Exercise training and cold exposure trigger distinct molecular adaptations to inguinal white adipose tissue

Abstract

Exercise training and cold exposure both improve systemic metabolism, but the mechanisms are not well established. Here, we tested the hypothesis that inguinal white adipose tissue (iWAT) adaptations are critical for these beneficial effects and determined the impact of exercise-trained and cold-exposed iWAT on systemic glucose metabolism and the iWAT proteome and secretome. Transplanting trained iWAT into sedentary mice improves glucose tolerance, while cold-exposed iWAT transplantation shows no such benefit. Compared to training, cold leads to more pronounced alterations in the iWAT proteome and secretome, downregulating >2,000 proteins but also boosting the thermogenic capacity of iWAT. In contrast, only training increases extracellular space and vesicle transport proteins, and only training upregulates proteins that correlate with favorable fasting glucose, suggesting fundamental changes in trained iWAT that mediate tissue-to-tissue communication. This study defines the unique exercise training- and cold exposure-induced iWAT proteomes, revealing distinct mechanisms for the beneficial effects of these interventions on metabolic health.

Keywords: CP: Metabolism; adipose tissue; cold; exercise; glucose; proteomics; secretome; spatial transcriptomics; transplantation.

Figure 1.. iWAT transplantation from exercise-trained but not cold-exposed mice improved glucose tolerance

(A) iWAT was collected from male mice after 11 days of exercise, cold exposure, or a sedentary lifestyle and transplanted into male C57BL6 recipient mice. All analyses were conducted 9 days post-transplantation. (B) Intraperitoneal GTT (ipGTT) and area under the curve (AUC) in recipient mice after 9 days from transplantation. Data are presented as mean ± SEM and were compared using 1-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.. Phenotypic responses of mice and their iWAT to exercise training and cold exposure

(A) Study design to perform quantitative proteomics of whole iWAT and secretome proteins from sedentary, exercise-trained, and cold-exposed mice. (B–D) Change in body weight (B), food intake (C), and glucose after 6 h of fasting (D) of sedentary (gray), exercise-trained (pink), and cold-exposed (green) mice (n = 12/group). (E) H&E-stained section of iWAT from sedentary, trained, and cold-exposed mice. Scale bar, 1,000 μm (4×, top) and 50 μm (20×, bottom). (F) Adipocyte cell count per field measurement of iWAT from sedentary (gray), exercise-trained (pink), and cold-exposed (green) mice (n = 5/group). (G–I) Protein concentration (G), iWAT mass weight (H), and total protein:tissue weight ratio (I) for iWAT from sedentary, exercise-trained, and cold-exposed mice used for tissue proteomics analysis (n = 3–4/group). Data are presented as mean ± SEM and were compared using 1-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3.. Distinct proteome and secretomic profiles of the exercise and cold-exposed iWATs

(A) PCA plot, including all protein quantified in iWAT from sedentary, exercise-trained, and cold-exposed mice (n = 4 sedentary, 4 training, 3 cold). (B and C) UpSet intersection diagram showing the upregulated (B) and downregulated (C) proteins compared to sedentary in iWAT from exercise-trained (pink) and cold-exposed (green) mice. (D) PCA plot, including all protein quantified in iWAT secretome from sedentary, exercise-trained, and cold-exposed mice (n = 3 sedentary, 4 training, 4 cold). (E and F) UpSet intersection diagram showing the upregulated (E) and downregulated (F) proteins compared to sedentary in iWAT secretome from exercise-trained (pink) and cold-exposed (green) mice. (G) Results of GSEA for cellular components of proteins that significantly change in iWAT following exercise training (pink) and cold exposure (green). The pathways shown here are considered significant after false discovery correction p < 0.05. (H) Results of GSEA for biological process of proteins that significantly change in iWAT following exercise training (pink) and cold exposure (green). The pathways shown here are considered significant after false discovery correction p < 0.05.

Figure 4.. Top 50 differentially expressed proteins in iWAT

(A and B) Heatmaps for the top 50 differentially expressed proteins in iWAT from exercise-trained (A) and cold-exposed mice (B). Rows represent the top 50 proteins, and columns represent the iWATs with their replicates. The colors follow the Z scores (blue, low; white, intermediate; red, high). (C) Correlation matrix of VEGFA and NEGR1 (known exercise-induced proteins) and UCP1 (known cold-induced protein) and the top 50 identified proteins with exercise and cold exposure, respectively. (D) Global correlation matrix of fasting glucose and the top 50 proteins changing in iWAT with exercise training (left) and cold exposure (right). The color of the circles in the matrices in (C) and (D) represents the level of correlation for the values that reach statistical significance (p < 0.05); blue represents positive correlation, and red represents negative correlation.

Figure 5.. Expression patterns of the Rilpl2 protein network in iWAT in response to exercise training

(A–C) Visium images (A and B) and relative individual violin plots (C) showing the Rilpl2 expression level across the cell clusters detected in iWAT from sedentary (left) and exercise (right). (D–F) Individual violin plots showing the expression levels and distribution of Myo5a (D), Rab8a (E), and Rab10 (F) across cell clusters detected in iWAT from sedentary (left) and exercise (right).