B). A similar picture was also obtained for glucose: While fasting blood glucose levels were comparable at 12 weeks of age (8.0±1.6 mM in Ldlr−/− and 7.6±1.4 mM in Mif−/−Ldlr−/− mice), with increasing age a significant difference emerged at week 35 because glucose levels increased in Ldlr−/− mice (10.2±2.0 mM) while remaining low in Mif−/−Ldlr−/− mice (7.3±1.0 mM) (Figure 1C).

Calculation of the HOMA index as a measure of IR in week 12 and 35 revealed a strong increase in Ldlr−/− mice (from 0.3±0.1 to 1.9±0.4). In contrast, HOMA hardly increased in Mif−/−Ldlr−/− (from 0.2±0.05 to 0.6±0.02) (Figure 1D). These data show that Mif−/− Ldlr−/− mice are protected from developing hyperinsulinemia and hyperglycemia, which suggests that MIF has a role in the development of IR.

There was no difference in food intake between the groups during the treatment and Mif−/−Ldlr−/− mice had a slightly lower body weight (not significant) (Table 1). Also when the treatment was prolonged (up to 52 weeks), there was no significant effect on body weight: both groups became obese and the average body weight was 46.9±5.6 g in Ldlr−/− and 44.1±7.2 g in Mif−/−Ldlr−/−. The mass of subcutaneous, visceral and epididymal fat was also comparable in Ldlr−/− and Mif−/−Ldlr−/− mice (subcutaneous: 1.45±0.37 g vs. 1.79±0.61 g; visceral: 0.71±0.12 g vs. 0.66±0.30 g; epididymal: 1.38±0.47 g vs. 1.26±0.43 g in week 52) and plasma leptin levels were similar in the two strains (Table 1).

In an independent experiment, the metabolic performance of Ldlr−/− and Mif−/−Ldlr−/− mice was analyzed in more detail. Mice were housed individually in computerized metabolic cages with free access to water and chow. There was no significant difference in voluntary activity, food intake, water consumption, O2 consumption and CO2 production (Online Figure I). In both groups, the respiratory exchange rate (RER) varied between 0.9 (night) and 1 (day) indicating that mice predominantly used glucose in chow as an energy substrate.

MIF-deficiency lowers chronic inflammation and reduces the magnitude of the inflammatory response

Serum amyloid A (SAA) is a circulating inflammation marker produced by liver and adipose tissue. SAA levels were significantly lower in Mif−/−Ldlr−/− mice already at week 12 (Table 1), i.e. at a time point at which Ldlr−/− and Mif−/−Ldlr−/− still had comparable levels of insulin and glucose. While SAA levels strongly increased in Ldlr−/− (up to 109±14 µg/mL at 35 weeks), they remained low in Mif−/−Ldlr−/− (10±7 µg/mL). Mif−/−Ldlr−/− also displayed significantly lower levels of fibrinogen, a liver-specific marker of inflammation (Table 1). Our finding that MIF influences the inflammatory status was also confirmed in normolipidemic C57BL/6 mice. Plasma SAA and fibrinogen concentrations were 70±11 µg/mL and 3.3±1.1 mg/mL in MIF-expressing C57BL/6 whereas MIF-deficient littermates displayed significantly lower levels (7±1 µg/mL and 2.7±0.8 mg/mL, P<0.05; not shown).

Stimulation experiments with a prototypic trigger of inflammation, IL-1β, revealed that MIF also determined the magnitude of an inflammatory response. Ldlr−/− and Mif−/−Ldlr−/− were intraperitoneally challenged with IL-1β (125.000 U/25 g body weight). Plasma SAA was quantified 18 h after IL-1β injection, which is a time point for which stimulation previously had been determined to be maximal (not shown). IL-1β stimulation resulted in an inflammatory response and significantly increased plasma SAA levels in Ldlr−/− mice (Figure 2A). In Mif−/−Ldlr−/− mice however, plasma SAA remained low, even at a later time point (not shown). Reconstitution of Mif−/−Ldlr−/− mice with recombinant MIF (single i.p. injection of 10 µg of LPS-free rMIF 15 h prior to IL-1βinduction) resulted in baseline and IL-1-stimulated SAA levels that were comparable to those observed in Ldlr−/− mice (Figure 2A).

Figure 2
A, Plasma SAA levels 18 h after stimulation with recombinant IL-1β (100.000 U/mouse). Plasma SAA levels started to increase 8 h after stimulation and returned to baseline after 30 h (not shown). In Mif−/−Ldlr−/− ...

