Table 1.
Metabolic characteristics of lean, obese and type 2 diabetic patients (n = 8–10) in fasting state.
Figure 1.
Skeletal muscle inflammation is increased in ob/ob mice.
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 P<0.05 and ** for P<0.01; n = 5.
Figure 2.
Macrophage recruitment in skeletal muscle of ob/ob mice.
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 P<0.05 and ** for P<0.01 for the comparison between CD45- and CD45+ fractions, and † stands for P<0.05 and †† for P<0.01 for the comparison between WT and ob/ob mice; n = 5.
Figure 3.
Inflammation is associated with insulin sensitivity in skeletal muscle of diet-induced obese and diabetic mice.
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) Ex-vivo 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 P<0.05 and ** for P<0.01; n = 5.
Figure 4.
Palmitate induces macrophages recruitment by C2C12 muscle cells.
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 figure 4A, ** stands for P<0.01 in palmitate vs vehicle condition. In figure 4C, * stands for P<0.05 and ‡ for P<0.01 for the respective comparison of CD68 and CD11c between Pal and NCD. NS, not significant; n = 5.
Figure 5.
Skeletal muscle overexpression of MCP1 induces local inflammation and alters insulin signaling and glucose metabolism in vivo.
A) QPCR analyses of MCP1, CD68, CD11c, TNFα, IL1β, RANTES and CXCL1 expression were performed on quadriceps. C–D) Ex-vivo 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 P<0.05 and ** for P<0.01; n = 6.
Figure 6.
Moderate metabolic alterations in MCK-MCP1 transgenic mice under standard chow diet.
A–B) Glycemia was evaluated in basal conditions (A, fed state) and following the insulin injection (ipITT, B). C–D) Fed plasma insulin (C) and TG (D) levels were measured in WT and MCP1- Tg mice E) Evolution of the glycemia during a glucose tolerance test in wt and MCK-MCP1 transgenic mice. F) mRNA quantification of G6Pase, PEPCK and SREBP1c levels in liver of wt and MCK-MCP1 transgenic mice. G–H) Representative images of Western blots (left) and quantification (right) of Serine 473 Akt phosphorylation in liver (G) and adipose tissue (H) of wt and MCK-MCP1 transgenic mice. The histograms on the right of the blots represent the ratio P-Akt (Ser 473)/Akt- total in the liver and the epididymal fat (eWAT) of the mice, respectively.
Figure 7.
Inflammation markers are increased in skeletal muscles of type 2 diabetic patients and correlate with HOMA-IR.
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 P<0.01 when comparing NIDM group with Control, † stands for P<0.05 and ‡ for P<0.01 when comparing NIDM with Obese.