Fig. 3: Myeloid Tshr deficiency alleviates macrophage infiltration and M1 polarization in liver, adipose tissues and skeletal muscle of HFD-fed mice and improves insulin resistance.

Male Tshr^MKO mice and age-matched Tshr^f/f littermates were fed with HFD for 9 weeks. Liver, eWAT and skeletal muscle were isolated from these mice. Flow cytometry was performed to analyze the effect of myeloid Tshr deficiency on macrophage infiltration and M1 polarization in liver, eWAT and skeletal muscle. a The percentage of CD11b⁺ F4/80⁺ macrophages from the CD45⁺ cell gate in liver, eWAT and skeletal muscle (n = 3). b The percentage of CD80⁺ macrophages from the CD11b⁺ F4/80⁺ cell gate in liver, eWAT and skeletal muscle (n = 3). The mRNA levels of Itgam, Adgre1 and Itgax in liver (c), eWAT (d) and skeletal muscle (e) were measured by qRT-PCR (n = 9). β-Actin was used as an internal control (n = 9). Primary hepatocytes, 3T3L1-differentiated adipocytes and L6-differentiated skeletal muscle cells were co-cultivated with Tshr^f/f- or Tshr^MKO-derived BMDMs for 48 h and then stimulated with 100 nM insulin for 15 min. f Western blotting analysis was performed to determine the levels of p-IRS1, IRS1, p-AKT, t-AKT, p-p65, p65, p-JNK and JNK in primary hepatocytes, 3T3L1-differentiated adipocytes and L6-differentiated skeletal muscle cells. β-Actin was used as a loading control. g Relative PEPCK1 activity of primary hepatocytes with the indicated treatments (n = 3). h Relative glucose uptake of 3T3L1-differentiated adipocytes with the indicated treatments (n = 3). Data are presented as mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student’s t-test for a and b; one-way ANOVA for c–e, g and h).