The expression of human CRP, which is a sensitive marker of chronic inflammation and a predictor of metabolic and cardiovascular disease, was induced by recombinant MIF (rMIF) as shown in Figure 2B: Mice transgenic for human CRP (CRPtg) responded to rMIF (10 µg; i.p.) with a significant increase (2.6-fold) in plasma CRP concentrations. The effect of MIF on CRP was time- and dose-dependent, and maximal 18 h after stimulation (not shown). MIF was less potent than IL-1β (9-fold increase of CRP, not shown), which is a well-established stimulator of CRP in this model. Protein mutants of MIF, i.e. C60S-MIF or P2A-MIF, which lack the intrinsic catalytic activity of MIF and have been found to also lack inflammatory activities20;21 did not stimulate CRP expression in CRPtg mice, thereby confirming that the effect of MIF on CRP was specific (Figure 2B). Consistent with this notion, rMIF but not the mutant proteins stimulated human CRP promoter activity in human HuH7 hepatoma cells transiently transfected with a plasmid containing a 300 bp fragment of the human CRP promoter cloned in front of a luciferase reporter gene. The CRP promoter-activating effect of MIF alone was about 2-fold (not shown) and additive to the stimulating effect of IL-1 (Online Figure II). Also, MIF stimulated the basal and IL-1-induced activity of the promoter of IL-6, the principle cytokine inducer of CRP (Online Figure II).

MIF-deficiency protects against the development of glucose intolerance and IR

To examine whether a reduction in chronic, low-grade inflammation by deleting mif would affect the development of IR, we subjected Ldlr−/− and Mif−/−Ldlr−/− mice (12 weeks of age) to glucose tolerance and insulin tolerance tests. In the presence of MIF, peak glucose levels normalized later and Ldlr−/− mice had a significantly higher AUC than Ldlr−/−Mif−/− (AUC: 817±353 vs. 507±174; P<0.05 Figure 3A). The difference in glucose tolerance became even more pronounced at later time points. At 35 weeks, the AUC was 1225±397 in Ldlr−/− but stayed at 519±194 in Ldlr−/−Mif−/−; P<0.001 (Figure 3B). Insulin levels did not differ significantly during glucose tolerance testing (not shown).

Figure 3
A, Glucose tolerance test in 12 w-old Ldlr−/− (open) and Mif−/−Ldlr−/− (solid) mice (2 g glucose per kg body weight; i.p.). Blood glucose levels were monitored at time points indicated. B, as A but 35 w ...

Subsequent insulin tolerance tests revealed that the clearance of plasma glucose occurred more efficiently (i.e. more rapidly within the first 15 min; P<0.05) in Ldlr−/−Mif−/− mice, (Figure 3C), which suggests that a difference in insulin sensitivity may exist. In line with this notion, hyperinsulinemic-euglycemic clamp analysis showed that the glucose infusion rate in Ldlr−/−Mif−/− mice was greater than in Ldlr−/− (14.0±3.4 vs 8.6±3.7 mg/kg.min; P<0.05) indicating that the presence of MIF may promote the development of IR (Figure 3D).

MIF-deficiency reduces the inflammatory state of WAT

Because the circulating levels of inflammatory markers were lower in Mif−/−Ldlr−/− mice, we analyzed the inflammatory status of liver and WAT. Western blot analysis of tissue homogenates showed that MIF is expressed in liver and WAT of Ldlr−/− mice (Figure 4A). A parallel IHC analysis demonstrated MIF immunoreactivity in all cell types present in these tissues (not shown).

Figure 4Figure 4
A, Western blot analysis of Mif in tissue homogenates of Ldlr−/− and Mif−/− Ldlr−/− mice (25–35 w). B, Computerized quantification of adipocyte size in Ldlr−/− (black bars) and Mif−/−Ldlr−/− ...

The liver tissue of Ldlr−/− mice showed slight c-Jun immunoreactivity but there was no significant difference in either c-Jun or p-c-Jun immunoreactivity when these mice were compared to Mif−/−Ldlr−/− littermates (not shown). The expression of hepatic genes encoding enzymes that control glucose homeostasis/de novo synthesis (phosphoenolpyruvate carboxykinase, glucose-6-phosphatase) also was comparable in both groups (not shown).

Histological analysis of WAT revealed smaller adipocytes in Mif−/−Ldlr−/− mice when compared to Ldlr−/−. This difference was observed already at young (12–18 w) age, i.e. prior to the infiltration of macrophages into WAT. Computerized quantification of adipocyte size demonstrated a significant difference in young (Online Figure III) and old (25–35 w) animals (Figure 4B).

