Preconditioning with ethyl 3,4-dihydroxybenzoate augments aerobic respiration in rat skeletal muscle

Abstract

Muscle respiratory capacity decides the amount of exertion one's skeletal muscle can undergo, and endurance exercise is believed to increase it. There are also certain preconditioning methods by which muscle respiratory and exercise performance can be enhanced. In this study, preconditioning with ethyl 3,4-dihydroxybenzoate (EDHB), a prolyl hydroxylase domain enzyme inhibitor, has been investigated to determine its effect on aerobic metabolism and bioenergetics in skeletal muscle, thus facilitating boost in physical performance in a rat model. We observed that EDHB supplementation increases aerobic metabolism via upregulation of HIF-mediated GLUT1 and GLUT4, thus enhancing glucose uptake in muscles. There was also a twofold rise in the activity of enzymes of tricarboxylic acid (TCA) cycle and glycolysis, ie, hexokinase and phosphofructokinase. There was an increase in citrate synthase and succinate dehydrogenase activity, resulting in the rise in the levels of ATP due to enhanced Krebs cycle activity as substantiated by enhanced acetyl-CoA levels in EDHB-treated rats as compared to control group. Increased lactate dehydrogenase activity, reduced expression of monocarboxylate transporter 1, and increase in monocarboxylate transporter 4 suggest transport of lactate from muscle to blood. There was a concomitant decrease in plasma lactate, which might be due to enhanced transport of lactate from blood to the liver. This was further supported by the rise in liver pyruvate levels and liver glycogen levels in EDHB-supplemented rats as compared to control rats. These results suggest that EDHB supplementation leads to improved physical performance due to the escalation of aerobic respiration quotient, ie, enhanced muscle respiratory capacity.

Keywords: cellular metabolism; ethyl 3,4-dihydroxybenzoate; exercise; prolyl hydroxylase enzyme.

Figure 1

Effect of EDHB on glycolysis. Notes: (A) Expression of glucose transporters (GLUT1 and GLUT4) was observed in muscle by Western blotting. Representative immunoblots are shown. (B) Glucose levels were estimated in plasma. (C) Hexokinase activity was estimated in rat muscle. (D) Muscle PFK activity. Values are mean ± SD (n=8 per group). **P<0.01 vs CS; *P<0.05 vs CS; ^#P<0.05 vs CT; and ^##P<0.01 vs CT. Abbreviations: EDHB, ethyl 3,4-dihydroxybenzoate; PFK, phosphofructokinase; CS, control sedentary; CT, control training; DS, drug sedentary; DT, drug training.

Figure 2

EDHB supplementation promotes energy production by promoting Krebs cycle enzymes activity. Notes: (A) Acetyl-CoA level in muscle. (B) CS activity. (C) SDH activity. (D) ATP level was estimated by phosphorescence. Values are mean ± SD (n=8 per group). **P<0.01 vs CS; *P<0.05 vs CS; ^##P<0.01 vs CT; and ^#P<0.05 vs CT. Abbreviations: EDHB, ethyl 3,4-dihydroxybenzoate; SDH, succinate dehydrogenase; CS, control sedentary; CT, control training; DS, drug sedentary; DT, drug training.

Figure 3

Effect of EDHB supplementation on lactate and lactate transporters. Notes: (A) Expression of lactate transporters by Western blotting. Representative immunoblots are shown. (B) Lactate level in plasma. (C) LDH activity in rat muscle. Values are mean ± SD (n=8 per group). **P<0.01 vs CS; *P<0.05 vs CS; ^##P<0.01 vs CT; and ^#P<0.05 vs CT. Abbreviations: EDHB, ethyl 3,4-dihydroxybenzoate; LDH, lactate dehydrogenase; CS, control sedentary; CT, control training; DS, drug sedentary; DT, drug training; MCT, monocarboxylate transporter.

Figure 4

Effect of EDHB supplementation on pyruvate (A) and glycogen (B) in rat liver and muscle. Notes: Values are mean ± SD (n=8 per group). **P<0.01 vs CS; *P<0.05 vs CS; ^##P<0.01 vs CT. Abbreviations: EDHB, ethyl 3,4-dihydroxybenzoate; CS, control sedentary; CT, control training; DS, drug sedentary; DT, drug training.

Figure 5

Effect of EDHB supplementation on fatty acid oxidation. Notes: Expression of proteins involved in fatty acid oxidation was studied by Western blotting in muscle homogenate. Representative immunoblots are shown. Values are mean ± SD (n=8 per group). **P<0.01 vs CS; ^##P<0.01 vs CT. Abbreviations: EDHB, ethyl 3,4-dihydroxybenzoate; CS, control sedentary; CT, control training; DS, drug sedentary; DT, drug training; PDK, pyruvate dehydrogenase kinase; CPTI, carnitine palmitoyltransferase I.

Figure 6

EDHB supplementation reduces fatigue. Notes: Level of inorganic phosphate in plasma. Values are mean ± SD (n=8 per group). **P<0.01 vs CS; ^##P<0.01 vs CT. Abbreviations: EDHB, ethyl 3,4-dihydroxybenzoate; CS, control sedentary; CT, control training; DS, drug sedentary; DT, drug training.

Figure 7

Scatter plot of hypoxia signaling PCR array studies for the metabolic genes listed. Abbreviations: PCR, polymerase chain reaction; min, minimum; avg, average; max, maximum.

Figure 8

Putative mechanism of EDHB in augmentation of physical performance. Notes: ↑ upregulation by EDHB supplementation and ↓ downregulation by EDHB. Abbreviations: EDHB, ethyl 3,4-dihydroxybenzoate; PDK, pyruvate dehydrogenase kinase; CPTI, carnitine palmitoyltransferase I; Pi, inorganic phosphate; SDH, succinate dehydrogenase; CS, control sedentary; HK, hexokinase; MCT, monocarboxylate transporter.