Insulin Resistance is Associated with MCP1-Mediated Macrophage Accumulation in Skeletal Muscle in Mice and Humans

<div><p>Inflammation is now recognized as a major factor contributing to type 2 diabetes (T2D). However, while the mechanisms and consequences associated with white adipose tissue inflammation are well described, very little is known concerning the situation in skeletal muscle. The aim of this study was to investigate, <i>in vitro</i> and <i>in vivo</i>, how skeletal muscle inflammation develops and how in turn it modulates local and systemic insulin sensitivity in different mice models of T2D and in humans, focusing on the role of the chemokine MCP1. Here, we found that skeletal muscle inflammation and macrophage markers are increased and associated with insulin resistance in mice models and humans. In addition, we demonstrated that intra-muscular TNFα expression is exclusively restricted to the population of intramuscular leukocytes and that the chemokine MCP1 was associated with skeletal muscle inflammatory markers in these models. Furthermore, we demonstrated that exposure of C2C12 myotubes to palmitate elevated the production of the chemokine MCP1 and that the muscle-specific overexpression of MCP1 in transgenic mice induced the local recruitment of macrophages and altered local insulin sensitivity. Overall our study demonstrates that skeletal muscle inflammation is clearly increased in the context of T2D in each one of the models we investigated, which is likely consecutive to the lipotoxic environment generated by peripheral insulin resistance, further increasing MCP1 expression in muscle. Consequently, our results suggest that MCP1-mediated skeletal muscle macrophages recruitment plays a role in the etiology of T2D.</p></div

Inflammation is associated with insulin sensitivity in skeletal muscle of diet-induced obese and diabetic mice.

<p>A–B) Glucose (A) and insulin (B) tolerance tests were performed on mice on fed a standard diet, a high fat diet (45% Kcal) for 16 weeks in absence or presence of rosiglitazone (200 mg/kg diet) for the last four weeks. Both tests were performed at week 15. C) <i>Ex-vivo</i> insulin signalling assay were performed on freshly isolated gastrocnemius. D–G) qPCR analyses of CD68 (D), CD11c (E), MCP1 (F) and CCR2 (G) expressions were performed on quadriceps of mice. * stands for <i>P</i><0.05 and ** for <i>P</i><0.01; n = 5.</p

Macrophage recruitment in skeletal muscle of ob/ob mice.

<p>A) Representative images (left panel) and quantitative analysis (right panel) of the immunostaining with F4/80 antibody in gastrocnemius of wild-type (wt) and ob/ob mice. B–D) Following digestion of skeletal muscles (gastrocnemius and quadriceps), CD45+ and CD45- cells were purified and qPCR were performed on the indicated genes in wt and ob/ob mice. The different histograms regroup the genes for the validation of cell fractionation (B), the genes more expressed in CD45+ cells (C) or the genes either more expressed in CD45- cells or similarly expressed in both fractions (D).* stands for <i>P</i><0.05 and ** for <i>P</i><0.01 for the comparison between CD45- and CD45+ fractions, and † stands for <i>P</i><0.05 and †† for <i>P</i><0.01 for the comparison between WT and ob/ob mice; n = 5.</p

Palmitate induces macrophages recruitment by C₂C₁₂ muscle cells.

<p>A) C₂C₁₂ differentiated myotubes were exposed to palmitate (pal, 500 µM) or vehicle (BSA) for 24 hours. MCP1, RANTES and CX3CL1 expression were subsequently determined by real-time PCR. B) Conditioned media from C₂C₁₂ cells treated as indicated in A) were collected and used for chemotaxis assay of Raw264.7 macrophages. Histogram represents the average number of Raw264.7 cells having performed chemotaxis. C) Wild-type C57Bl6 males were either fed a normal chow diet (NCD, low fat) or the same diet enriched with specific lipids sources (20% w/w. Sunflower (Sun), Rapeseed (Rap) and palm oil (Pal). CD11c and CD68 expression in gastrocnemius were subsequently determined by real-time PCR. D) F4/80 immunostaining was performed on muscle sections of mice either fed a normal chow diet, a Sunflower or a Palmitate enriched Diet. Macrophages quantification is shown in the lower right hand panel. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110653#pone-0110653-g004" target="_blank">figure 4A</a>, ** stands for <i>P</i><0.01 in palmitate vs vehicle condition. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110653#pone-0110653-g004" target="_blank">figure 4C</a>, * stands for <i>P</i><0.05 and ‡ for <i>P</i><0.01 for the respective comparison of CD68 and CD11c between Pal and NCD. NS, not significant; n = 5.</p

Skeletal muscle overexpression of MCP1 induces local inflammation and alters insulin signaling and glucose metabolism <i>in vivo</i>.

<p>A) QPCR analyses of MCP1, CD68, CD11c, TNFα, IL1β, RANTES and CXCL1 expression were performed on quadriceps. C–D) <i>Ex-vivo</i> insulin signaling assay were performed on freshly isolated gastrocnemius. The histogram (D) represents the fold response to insulin on Akt phosphroylation in Wild-Type (WT) and MCK-MCP1-Tg mice. E) Genomic mitochondrial DNA was evaluated by qPCR in skeletal muscle of WT and transgenic mice. * stands for <i>P</i><0.05 and ** for <i>P</i><0.01; n = 6.</p

Metabolic characteristics of lean, obese and type 2 diabetic patients (n = 8–10) in fasting state.

<p>** p<0.01 obese Vs control; p<0.01 NIDM Vs Control, ¥p<0.01 obese Vs NIDM; NS: Not Significant. (NIDM, Non Insulin-dependent Diabetes Mellitus).</p><p>Metabolic characteristics of lean, obese and type 2 diabetic patients (n = 8–10) in fasting state.</p

Skeletal muscle inflammation is increased in ob/ob mice.

<p>Epidydimal fat (eWAT), quadriceps (quads) and gastrocnemius (gastrocs) of wild –type (wt) and ob/ob mice were collected prior to analysis with qPCR of CD68 (A), CD11c (B), TNFα (C) and MCP1 D) qPCR analysis was performed for F4/80, IL1β, RANTES, TLR4, Foxp3, CD4 and CD8a in quadriceps from wt and ob/ob mice. E) qPCR analysis was performed for CX3CL1, CX3CR1 and IL10 in wt and ob/ob mice quadriceps. F–G) TNFα (E) and MCP1 (F) protein concentration in gastrocnemius of wt and ob/ob mice were quantified by ELISA. * stands for <i>P</i><0.05 and ** for <i>P</i><0.01; n = 5.</p

Inflammation markers are increased in skeletal muscles of type 2 diabetic patients and correlate with HOMA-IR.

<p>Biopsies from vastus lateralis of control subjects (n = 8), obese non-diabetic patients (n = 9) and obese type 2 diabetic patients (NIDM, Non Insulin-dependent Diabetes Mellitus) (n = 10) were collected and qPCR were performed for CD68 (A) and TNFα (B). Correlation analyses between the expression of CD68 and TNFα (C) and MCP1 and CD68 in the vastus lateralis of patients (D). E–F) Correlation analyses between plasma FFA and HOMA-IR levels of subjects and CD68 expression, respectively. ** stands for <i>P</i><0.01 when comparing NIDM group with Control, † stands for <i>P</i><0.05 and ‡ for <i>P</i><0.01 when comparing NIDM with Obese.</p