Hypothalamus-Muscle Parallel Induction of Metabolic Pathways Following Physical Exercise

Abstract

The modern lifestyle requires less physical activity and skills during our daily routine, leading to multiple pathologies related to physical disabilities and energy accessibility. Thus, exploring the mechanisms underlying the metabolic regulation of exercise is crucial. Here, we characterized the effect of forced and voluntary endurance exercises on three key metabolic signaling pathways, sirtuins, AMPK, and mTOR, across several metabolic tissues in mice: brain, muscles, and liver. Both voluntary and forced exercises induced AMPK with higher intensity in the first. The comparison between those metabolic tissues revealed that the hypothalamus and the hippocampus, two brain parts, showed different metabolic signaling activities. Strikingly, despite the major differences in the physiology of muscles and hypothalamic tissues, the hypothalamus replicates the metabolic response of the muscle in response to physical exercise. Specifically, muscles and hypothalamic tissues showed an increase and a decrease in AMPK and mTOR signaling, respectively. Overall, this study reveals new insight into the relation between the hypothalamus and muscles, which enhances the coordination within the muscle-brain axis and potentially improves the systemic response to physical activity performance and delaying health inactivity disorders.

Keywords: exercise; hippocampus; hypothalamus; metabolic pathways; muscle.

Figure 1

Voluntary physical exercise has a greater effect on body composition. (A) Schematic figure of experimental design. Drawn by Biorender. (B) Percentage change in body weight measurements before exercise (D1), on exercise day 6 (D6), and at the end of the training session, day 10 (D10). (C) Percentage average body fat of young (3 months) WT male mice from each group (control and treadmill). (D) Percentage change in body weight measurements, before exercise (start), after acclimation (W1), and once a week during exercise (W). (E) Epididymal WAT weight normalized to body weight at the end of the training session in young (4 months) WT male mice from each group control (CT), running wheel (RW), and treadmill (TM). The values shown are mean ± s.e.m.; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001 (two-way ANOVA, followed by Tukey post hoc test adjusted for multiple comparison for weight, and two-tailed t-test for fat (%); in (A,B) n = 6 for CT and TM; In (C,D) n = 6 for CT, RW, and TM.

Figure 2

Activation of skeletal muscle AMPK signaling pathway is stronger under voluntary exercise. (A) Relative gene expression levels of Pgc1α. (B,C) Western blot analyses of pAMPK, AMPK, pAKT, AKT, SIRT1, SIRT6, H3K56Ac, and LC3B. (D,E) Densities of the bands were normalized to actin or Ponceau staining which served as an internal control in the gastrocnemius muscle of young (4-6 months) WT male mice. Ratios of phosphorylated AMPK to unphosphorylated AMPK, phosphorylated AKT to unphosphorylated AKT, LC3B II normalized to LC3B I, and SIRT1 and SIRT6 protein expression normalized to actin of each blot. H3K56Ac is normalized to H4. The values shown are mean ± s.e.m., *p < 0.05; **p < 0.01 (ordinary one-way ANOVA for (A), two-tailed t-test for (D,E); n = 5, 6 for CT, TM (black), and RW (gray).

Figure 3

Unique hippocampal metabolic signaling pathway activation under forced exercise. (A) Relative gene expression levels of Sirt6, Sirt1, Pgc1α, Tfam, Nrf2, and Bdnf at rest (CT) or under short-term forced (TM) exercise. (B) Western blot analysis of p-AMPK, AMPK, pAKT, AKT, pS6, S6, LC3B, SIRT1, SIRT6, and H3K56Ac, in the hippocampi of young (4–6 months) WT male mice at rest (CT) or under long-term forced exercise (TM). Tubulin served as the loading control. (C) Expression ratios: Phosphorylated AMPK to unphosphorylated AMPK, (D) phosphorylated AKT to unphosphorylated AKT, (E) phosphorylated S6 to unphosphorylated S6, (F) LC3B II protein expression normalized to LC3B I protein expression, (G) SIRT1 and (H) SIRT6 protein expression normalized to tubulin, and (I) H3K56Ac normalized to H3 in young (4–6 months) WT male mice in the hippocampus. The values shown are mean ± s.e.m., *p < 0.05 (two-way ANOVA, followed by Bonferroni post hoc test in (A), two-tailed t-test for (C–I); in (A), n = 3 for CT and TM; In (B–I) n = 6 for CT and TM.

Figure 4

Unique hippocampal AMPK and AKT signaling pathway activation under voluntary exercise. (A) Western blot analysis of pAMPK, AMPK, pAKT, AKT, pS6, S6, LC3B, SIRT1, SIRT6, and H3K56Ac, in the hippocampi of young (4–6 months) WT male mice, at rest (CT) or under long-term voluntary exercise (RW). Ponceau staining served as loading control. (B) Phosphorylated AMPK to unphosphorylated AMPK ratio (C) phosphorylated AKT to unphosphorylated AKT ratio. (D) Phosphorylated S6 normalized to unphosphorylated S6; (E) LC3B II protein expression normalized to LC3B I protein expression. (F) SIRT1 and (G) SIRT6 protein expression levels normalized to actin. (H) H3K56Ac normalized to H4. The values shown are mean ± s.e.m., *p < 0.05 (two-tailed t-test); in (A–H) n = 6 for CT and RW.

Figure 5

Forced exercise activates AMPK and SIRT6 pathways in the hypothalamic region. (A) Representative Western blot analysis of p-AMPK, AMPK, pAKT, AKT, p-S6, S6, LC3B, SIRT1, SIRT6, and H3K56Ac in young (4 months) WT male mice in the hypothalamus, at rest (CT) or under long-term forced exercise (TM). Densities of the bands were normalized to actin, which served as an internal control. (B) Phosphorylated AMPK vs. unphosphorylated AMPK. (C) Phosphorylated AKT normalized to unphosphorylated AKT. (D) Phosphorylated S6 normalized to unphosphorylated S6. (E) LC3B II protein expression normalized to LC3B I protein expression. (F) SIRT1 and (G) SIRT6 protein expression normalized to actin. (H) H3K56Ac normalized to H4 in young H4 in young (4 months) WT male mice in the hypothalamus. The values shown are mean ± s.e.m., *p < 0.05, **p < 0.01; (two-tailed t-test). In (A–H) n = 6.

Figure 6

Voluntary exercise activates AMPK, SIRT6, and autophagy in the hypothalamic region. (A) Representative Western blot analysis of p-AMPK, AMPK, pAKT, AKT, p-S6, S6, LC3B, SIRT1, SIRT6, and H3K56Ac in young (4 months) WT male mice in the hypothalamus at rest (CT) or under long-term voluntary exercise (RW). Densities of the bands were normalized to actin, which served as an internal control. (B) Phosphorylated AMPK normalized to unphosphorylated AMPK; (C) phosphorylated AKT normalized to unphosphorylated AKT. (D) Phosphorylated S6 normalized to unphosphorylated S6. (E) LC3B II protein expression normalized to LC3B I protein expression. (F) SIRT1 and (G) SIRT6 protein expression normalized to actin. (H) H3K56Ac normalized to H4 in the hypothalamus of young H4 in young (4-month-old) WT male mice. The values shown are mean ± s.e.m., ^*p < 0.05, ^**p < 0.01; (two-tailed t-test). In (A–H) n = 4 for CT and n = 6 RW.