SUPPLEMENTAL DATA Supplemental Table 1: Demographics and characteristics of study participants Lean (n=15) Obese (n=12) Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± 9.5 44.8 ± 9.1 White, n (%) 13 (87%) 9 (75%) BMI, kg/m² [mean ± SD] 21.7 ± 1.7 36.0 ± 4.8 Supplemental Table 2: Adipose tissue in lean (n=4) and obese (n=4) mice as quantified by micro- CT imaging Adipose Tissue, mm² Lean Mice (ND-fed) Obese Mice (60% HFD-fed) P-Value Thigh IMAT/PMAT 2.14 (1.7 2.5) 47.94 (28.6 65.8) 0.021 Calf IMAT/PMAT 0.18 (0.11 0.31) 8.29 (3.8 14.5) 0.043 Total IMAT/PMAT 2.3 (1.5 3.3) 56.4 (34.9 77.4) 0.021 VAT 45.3 (28.1 68.7) 288.8 (270.2 295.2) 0.021 SAT 13.8 (10.5 18.9) 125.3 (100.3 147.1) 0.021 IMAT/PMAT: intermuscular adipose tissue/perimuscular adipose tissue; VAT: abdominal visceral adipose tissue; SAT: abdominal subcutaneous adipose tissue. Values are reported as median (interquartile range). P-values were computed using the nonparametric Kruskal Wallis test (SAS 9.3). All tests were 2-sided.
Supplemental Figure Legends Supplemental Figure 1: Quantitative RT-PCR analysis of inflammatory gene expression in SM. The entire quadriceps SM (including muscle fibers, IMAT, and PMAT) harvested from mice fed normal diet (ND; n=6 8 mice/group) or high-fat diet (HFD; n=8 10 mice/group) for 12 weeks were analyzed by quantitative RT-PCR. *P<0.05, **P<0.01, P<0.001 compared with ND group. Supplemental Figure 2: Flow cytometric analysis of macrophages and T cells in SM. (A) Gating example of F4/80+ macrophages and F4/80+CD11c+ M1 macrophages. (B) Example of gating total CD3+, CD4+, and CD8+ T cells in SM of ND-fed mice. Supplemental Figure 3: Immunohistochemical staining of sections of quadriceps isolated from ND-fed mice under 10x magnification Supplemental Figure 4: Representative images of extramyocellular adipose tissue in mouse fed lower-fat diet. Cross-sectional micro-ct images of the proximal, mid, and distal thigh and midcalf for a mouse fed lower-fat diet (panels A D) with corresponding adipose tissue superimposed in red (panels E H). Supplemental Figure 5: Representative images of extramyocellular adipose tissue in 60% HFD-fed mouse. Cross-sectional micro-ct images of the proximal, mid, and distal thigh and
midcalf for a mouse fed 60% HFD (panels A D) with corresponding adipose tissue superimposed in red (panels E H). Supplemental Figure 6: Representative images of visceral adipose tissue in lean and obese mice. Cross-sectional micro-ct images of the abdomen and adipose tissue for a mouse fed lower-fat diet (panels A and B) and a mouse fed 60% HFD (panels C and D). VAT is shown in red, and SAT is shown in yellow. Supplemental Figure 7: Obesity-induced SM and systemic insulin resistance. (A) Homeostasis model assessment of insulin resistance (HOMA-IR) in lean and 60% HFD fed WT mice (n=4 mice/group). (B) Representative immunoblot and quantification of serinephosphorylated Akt [P-Akt (S473)] protein expression in SM of mice fed ND or 60% HFD and injected with 1.5 U/kg body weight regular human insulin or PBS 10 minutes prior to sacrifice. The levels of P-Akt (S473) were expressed as P-Akt/Akt ratio. Insulin-stimulated P-Akt/Akt ratio was compared between lean and obese groups. *P<0.05, P<0.001 compared with lean controls. Supplemental Figure 8: The effect of T H 1 cells on myotube inflammation and metabolic functions and the involvement of the JAK/STAT pathway. (A) Representative immunoblot and quantification of total STAT1 protein and tyrosine 701 phosphorylated STAT1 (P-STAT1) protein in SM of mice fed ND or HFD for 12 weeks. (B) Quantitative RT-PCR analysis of proinflammatory cytokine and chemokine expression in differentiated C2C12 myotubes treated with T H 1 supernatant with or without antibody neutralization of IFNγ and T H 1 supernatant in the
presence of JAK inhibitor I for 48 hours (n=6 10 samples/group). (C) mrna level (n=4 6 samples/group) and the amount of P-STAT1 protein (n=5 samples/group) in C2C12 cells treated with T H 1-conditioned medium with or without antibody neutralization of IFNγ. (D) P-Akt (S473)/Akt protein levels in differentiated C2C12 cells treated with T H 1 for 48 hours and exposed to 100 nm insulin for 15 minutes (n=4 5 samples/group). Results are shown as mean SEM of 2 separate experiments, each with 2 3 samples/group. *P<0.05, **P<0.01, P<0.001 for comparison between control and each treatment group.
Supplemental Figure 1 T cell and macrophage marker expression in SM of lean and obese mice 40 ND 30 ** ** HFD 20 * ** Relative mrna 10 5 4 ** * ** * ** 3 * 2 * 1 0 F4/80 CD11c MCP1 IL-12b IL-18 T N F α C D 3 C D 8 C D 4 R A N T E S IF N g
Supplemental Figure 2 A B
Supplemental Figure 3 Isotype control Mac3 macrophages CD3 T cells
Supplemental Figure 4
Supplemental Figure 5
Supplemental Figure 6
Supplemental Figure 7 A B HOMA-IR 150 100 50 * Insulin Lean Obese - + + + - + + + 0.8 P-Akt Akt 0 Lean Obese P-Akt (S473)/Akt 0.6 0.4 0.2 0.0 Insulin - + - + Lean Obese
Supplemental Figure 8 A ND HFD P-Stat1 Stat1 Tubulin 2.0 STAT1 ns 2.0 P-STAT1 * STAT1/Tubulin 1.5 1.0 0.5 P-STAT1/STAT1 1.5 1.0 0.5 0.0 N D H F 0.0 N D H F D
Supplemental Figure 8 B Relative mrna 4 3 2 1 IL-6 Relative mrna 2.5 2.0 1.5 1.0 0.5 ** TNFα ns ns 0 Control T H 1 T H 1+Anti-IFNγ T H 1+Jak inh. 0.0 Control T H 1 T H 1+Anti-IFNγ T H 1+Jak inh. Relative mrna 25 20 15 10 5 MCP-1 Relative mrna 20 15 10 5 RANTES 0 Control T H 1 T H 1+Anti-IFNγ T H 1+Jak inh. 0 Control T H 1 T H 1+Anti-IFNγ T H 1+Jak inh.
Supplemental Figure 8 C Control TH1 TH1+Anti-IFNg P-STAT1 (Y701) β-actin Relative mrna 20 15 10 5 STAT1 P-STAT1/β-actin 0.8 0.6 0.4 0.2 P-STAT1 ** ** 0 Control T H 1 T H 1+Anti-IFNγ 0.0 Control T H 1 T H 1+Anti-IFNγ
Supplemental Figure 8 D Control Naive CD4 TH1 TH1+Jak P-Akt Insulin inhibitor + + + + + + + + + 1.5 ** P-STAT1 (Y701) P-Akt (S473) P-Akt/Akt 1.0 0.5 Akt 0.0 Control Naive CD4 T H 1 T H 1+Jak inh.