Macrophage Migration Inhibitory Factor Deficiency Ameliorates High-Fat Diet Induced Insulin Resistance in Mice with Reduced Adipose Inflammation and Hepatic Steatosis

<div><p>Macrophage infiltration is a critical determinant of high-fat diet induced adipose tissue inflammation and insulin resistance. The precise mechanisms underpinning the initiation of macrophage recruitment and activation are unclear. Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, displays chemokine-like properties. Circulating MIF levels are elevated during obesity however its role in high-fat diet induced adipose inflammation and insulin resistance remains elusive. Wildtype and MIF^−/− C57Bl\6J mice were fed chow or high-fat diet. Body weight and food intake was assessed. Glucose homeostasis was monitored by glucose and insulin tolerance tests. Adipose tissue macrophage recruitment and adipose tissue insulin sensitivity was evaluated. Cytokine secretion from stromal vascular fraction, adipose explants and bone marrow macrophages was measured. Inflammatory signature and insulin sensitivity of 3T3-L1-adipocytes co-cultured with wildtype and MIF^−/− macrophage was quantified. Hepatic triacylglyceride levels were assessed. MIF^−/− exhibited reduced weight gain. Age and weight-matched obese MIF^−/− mice exhibited improved glucose homeostasis coincident with reduced adipose tissue M1 macrophage infiltration. Obese MIF^−/− stromal vascular fraction secreted less TNFα and greater IL-10 compared to wildtype. Activation of JNK was impaired in obese MIF^−/−adipose, concomitant with pAKT expression. 3T3-L1-adipocytes cultured with MIF^−/− macrophages had reduced pro-inflammatory cytokine secretion and improved insulin sensitivity, effects which were also attained with MIF inhibitor ISO-1. MIF^−/− liver exhibited reduced hepatic triacyglyceride accumulation, enhanced pAKT expression and reduced NFκB activation. MIF deficiency partially protects from high-fat diet induced insulin resistance by attenuating macrophage infiltration, ameliorating adipose inflammation, which improved adipocyte insulin resistance <i>ex vivo</i>. MIF represents a potential therapeutic target for treatment of high-fat diet induced insulin resistance.</p></div

MIF^−/− macrophages have altered adipocyte-macrophage crosstalk compared to WT macrophages, while ISO-1 blocks MIFs insulin desensitizing capacity in adipocytes.

<p>(a) Chronic treatment of 3T3-L1 adipocytes with rMIF (100 ng) and its effect on insulin (100 nM)-stimulated ³H-glucose uptake was evaluated. Data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t untreated +Insulin. (b) The effect of WT and MIF^−/− BMM on insulin (100 nM)-stimulated ³H-glucose transport into 3T3-L1-adipocytes was evaluated. Fold increase in ³H-glucose transport into adipocytes in response to insulin over basal (non-insulin-stimulated) is presented (white bars =  -insulin, black bars =  + insulin). Data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t adipocytes co-cultured with WT BMM+insulin. The effect of BMM co-culture on adipocyte on (c) <i>Glut-4</i> and (d) <i>Irs-1</i> mRNA expression was determined by real-time PCR, n = 4/group. (e) Immunoblot analysis of phosphorylated AKT, whole-cell AKT and β-actin in co-cultured adipocytes stimulated with insulin (100 nM). Levels of (f) TNFα (g) IL-6, were measured in media from co-cultured cells. N = 4/group, data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t adipocytes co-cultured with WT BMM. (h) The effect of unstimulated J774.2 CM, MIF (100 ng/ml)-stimulated (CM+M), ISO-1 (50 µMl) treated (CM+I) and cells pretreated with ISO-1 (50 µM) 1 hour prior MIF-stimulation (CM+M+I) on insulin (100 nM)-stimulated ³H-glucose transport into 3T3-L1adipocytes was evaluated and expressed in DPM. N = 4/group, data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t adipocytes treated with (CM+M), #p&lt;0.05, ##p&lt;0.01 and ###p&lt;0.001 w.r.t control.</p

Obese MIF^−/− mice exhibit reduced adipose tissue inflammation and improved adipose tissue insulin sensitivity compared to obese WT mice.

<p>Levels of pro-inflammatory (a) TNFα and (b) IL-1β secretion into media from adipose tissue explants was measured by ELISA. Lean = 17, obese n = 17. Gene expression analysis of (c) <i>Tnfα</i>, (d) <i>Il-1β</i> in lean and obese adipose tissue from WT and MIF^−/− mice. Lean = 5, obese n = 5. (e) Immunoblot analysis of phosphorylated JNK and control β-actin and corresponding densitometry analysis expressed in arbitrary units (AU) Lean = 3, obese n = 3. (f). <i>Ex vivo</i> insulin (100 nM)-stimulated ³H-glucose transport into whole adipose tissue (50 mg) harvested from obese WT and MIF^−/− mice was evaluated. Fold increase in ³H-glucose transport into adipose in response to insulin over basal (non-insulin-stimulated) is presented (white bars =  −insulin, black bars =  + insulin) n = 6. Gene expression analysis of (g) <i>Glut-4</i> in adipose tissue from lean and obese WT and MIF^−/− mice. Lean = 5, obese n = 5. (h) Immunoblot analysis of phosphorylated AKT levels and control β-actin. Lean = 3, obese n = 3. WT mice represented by white bars, MIF^−/− mice represented by black bars in all graphs. Data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t obese WT.</p

MIF^−/− bone marrow macrophages have reduced inflammatory signature compared to WT mice.

