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. The proinflammatory cytokine MIF (macrophage migration inhibitory factor) 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 weeks, whereas 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 serum amyloid A and fibrinogen levels at baseline and under inflammatory conditions. Conversely, MIF stimulates the in vivo expression of human C-reactive protein, 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 (intercellular adhesion molecule-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.

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 D, HOMA values of Ldlr−/− (open) and Mif−/−Ldlr−/− (solid) mice at the age of 12 and 35 w. Data are expressed as means±SD (n≥8 per genotype); *P<0.05.

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−/− mice, MIF was reconstituted by i.p. administration of recombinant human MIF protein (rhMIF; 10 µg free of LPS (<1pg endotoxin/µg rhMIF protein as assessed by Limulus Amoebocyte Lysate assay). B, Human CRP levels in human CRP transgenic mice 18 h after stimulation with either rhMIF or P2 A-rhMIF lacking tautomerase activity or C60S-rhMIF lacking oxidoreductase activity (all 10 µg; i.p.). Data are means±SD (n≥9 per treatment group); *P<0.05.

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 old mice. C, Insulin tolerance test in 12 w-old Ldlr−/− (open circles) and Mif−/−Ldlr−/− (solid squares) mice. Animals were treated with insulin (0.5 U per 25 g body weight; i.p.). D, Assessment of whole body insulin resistance by euglycemic hyperinsulinemic clamp analysis at 12 w of age. Glucose infusion rate (GIR) is shown. Data are means±SD (n≥10 per genotype); *P<0.05.

Figure 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−/− (red bars) mice. Adipocytes were classified into groups on basis of their size (x-axis). An identical total area was analyzed by the software. The peak size of adipocytes in Ldlr−/− was 1500 to 1800 µm², whereas in Mif−/−Ldlr−/− mice it was only 600 to 900 µm². Data shown are means±SD (n≥8); *P<0.05. C, Representative photomicrographs of epididymal adipose tissue from Ldlr−/− and Mif−/− Ldlr−/− mice (n≥7 per genotype; 4 cross-sections per mouse) stained with a macrophage-specific antibody (MAC-3) or D, a c-Jun specific antibody detected with Nova Red or E, a c-Jun specific antibody detected with fluorescent-labelled secondary antibody or F, with DAPI (for nuclei); G, Merged photomicrographs showing that c-Jun-immunoreactivity (IR) is nuclear.

Figure 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−/− (red bars) mice. Adipocytes were classified into groups on basis of their size (x-axis). An identical total area was analyzed by the software. The peak size of adipocytes in Ldlr−/− was 1500 to 1800 µm², whereas in Mif−/−Ldlr−/− mice it was only 600 to 900 µm². Data shown are means±SD (n≥8); *P<0.05. C, Representative photomicrographs of epididymal adipose tissue from Ldlr−/− and Mif−/− Ldlr−/− mice (n≥7 per genotype; 4 cross-sections per mouse) stained with a macrophage-specific antibody (MAC-3) or D, a c-Jun specific antibody detected with Nova Red or E, a c-Jun specific antibody detected with fluorescent-labelled secondary antibody or F, with DAPI (for nuclei); G, Merged photomicrographs showing that c-Jun-immunoreactivity (IR) is nuclear.

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−/− and Ldlr−/−Mif−/− mice assessed by Western blotting (a representative blot is shown). Data are means±SD (n≥7 per genotype); *P<0.05.

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 of epididymal adipose tissue from Ldlr−/− and Mif−/−Ldlr−/− mice (n≥7 each) stained with an antibody specific for ICAM-1 or D, an antibody specific for CD44.

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 the aortic valve area of the aortic root. Data are means±SD (n=7 per genotype);*P<0.05.