Disrupting AMPK-Glycogen Binding in Mice Increases Carbohydrate Utilization and Reduces Exercise Capacity

Abstract

The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy balance and metabolism and binds glycogen, the primary storage form of glucose in liver and skeletal muscle. The effects of disrupting whole-body AMPK-glycogen interactions on exercise capacity and substrate utilization during exercise in vivo remain unknown. We used male whole-body AMPK double knock-in (DKI) mice with chronic disruption of AMPK-glycogen binding to determine the effects of DKI mutation on exercise capacity, patterns of whole-body substrate utilization, and tissue metabolism during exercise. Maximal treadmill running speed and whole-body energy utilization during submaximal running were determined in wild type (WT) and DKI mice. Liver and skeletal muscle glycogen and skeletal muscle AMPK α and β2 subunit content and signaling were assessed in rested and maximally exercised WT and DKI mice. Despite a reduced maximal running speed and exercise time, DKI mice utilized similar absolute amounts of liver and skeletal muscle glycogen compared to WT. DKI skeletal muscle displayed reduced AMPK α and β2 content versus WT, but intact relative AMPK phosphorylation and downstream signaling at rest and following exercise. During submaximal running, DKI mice displayed an increased respiratory exchange ratio, indicative of greater reliance on carbohydrate-based fuels. In summary, whole-body disruption of AMPK-glycogen interactions reduces maximal running capacity and skeletal muscle AMPK α and β2 content and is associated with increased skeletal muscle glycogen utilization. These findings highlight potential unappreciated roles for AMPK in regulating tissue glycogen dynamics and expand AMPK's known roles in exercise and metabolism.

Keywords: AMP-activated protein kinase; carbohydrate binding module; energy utilization; exercise; glycogen; metabolism; skeletal muscle.

FIGURE 1

The double knock-in (DKI) mutation used to disrupt whole-body AMPK-glycogen binding in mice reduces maximal running speed but does not alter time to exhaustion at submaximal running speed. (A) Maximal running speed at a 0° treadmill incline in wild type (WT) and DKI mice (n = 26); (B) Time to exhaustion at 70% of individual maximal running speed at a 0° incline (n = 10–13). Male mice, 17–21 wk ****p < 0.0001.

FIGURE 2

DKI mice have increased respiratory exchange ratio (RER) and carbohydrate (CHO) oxidation rates during exercise calorimetry experiments. Respiratory gases (VO₂ and VCO₂) were measured every min and used to calculate RER, total energy expenditure (TEE), and substrate utilization during exercise calorimetry treadmill running at 60% of individual maximal running speed. Data presented are the average over the final 5 min of exercise. (A) VO₂; (B) TEE; (C) RER; (D) CHO oxidation rates; (E) Fat oxidation rates; (F) Percent contribution of fat and CHO oxidation rates to TEE. Male mice, 17–20 weeks, n = 11–13. *p < 0.05.

FIGURE 3

DKI and WT mice have similar circulating glucose, lactate, and non-esterified fatty acid (NEFA) levels following a maximal running test at a 5° incline. (A) Blood glucose and (B) lactate were measured via tail tip bleeding the morning before and immediately following the maximal exercise test at a 5° incline. Fed male mice, 17–20 weeks, n = 12–18. (C) Blood was collected via retro-orbital bleed from mice immediately before and after completing the maximal running test, and serum NEFA levels were assessed. Male mice, 17–20 weeks, n = 7–10. **p < 0.01, ****p < 0.0001.

FIGURE 4

DKI and WT mice display similar changes in liver and skeletal muscle glycogen following a maximal running test at a 5° incline. Glycogen content was assessed in (A) liver and (C) skeletal muscle from rested mice and maximally exercised mice. Change in glycogen (Δglycogen content) in (B) liver and (D) skeletal muscle was determined by the difference between individual glycogen content of maximally exercised mice and the average glycogen content of rested mice from the same genotype. Male mice, 17–20 weeks, n = 7–10. **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Maximal running speed at a 5° incline is positively correlated with skeletal muscle glycogen utilization following maximal running in WT but not DKI mice. (A) Correlation between maximal running speed and change in liver glycogen was not significant in either WT or DKI mice. (B) WT mice display a significant positive correlation between maximal running speed and change in skeletal muscle glycogen between the rested state and following maximal treadmill running, which was not observed in DKI mice. Male mice, 17–20 weeks, n = 8–9. *p < 0.05.

FIGURE 6

DKI mouse skeletal muscle displays similar content of proteins associated with glucose uptake and protein markers of mitochondrial content relative to WT. (A) Representative immunoblots and stain free image of GLUT4, CPT1b, and citrate synthase in WT and DKI skeletal muscle; (B) Quantified total GLUT4; (C) Quantified total CPT1b; (D) Quantified total citrate synthase; (E) Representative immunoblots and stain free image of mitochondrial oxidative phosphorylation (OXPHOS) complex proteins in WT and DKI skeletal muscle; (F) Quantified OXPHOS complex protein content. Male mice, 20–32 weeks, n = 7–9.

FIGURE 7

DKI mice have reduced skeletal muscle AMPK α and β2 content but intact AMPK, ACC and TBC1D1 signaling in response to a maximal running test at a 5° incline versus WT. Skeletal muscles were collected from rested and maximally exercised male WT and DKI mice, and phosphorylation of AMPK and downstream substrates was assessed using Western blotting. (A) Representative immunoblots of p-T172 and total AMPK, p-S182 and total AMPK β, p-S79 and total acetyl-CoA carboxylase (ACC) and p-S660 and total TBC1 domain family member 1 (TBC1D1) with representative stain free image. Quantified relative (B) AMPK p-T172, (C) AMPK β p-S182, (D) ACC p-S79, and (E) TBC1D1 p-S660; (F) Total AMPK α; (G) Total AMPK β. Male mice, 17–20 weeks, n = 6–8. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.