Specific immunostaining of macrophages (anti-MAC3) showed that the WAT of 25–35 weeks old Mif−/−Ldlr−/− contained significantly fewer macrophages and fewer crown-like structures than the WAT of Ldlr−/− (Figure 4C and Online Figure IV). Analysis of the expression of genes that are characteristic for M1-type (CXCR4; CCR2) and M2-type (Ly-6C; Mrc1) macrophage responses in WAT by Affymetric microarray (Online Table I) revealed that the M1/M2 ratio tended to be lower in Mif−/−Ldlr−/− mice (not shown). In an independent RT-PCR analysis (n=6) we found that the CCR2/Ly-6C mRNA expression ratio is 54% (P<0.05) lower in Mif−/−Ldlr−/−. Plasma adiponectin levels were comparable in Mif−/−Ldlr−/− (10.4±1.8 µg/mL) and Ldlr−/− mice (11.2±2.6 µg/mL).

In Ldlr−/− mice, pronounced c-Jun immunoreactivity (Figure 4D) was observed in MAC3- positive areas as well as in adipocytes. Merging the immunofluorescent signals of c-Jun and nuclear DAPI revealed that a substantial amount of c-Jun was associated to the nucleus (Figure 4E–G). Nuclear c-Jun immunoreactivity was predominantly found in adipocytes that were in close proximity to macrophages/crown-like structures but such staining was less evident in more distant adipocytes. In WAT from Ldlr−/−Mif−/− mice, c-Jun immunoreactivity was less intense and mainly cytosolic.

To study a role of MIF in the inflammatory responsiveness of macrophages, thioglycollate-elicited macrophages were isolated from wild-type and MIF−/− mice and cells were stimulated with LPS. Supernatants were harvested after 6 h and 12 h, and assayed for IL-6 by ELISA. MIF-deficient macrophages showed a significantly impaired responsiveness as demonstrated by significantly less IL-6 production compared to wild-type macrophages (Online Figure V).

A comparison of WAT from Mif−/−Ldlr−/− and Ldlr−/− by functional microarray analysis across pathways showed that the major changes in gene expression occur in the functional categories ‘cell signaling’, ‘cell cycle control’, ‘immune response’ and ‘lipid metabolism’. Within the category ‘cell signaling’, the insulin-sensitive processes ‘leptin signaling via JAK/STAT and MAPK cascades’ and ‘IGF-R1 signaling’ were differentially affected (Online Table I). These data, considered together with the IHC analysis of c-Jun, suggest an effect of MIF on insulin sensitivity.

As a direct test of the influence of MIF on the insulin signaling cascade, we injected insulin (i.p.; 0.5 U insulin/25 g body weight) into Ldlr−/− and Mif−/−Ldlr−/− littermates. Mice were sacrificed 10 min after injection (insulin dose and time point of sacrifice had been optimized in previous scouting experiments) and the IRS1-associated PI3-kinase activity was determined as a functional measure of the insulin signaling route. PI3-kinase activity was significantly higher in WAT of Mif−/−Ldlr−/− mice when compared to Ldlr−/− mice (Figure 5A). The biological relevance of this effect was supported further by the higher levels of phospho-AKT, a downstream effector of PI3-kinase, in WAT of Mif−/−Ldlr−/− mice (Figure 5B). No difference in PI3-kinase activity and phospho-AKT levels in liver and muscle were observed (Online Figure VI).

Figure 5
A, PI3-kinase activity in WAT from 25–35 w old Ldlr−/− and Ldlr−/−Mif−/− mice sacrificed precisely 10 min after treatment with 0.5 U insulin per 25 g body weight. B, p-AKT levels in WAT of Ldlr−/− ...

MIF-deficiency blocks macrophage infiltration into WAT

Pathway analysis of the WAT transcriptome dataset showed that the presence of MIF was significantly associated with the inflammatory processes ‘IL-1 and IL-6 signaling’, ‘ERK activation’, ‘IL-3 activation and signaling’ and ‘cell adhesion’ (all P<0.05; Online Table I). Consistent with the enhanced inflammatory status of MIF-expressing Ldlr−/− mice the genes encoding for chemokines (Ccl2, Ccl9, Ccr5, Ccl6), proteases (Mmp12), complement components (C1qb, C1qa, C3ar3, C3ar1), acute phase proteins (Mup-1, Orm2, SAA3), cell adhesion/immune cell recruitment factors (Cd9, Cd44, Cd84, Cd72) also were significantly (P<0.01) upregulated, suggesting that MIF promotes the recruitment of immune cells into adipose tissue. To substantiate this observation, we measured the expression level of proteins that mediate monocyte/macrophage recruitment into WAT. Circulating levels of VCAM-1 and ICAM-1 were significantly lower in Mif−/− Ldlr−/− mice compared to Ldlr−/− (Figure 6A,B). IHC staining of the cell adhesion molecules ICAM and CD44 in cross-sections of WAT showed pronounced ICAM- and CD44 levels in Ldlr−/− mice. CD44 was predominantly detected in MAC3-positive cells of crown-like structures. ICAM-1 and CD44 staining was markedly and significantly reduced in Mif−/−Ldlr−/− mice (Figure 6C, D and Online Figure IV) providing a molecular rationale for the lower macrophage content in WAT.

