Dynamics of training and acute exercise-induced shifts in muscular glucose transporter (GLUT) 4, 8, and 12 expression in locomotion versus posture muscles in healthy horses

Abstract

Important changes in glucose transporter (GLUT) expression should be expected if the glucose influx plays a pivotal role in fuelling or connecting metabolic pathways that are upregulated in response to exercise. The aim was to assess GLUT4, 8, and 12 dynamics in response to training and acute exercise. Methods: Sixteen untrained Standardbred mares (3-4 year) performed an incremental SET at the start and end of 8 weeks harness training. M. pectoralis (PM) and M. vastus lateralis (VL) muscle biopsies were taken before and after each SET, allowing for comparing rest and acute samples in untrained (UT) and trained (T) condition using Western Blot for GLUT quantification and Image Pro v.10 for Blot analysis. Data were normalized against GAPDH. Basal GLUT-levels of PM versus VL were analysed with the Wilcoxon matched-pairs signed rank test. The effect of acute exercise or training was assessed using the Friedman test with a post hoc Dunn's. Results: Basal GLUT4 and GLUT12 protein expression were significantly higher in the VL compared to the PM (P_GLUT4 = 0.031 and P_GLUT12 = 0.002). Training had no effect on basal GLUT4 expression, neither in the VL (p > 0.9999), nor the PM (p > 0.9999). However, acute exercise in trained condition significantly decreased GLUT4 expression in the VL (p = 0.0148). Neither training nor acute exercise significantly changed total GLUT8 protein expression. Training significantly decreased total GLUT12 protein expression in rest biopsies, only visible in the VL (p = 0.0359). This decrease was even more prominent in the VL after acute exercise in trained condition (P_VL = 0.0025). Conclusion: The important changes seen in GLUT12 expression downregulation, both in response to training and acute exercise in the horse, the downregulation of GLUT4 expression after acute exercise in trained condition and the lack of differential shifts in GLUT8 expression in any of the studied conditions, questions the importance of glucose as substrate to fuel training and exercise in healthy horses. These findings encourage to further explore alternative fuels for their involvement in equine muscular energetics.

Keywords: energy; equine; exercise; fuel; glucose; glycogen; metabolism; training.

FIGURE 1

Insulin and muscle contraction mediated translocation of GLUT4 in human skeletal muscles. Once insulin binds to its receptor, it leads to the autophosphorylation of the transmembrane units, which in turn activates the downstream signaling proteins insulin receptor substrate 1 and 2 (IRS1/2) and adapter protein with Pleckstrin and Src homology domains (APS). (A) Phosphorylated IRS1/2 proteins serve as an activator of the phosphatidylinositol (PI)- 3-kinase, which converts PIP2 to PIP3 at the plasma membrane. Further, PIP3 activates the phosphoinositide-dependent protein kinases 1 and 2 (PDK1/2), which phosphorylate protein kinase B (AKT) and protein kinase C (PKC). Both kinases contribute to the trafficking of GLUT4-containing vesicles toward the plasma membrane. (B) Activation of the insulin receptor also increases phosphorylation of the proto-oncogene B-lineage lymphoma (Cbl), a process that requires the presence of the adaptor proteins APS and CAP. The Cbl-APS/CAP complex enhances the recruitment of CT10 regulator of kinase (Crk), which has been proposed to promote GLUT4 translocation to the plasma membrane. (C) In order to fulfill the increased energy demands of skeletal muscle during exercise, muscle contraction increases 5′ adenosine monophosphate (AMP) levels, leading to the activation of AMP kinase (AMPK), which translocates GLUT4-containing vesicles towards the plasma membrane and the T-tubules. Image generated with biorender.com.

FIGURE 2

Overview training trial protocol and sample collection timepoints. SET: standardized exercise test; UT-rest: untrained at rest; UT-acute: untrained after execution of SET; T-rest; trained at rest; T-acute: trained after execution of SET (Standardized Exercise Test).

FIGURE 3

Basal GLUT4 (A), GLUT8 (B), and GLUT12 (C) protein expression in the m. pectoralis (PM) versus the m. vastus lateralis (VL). Graphs represent mean ± standard deviation of total protein expression in arbitrary units. *p < 0.05; **p < 0.01. a.u. arbitrary units.

FIGURE 4

Total GLUT4 (A), GLUT8 (B), and GLUT12 (C) protein expression in the m. vastus lateralis (VL) in response to acute exercise and 8 weeks of harness training. Graphs represent mean ± standard deviation of total protein expression, relative to basal levels (UT-rest) in fold change. *p < 0.05; **p < 0.01. UT: untrained; T: trained.