HIIT Promotes M2 Macrophage Polarization and Sympathetic Nerve Density to Induce Adipose Tissue Browning in T2DM Mice

Abstract

Browning of white adipose tissue (WAT) is a focus of research in type 2 diabetes mellitus (T2DM) and metabolism, which may be a potential molecular mechanism for high-intensity interval training (HIIT) to improve T2DM. In this study, male C57BL/6J wild-type mice were subjected to an 8-week HIIT regimen following T2DM induction through a high-fat diet (HFD) combined with streptozotocin (STZ) injection. We found that HIIT improved glucose metabolism, body weight, and fat mass in T2DM mice. HIIT also decreased adipocyte size and induced browning of WAT. Our data revealed a decrease in TNFα and an increase in IL-10 with HIIT, although the expression of chemokines MCP-1 and CXCL14 was increased. We observed increased pan-macrophage infiltration induced by HIIT, along with a simultaneous decrease in the expression of M1 macrophage markers (iNOS and CD11c) and an increase in M2 macrophage markers (Arg1 and CD206), suggesting that HIIT promotes M2 macrophage polarization. Additionally, HIIT upregulated the expression of Slit3 and neurotrophic factors (BDNF and NGF). The expression of the sympathetic marker tyrosine hydroxylase (TH) and the nerve growth marker GAP43 was also increased, demonstrating the promotion of sympathetic nerve growth and density by HIIT. Notably, we observed macrophages co-localizing with TH, and HIIT induced the accumulation of M2 macrophages around sympathetic nerves, suggesting a potential association between M2 macrophages and increased density of sympathetic nerves. In conclusion, HIIT induces adipose tissue browning and improves glucose metabolism in T2DM mice by enhancing M2 macrophage polarization and promoting sympathetic nerve growth and density.

Keywords: high-intensity interval training; innervation; macrophage; sympathetic nervous system; type 2 diabetes mellitus; white adipose tissue browning.

Figure 1

The effects of exercise intervention on glucose metabolism in T2DM mice. (A) Fasting blood glucose of the three groups at the end of 8 weeks of exercise. (B,C) Blood glucose levels and their AUC at 0, 15, 30, 60, 90, and 120 min after glucose injection. (D,E) Blood glucose levels and their AUC at 0, 15, 30, 60, 90, and 120 min after insulin injection. * p < 0.05, ** p < 0.01. Data are expressed as mean ± SEM. n = 8 per group.

Figure 2

The effects of exercise intervention on body weight and fat weight in T2DM mice. (A) Daily water intake in CON and T2DM mice and T2DM-EX mice. (B) Daily food intake in CON and T2DM mice and T2DM-EX mice. (C) Body weight of CON, T2DM, and T2DM-EX mice after intervention. (D,E) Total weight of inguinal, epididymal, and perirenal adipose tissue and its ratio to body weight. “ns” indicates no significance, ** p < 0.01, *** p < 0.001. Data are expressed as mean ± SEM. n = 8 per group.

Figure 3

The effects of exercise intervention on browning-related markers. (A) Representative images of iWAT with hematoxylin and eosin staining of histological sections from control, T2DM, and T2DM-EX mice. (B,C) Analysis of adipocyte size and frequency of distribution in HE-stained sections by ImageJ. Three samples per group, 6 fields per section, minimum 80 cells counted per image. (D,E) Representative images of UCP1 immunohistochemical staining of iWAT in control, T2DM, and T2DM-EX mice and their integrated optical density (IOD) value statistics. Three samples per group, 6 fields per section. (F) Thermogenesis gene expression in iWAT, n = 6 per group. * p < 0.05, ** p < 0.01. Scale bar = 50 μm; data are expressed as mean ± SEM.

Figure 4

The effects of exercise and T2DM on inflammatory factors in iWAT. (A–D) The mRNA expression levels of TNFα, IL-10, MCP-1, and CXCL14, n = 6. * p < 0.05, ** p < 0.01. Data are expressed as mean ± SEM.

Figure 5

The effects of exercise intervention on macrophage infiltration and polarization in T2DM mice. (A) Representative images of Mac2 immunofluorescence staining of iWAT in control, T2DM, and T2DM-EX mice, scale bar = 50 μm. (B) Percentage of Mac2-positive area in total image, 3 samples per group, 6 fields per section. (C–E) The mRNA expression levels of pan-macrophage markers (Mac2, F4/80, and CD11b). (F) Representative images showing the double-positive staining of Mac2 and iNOS, Mac2 and Arg1. Scale bar = 50 μm. White arrows indicate Mac2 single-positive cells; red arrows indicate the double-positive cells. Green: Mac2; red: iNOS or Arg1; blue: nucleus stained by DAPI. (G) The ratio of double-positive cells to Mac2 single-positive cells. M1 represents Mac2⁺iNOS⁺ and M2 represents Mac2⁺Arg1⁺. Three samples per group, 6 fields per section. (H) The mRNA levels of M1-type macrophage markers (iNOS, CD11c) in iWAT. (I) The mRNA levels of M2-type macrophage markers (CD206, Arg1) in iWAT. * p < 0.05, ** p < 0.01. Data are expressed as mean ± SEM.

Figure 6

The effects of exercise and T2DM on local sympathetic nerves in iWAT. (A–C) The mRNA expression levels of Slit3, BDNF, and NGF. (D) Representative images of TH immunofluorescence staining of iWAT in control, T2DM, and T2DM-EX mice, scale bar = 50 μm. (E) Ratio of the number of TH-positive cells to the number of DAPI-stained nuclei. Three samples per group, 6 fields per section. (F) TH protein expression was analyzed by Western blot and quantified using ImageJ in iWAT collected from CON, T2DM, and T2DM-EX group mice. n = 6 per group. (G,H) The mRNA expression levels of TH and GAP43, n = 6 per group. * p < 0.05, ** p < 0.01. Data are expressed as mean ± SEM. Original images of (F) can be found in Supplementary Materials.

Figure 7

Exercise-induced aggregation of M2-like macrophages surrounding sympathetic nerves. Immunofluorescence staining of iWAT for TH (white), Mac2 (green), and iNOS (red, A) or Arg1 (red, B), and stained nuclei with DAPI (blue). Scale bar = 50 μm. (C–E) In pan-macrophages (Mac2⁺), the proportion of TH⁺Mac2⁺iNOS⁺ cells or TH⁺Mac2⁺Arg1⁺ cells and the ratio of the two. Three samples per group, 6 fields per section. * p < 0.05, ** p < 0.01. Data are expressed as mean ± SEM.