Figure 6
Plasma levels of A, ICAM-1 and B, VCAM-1 in 25–35 w old Ldlr−/− and Mif−/− Ldlr−/− mice. Data are means±SD (n≥8 per genotype); *P<0.05. C, Representative photomicrographs ...

MIF-deficiency reduces atherosclerosis associated with IR

Atherosclerosis developed after glucose intolerance/IR and was analyzed in mice maintained on a chow diet for 52 weeks. The aortic plaque load (determined by en face Oil-Red O-staining) of Mif−/−Ldlr−/− mice was lower than in Ldlr−/− (Figure 7A). Mif−/−Ldlr−/− also displayed significantly less atherosclerosis in the aortic valve area of the aortic root (Figure 7B). Analysis of the lesional content of monocytes/macrophages in cross-sections of the aortic root demonstrated a significant reduction in the numbers of these cells in Ldlr−/−Mif−/− mice (reduced by 5.1-fold P<0.05; Figure 7C and data not shown)

Figure 7
A, Representative photomicrographs of longitudinally opened en face Oil-red O-stained aortas from Ldlr−/− and Mif−/−Ldlr−/− mice (52 w of age). B, Analysis of atherosclerosis and C, monocyte adhesion in ...

These data are consistent with the observed effects of MIF in WAT, and they support the conclusion that MIF-deficiency impairs the accumulation of monocytes/macrophages in the vascular wall, which is a fundamental, pathologic feature necessary for the development of atherosclerosis.

Discussion

MIF plays pivotal roles in inflammatory diseases and atherogenesis13;14 but it has remained unclear whether MIF is causally involved in the development of metabolic disorders associated with obesity and the metabolic syndrome. We show herein that genetic deletion of MIF blocks the development of glucose intolerance, IR and associated atherosclerotic disease. Importantly, MIF-deficiency reduces macrophage infiltration into WAT and lowers both tissue-specific and systemic chronic inflammation without affecting obesity (adiposity) and lipid risk factors. The data indicate that the adipocyte and the macrophage are of importance to the effects observed by MIF-deficiency. To our knowledge, the present study provides the first experimental evidence for the direct involvement of MIF in the evolution of IR/glucose intolerance and it is consistent with previous reports showing that MIF is a key element in atherogenesis13;14. Our observation that MIF-deficiency reduces WAT inflammation and selectively improves the insulin sensitivity of this tissue is consistent with the finding that glucose uptake into WAT is increased in Mif−/− mice under conditions of severe inflammation (LPS-induced endotoxemia) with glucose uptake of skeletal muscle and hepatic glucose production being unaffected22.

Chronic low-grade inflammation is considered to be an important risk factor of metabolic and cardiovascular diseases but it is unclear how it can be manipulated without severe consequences to the organism23. Metabolic and immune response pathways are evolutionarily linked and therefore modulation of inflammatory risk factors often affects metabolic risk factors and vice versa7;24. For example, deletion of inflammatory cytokines such as IL-1α, IL-6, IL-18 and TNFα can result in a significant increase in plasma cholesterol24. Here, we show that MIF-deficiency lowers the inflammatory reactivity without affecting typical metabolic risk factors including plasma triglycerides, free fatty acids, VLDL, LDL, HDL, body weight, adipose mass, voluntary activity and metabolic performance. Compared to Ldlr−/− mice, Mif−/−Ldlr−/− mice display lower levels of systemic (SAA, fibrinogen) and vascular (ICAM-1, VCAM-1) inflammation markers, and their WAT contains less macrophages, nucleus-associated c-Jun, ICAM-1 and CD44. One reason for the observed selective reduction in inflammation may be that MIF does not participate in the interface that links metabolic to inflammatory pathways (‘metaflammation’ pathways23) and that MIF’s role within the inflammatory cascade is mainly to amplify and enhance existing inflammatory signals. This amplifier function may explain the large differences in local WAT-specific inflammation observed in this study. Adipose tissue is considered to be an important site for the production of inflammatory mediators 1;23, and it is possible that the lower WAT inflammation observed in the setting of MIF-deficiency is due to both a lower systemic inflammatory response and to a loss of amplifying signals for cytokine (IL-6) and acute phase (CRP, SAA, fibrinogen) responses in the liver. This also illustrates that difference in inflammatory status in Mif−/−Ldlr−/− and Ldlr−/− can either be primary (i.e. a direct effect of MIF), or secondary (i.e. a consequence of a MIF effect on another factor).