<p>Levels of pro-inflammatory (a) IL-6, (b) IL-1β (c) MCP-1 secretion into media from lean WT and MIF^−/− BMM stimulated ±LPS. (d) Gene expression analysis of <i>Il-6</i> in WT and MIF^−/− BMM±LPS, n = 5/group. (e) Immunoblot analysis of phosphorylated and whole cell ERK, JNK, p38, NFkB and β-actin stimulated ±LPS, n = 3/group. WT mice represented by white bars, MIF^−/− mice represented by black bars in all graphs. Data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t WT+LPS.</p

Lack of MIF improves HFD-induced hepatic steatosis and hepatic insulin sensitivity.

<p>Liver tissue was harvested from lean and obese WT and MIF^−/− mice. (a) Weight of liver tissue expressed as a percentage of total body weight. Lean = 4, obese = 4. (b) Fasting plasma ALT levels in lean and obese mice. (c) Hepatic triacylglyceride levels, lean = 4, obese = 4. (d) Hematoxylin &amp; eosin staining to visualize hepatic lipid accumulation (representative of n = 3 images per group). (e) Gene expression analysis of markers of lipogenesis and lipid storage as determined by RT-PCR. Lean = 4, obese = 4. (f) Immunoblot analysis of phosphorylated AKT and β-actin from livers of WT and MIF^−/− mice ± insulin. Lean = 3, obese = 3. (g) Unstimulated tissue phosphorylated-NFkB were determined by immunoblot analysis. Lean = 3, obese = 3. WT mice represented by white bars, MIF^−/− mice represented by black bars in all graphs. Data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t obese WT.</p

MIF deficiency partially protects from HFD-induced obesity and improves glucose homeostasis.

<p>(a) GTT (1.5 g glucose/kilogram (kg) body weight (BW)) in fasted lean and obese WT and MIF^−/− mice (white circles = WT lean; black circles =  MIF^−/− lean; white squares = WT obese; black squares = MIF^−/− obese). Lean n = 9, obese n = 9. (b) ITT (0.75 U insulin/kg BW) in fasted lean and obese WT and MIF^−/− mice (white circles = WT lean; black circles = MIF^−/− lean; white squares = WT obese; black squares = MIF^−/− obese). Lean n = 18, obese n = 18–33. (c&amp;d) AUC for WT (white bars) and MIF^−/− (black bars) mice over the course of GTT and ITT expressed as arbitrary units (AU). Lean n = 9, obese n = 18–33. (e) Plasma insulin levels over time in response to glucose challenge (white circles = WT lean; black circles = MIF−/− lean; white squares = WT obese; black squares = MIF−/− obese). Lean n = 9, obese n = 9. (f) Accumulative weight of WT (white circles) and MIF^−/− (black circles) mice. (g) Accumulative food intake of WT (white circles) and MIF^−/− (black circles). (h) Body mass composition (white bars = lean fat mass; black bars = fat fat mass). Lean n = 3–4, obese n = 8–9. Data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t obese WT.</p

MIF mRNA is elevated in adipose tissue of obese WT mice, while adipose tissue lack of MIF reduced ATM recruitment into adipose tissue following HFD.

<p>(a) Immunoblot analysis of MIF and control β-actin in adipose of lean and obese WT mice (n = 4). (b) Densitometry analysis quantifying phosphorylated MIF protein expression relative to β-actin expressed in arbitrary units (AU) in lean (white bars) and obese (black bars) mice. (c) Gene expression analysis determined by RT-PCR of <i>Mif</i> in the epididymal adipose tissue (EAT), visceral adipose tissue (VAT), stromal vascular fraction (SVF), adipocyte fraction and liver of lean (white bars) and obese (black bars) WT mice. Lean n = 3–6, obese n = 4–7. (c) Gene expression analysis of known MIF receptors <i>Cxcr4, Cxcr2, Cd74</i> in EAT of lean (white bars) and obese (black bars) mice, lean = 4–8, obese n = 3–5. (d) Recruitment of ATM into adipose tissue: cells triple positive (F4/80⁺/CD11B⁺/CD11C⁺) were classified as M1 macrophages. Cells double positive (F4/80⁺/CD11B⁺/CD11C⁻) were classified as M2 macrophages. (e) Recruitment of T-cells into adipose tissue: Cells double positive (CD3⁺/CD4⁻/CD8⁺) were classified as cytotoxic T-cells. Cells double positive (CD3⁺/CD4⁺/CD8⁻) were classified as helper T-Cells. Recruitment of cells is presented as percentage of total SVF cells. Lean WT (white bars) = 9, lean MIF^−/− (grey bars) = 9, obese WT (dark grey bars) n = 15–17, obese MIF^−/− (black bars) n = 15–17. (f) TNFα and (g) IL-10 cytokine secretion from SVF and adipocyte fraction from lean and obese WT (white bars) and MIF^−/− (black bars). Lean = 12, obese n = 12. Data are mean ± SEM, *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 w.r.t obese WT.</p

Plasma metabolic profile and adipose tissue depot weights.

<p>Plasma was isolated from overnight fasted WT and MIF^−/− mice by cardiac puncture and metabolic markers were analyzed enzymatically. (*p&lt;0.05, **p&lt;0.01 w.r.t. WT obese; #p&lt;0.05, ##p&lt;0.01 and ###p&lt;0.001 w.r.t. w.r.t. respective lean).</p><p>Plasma metabolic profile and adipose tissue depot weights.</p