Creatine supplementation increases glucose oxidation and AMPK phosphorylation and reduces lactate production in L6 rat skeletal muscle cells

Abstract

Recent observations have suggested that creatine supplementation might have a beneficial effect on glucoregulation in skeletal muscle. However, conclusive studies on the direct effects of creatine on glucose uptake and metabolism are lacking. The objective of this study was to investigate the effects of creatine supplementation on basal and insulin-stimulated glucose transporter (GLUT4) translocation, glucose uptake, glycogen content, glycogen synthesis, lactate production, glucose oxidation and AMP-activated protein kinase (AMPK) phosphorylation in L6 rat skeletal muscle cells. Four treatment groups were studied: control, insulin (100 nM), creatine (0.5 mM) and creatine + insulin. After 48 h of creatine supplementation the creatine and phosphocreatine contents of L6 myoblasts increased by approximately 9.3- and approximately 5.1-fold, respectively, but the ATP content of the cells was not affected. Insulin significantly increased 2-deoxyglucose uptake ( approximately 1.9-fold), GLUT4 translocation ( approximately 1.8-fold), the incorporation of D-[U-(14)C]glucose into glycogen ( approximately 2.3-fold), lactate production ( approximately 1.5-fold) and (14)CO(2) production ( approximately 1.5-fold). Creatine neither altered the glycogen and GLUT4 contents of the cells nor the insulin-stimulated rates of 2-DG uptake, GLUT4 translocation, glycogen synthesis and glucose oxidation. However, creatine significantly reduced by approximately 42% the basal rate of lactate production and increased by approximately 40% the basal rate of (14)CO(2) production. This is in agreement with the approximately 35% increase in citrate synthase activity and also with the approximately 2-fold increase in the phosphorylation of both alpha-1 and alpha-2 isoforms of AMPK after creatine supplementation. We conclude that 48 h of creatine supplementation does not alter insulin-stimulated glucose uptake and glucose metabolism; however, it activates AMPK, shifts basal glucose metabolism towards oxidation and reduces lactate production in L6 rat skeletal muscle cells.

Figure 1. Creatine (Cr), phosphocreatine (PCr) (A) and ATP contents (B) of L6Glut4-myc myoblasts after incubation for 0, 24 and 48 h either in the absence (control) or presence of 0.5 mm creatine (creatine)

Data are representative of four independent experiments, with quadruplicate samples in each experiment. Data are expressed as means ±s.e.m.*P < 0.05versus PCr control at 0, 24 and 48 h; **P < 0.05versus Cr control at 0, 24 and 48 h; #P < 0.05versus Cr control at 0, 24 and 48 h and Cr at 24 h (two-way ANOVA).

Figure 2. Effects of creatine (Cre), insulin (Ins) and insulin plus creatine (Ins + Cre) on 2-deoxyglucose uptake (A), GLTU4 translocation (B) and glycogen synthesis (C)

D, representative blot of GLUT4 (55 kDa) in L6 rat skeletal muscle cells. Prior to insulin stimulation (20 min, 100 nm where indicated), cells were cultivated for 48 h either in the presence or absence of creatine. Data representative of 4 independent experiments with quadruplicate samples in each experiment (A, B and C). For GLUT4 content we performed 4 independent experiments with duplicates in each experiment (D). *P < 0.05versus control (Con) and creatine (one-way ANOVA).

Figure 3. Glycogen content of L6 rat skeletal muscle cells incubated for 0, 24 and 48 h either in the absence (control) or presence of creatine

Data representative of 4 independent experiments with quadruplicate samples in each experiment. *P < 0.05versus control and creatine time 0 h (two-way ANOVA).

Figure 4. Effects of creatine (Cre), insulin (Ins) and insulin plus creatine (Ins + Cre) on the production of lactate (A) and ¹⁴CO₂ (B) from d-[U-¹⁴C]glucose in L6 rat skeletal muscle cells

Prior to insulin stimulation (2 h, 100 nm), cells were cultivated for 48 h either in the absence or presence of creatine (0.5 mm). Data representative of 4 independent experiments with quadruplicates in each experiment. *P < 0.05versus control (Con). #P < 0.05versus control, insulin and insulin + creatine (one-way ANOVA).

Figure 5. Maximum activity of citrate synthase in L6 rat skeletal muscle cells incubated for 0, 24 and 48 h either in the absence (control) or presence of creatine

Data representative of 4 independent experiments with quadruplicates in each experiment. *P < 0.05versus control 0, 24 and 48 h (two-way ANOVA).

Figure 6. Representative blots and their respective densitometric quantification of the effects of insulin (Ins) and creatine (Cre) on AMPK and ACC phosphorylation

P-AMPKα (62 kDa, A), P-ACC (257 kDa, B), P-AMPK α-1 (62 kDa) and AMPK α-1 (62 kDa) (C), P-AMPK α-2 and AMPK α-2 (D) protein content. P-AMPK α-1 (C) and P-AMPK α-2 (D) are expressed as relative to AMPK α-1 and AMPK α-2 contents, respectively. Data representative of 4 independent experiments with duplicates in each experiment. AICAR (1 mm, 30 min) was used as a positive control for ACC phosphorylation. *P < 0.05versus control and insulin (A and B, ANOVA) and versus control (C and D, t test).

Figure 7. Schematic representation of the effects of creatine on glucose uptake and metabolism in L6 rat skeletal muscle cells

Creatine supplementation did not alter either basal or insulin-stimulated GLUT4 translocation (1), glucose uptake (2), or glycogen synthesis (3). The GLUT4 and glycogen contents of the cells were not altered by creatine supplementation either. However, the basal production of lactate was reduced while the maximum activity of citrate synthase and the production of CO₂ were increased after creatine supplementation. ⇑= increase; ⇓= reduction; ⇔= no effect; PM = plasma membrane; CT = creatine transporter.