The specific effect of MIF-deficiency on the inflammatory state (but not on lipid/metabolic risk factors) enabled us to study the consequences of a prolonged, selective reduction of inflammation. Our results clearly demonstrate that lowering chronic inflammation per se is an effective strategy to block the development of metabolic as well as cardiovascular disease. Genetic deletion of MIF thus produces a different phenotype than that resulting from genetic deficiency of CCR2, which encodes a high-affinity ligand of CCL2/MCP-1 that also regulates macrophage infiltration into WAT in the context of IR25. Genetic deficiency in CCR2 reduces food intake and adiposity thereby attenuating the development of obesity. Our data show that WAT inflammation and the development of IR can be reduced significantly without affecting the development of obesity.

Recent epidemiological data provide support for a role for MIF in the development of IR in humans. Herder and coworkers reported a strong positive association between systemic concentrations of MIF and impaired glucose tolerance and T2D26. They also showed that the MIF genotype rs1007888CC is associated with increased circulating MIF levels and an increased T2D risk27. Interestingly, in male participants, MIF levels were significantly associated with high CRP and IL-6 levels. We found that MIF-expressing mice (independent of the Ldlr−/− background) display higher levels of fibrinogen, an IL-6-inducible liver-derived acute phase protein (APP), and SAA, which can be viewed as the murine counterpart of CRP. We also demonstrate that MIF is involved in the constitutive and IL-1-induced expression of SAA. This effect and the finding that MIF stimulates the expression of human CRP in vivo as shown in human CRP transgenic mice have not been reported so far.

The role of MIF in the regulation of acute phase genes (e.g. CRP, SAA, fibrinogen) has not been analyzed systematically. A positive effect of MIF on APP is of great importance because these proteins are not only powerful predictors of disease but also participate in pathophysiological processes leading to the formation of atherosclerotic lesions8. Possible sources of pro-atherogenic APP production are WAT and liver. It is well established that very powerful cytokine inducers of hepatic APP expression are IL-1β and IL-69;28, both of which are increasingly expressed during ageing in mouse WAT9. It is thus possible that the observed differences in APP levels are a consequence of a local effect of MIF in WAT (e.g. on IL-6 release by this tissue). However, from a mechanistic point of view, the positive association between MIF and APP plasma levels remain unclear because studies that could provide a molecular rationale are lacking. A possible explanation may be that MIF controls a transcription factor shared by the various APP. We have shown that immunoneutralization of MIF lowers plasma fibrinogen and IL-6 levels and reduces the expression level of C/EBPβ, a common transcription factor29. C/EBPβ is not only a positive regulator of IL-6, fibrinogen, SAA and CRP but also a transcription factor for the adhesion molecules VCAM-1 and ICAM-130, each of which was affected in this study in a MIF-dependent way.

Taken together, our observations support the overall physiological importance of chronic inflammation in the pathogenesis of IR and associated atherosclerosis. Given that the metabolic parameters studied (e.g. triglycerides, free fatty acids, VLDL, LDL, HDL, body weight and adiposity) were unchanged in Ldlr−/− and Mif−/−Ldlr−/− mice, MIF may represent a unique therapeutic target for the specific reduction of WAT inflammation and the ensuing development of cardiovascular and metabolic diseases.

Acknowledgements

We thank Annie Jie and Karin Toet for excellent bioinformatical and analytical help.

Sources of funding

This study was supported by the Dutch Organization for Scientific Research (NOW-Zon-MW; VENI-016.036.061 to R.K.; VENI-916-36-071 and VIDI-917.76.301 to P.J.V), the Dutch-Heart-Foundation (grant 2002B102 to L.V.), the TNO research-program Personalized-Health (to R.K., M.E., T.K.), the Nutrigenomics Consortium, the Centre for Medical Systems Biology (grant-115) in the framework of the Netherlands-Genomics-Initiative (to J.d.V.v.d.W. and K.W.v.D), the US National Institutes of Health (R.B.), the German-Research-Council (DFG) (SFB 542/TP-A7 and DFG-FOR809/P1 to J.B.;Fi712/2-1 to G.FR.), the Cologne-Fortune-Program of the Medical Faculty of Cologne University (to G.FR.) and from the European Commission (grant COST-BM0602 to D.M.O.)

Footnotes

Disclosures: None.

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