Nutrient Excess and AMPK Downregulation in Incubated Skeletal Muscle and Muscle of Glucose Infused Rats

Abstract

We have previously shown that incubation for 1h with excess glucose or leucine causes insulin resistance in rat extensor digitorum longus (EDL) muscle by inhibiting AMP-activated protein kinase (AMPK). To examine the events that precede and follow these changes, studies were performed in rat EDL incubated with elevated levels of glucose or leucine for 30min-2h. Incubation in high glucose (25mM) or leucine (100μM) significantly diminished AMPK activity by 50% within 30min, with further decreases occurring at 1 and 2h. The initial decrease in activity at 30min coincided with a significant increase in muscle glycogen. The subsequent decreases at 1h were accompanied by phosphorylation of αAMPK at Ser485/491, and at 2h by decreased SIRT1 expression and increased PP2A activity, all of which have previously been shown to diminish AMPK activity. Glucose infusion in vivo, which caused several fold increases in plasma glucose and insulin, produced similar changes but with different timing. Thus, the initial decrease in AMPK activity observed at 3h was associated with changes in Ser485/491 phosphorylation and SIRT1 expression and increased PP2A activity was a later event. These findings suggest that both ex vivo and in vivo, multiple factors contribute to fuel-induced decreases in AMPK activity in skeletal muscle and the insulin resistance that accompanies it.

Fig 1. Incubation of EDL with 25mM glucose diminishes AMPK Thr¹⁷² phosphorylation, ACC Ser⁷⁹ phosphorylation, and α2 AMPK activity, and increases AMPK Ser^485/491 phosphorylation.

EDL muscles were incubated in Krebs-Henseleit solution containing 25 mM glucose for 30, 60 and 120 min. Phosphorylation of AMPK Thr¹⁷² (A), ACC Ser⁷⁹ (B), AMPK Ser^485/491 (C) were measured by western blot and AMPK activity (D) was determined using the SAMS peptide assay. Results are means + SE (n = 6). *P < 0.05 relative to 30 min incubation with 5.5 mM glucose.

Fig 2. Incubation of muscle in 25mM diminishes SIRT1 protein abundance, NAMPT activity, and NAD/NADH ratio after 1 or 2h.

EDL were incubated with 25 mM glucose for 30, 60 or 120 min. Western blot analysis and quantification of representative blots are shown. SIRT1 protein expression (A) NAMPT activity (B) and NAD/NADH (C) were determined as described in the methods section. Results are means + SE (n = 6). *P < 0.05 compared to 30 min incubation with 5.5 mM glucose.

Fig 3. Incubation with elevated glucose levels or leucine increases muscle glycogen content at 30 min and 1h.

Glycogen content was measured in EDL muscles incubated for 30 or 60 min in media containing 5.5 or 25 mM glucose (A) or 5.5mM glucose with or without 100μM leucine (B). Since no changes were found between 5.5mM glucose at 30 and 60min, only 30min values are shown. Results are means + SE (n = 4–6). *P <0.05 compared to incubation with 5.5 mM glucose.

Fig 4. Incubation with elevated glucose levels or leucine or and in vivo glucose infusion increases PP2A activity at 2h (ex vivo) or 8h (in vivo).

EDL muscles incubated for 30, 60 and 120 min in media containing 5.5 or 25 mM glucose (A) or 5.5mM glucose with or without 100μM leucine (C) and red gastrocnemius muscles from rats infused with glucose for 0, 3, 5, or 8h (B) were analyzed for PP2A activity as described in the methods section. Results are means + SE (n = 4–6). *P <0.05 compared to 30 min incubation with 5.5 mM glucose (A) and (C) or the 0h group for infusions (B).

Fig 5. Inhibition of glucose-induced mTOR/p70S6K phosphorylation does not affect AMPK phosphorylation.

EDL were preincubated in the presence of rapamycin (100μM) for 30 min and then with 5.5 or 25 mM glucose for 1hr. Muscle lysates were analyzed for P-AMPK Thr¹⁷² (A), P-mTOR Ser²⁴⁴⁸ (B) and AMPK Ser^485/491 (C) by western blot. Results show quantification of western blots by densitometry. Results are means ± SE (n = 5). *, p<0.05 compared to values for 5.5 mM glucose.

Fig 6. Incubation of EDL with 100 μM leucine diminishes AMPK Thr¹⁷² phosphorylation, SIRT1 abundance, and NAD/NADH ratio, and increases AMPK Ser^485/491 phosphorylation.

EDL muscles were incubated in Krebs-Henseleit solution containing 5.5 mM of glucose with or without 100 μM of leucine for 30, 60 and 120 min. Phosphorylation of AMPK Thr¹⁷² (A) and AMPK Ser^485/491 (B), SIRT1 protein expression (C) and the NAD/NADH ratio (D) were measured. Results are means + SE (n = 6). *P < 0.05 relative to 30 min incubation with 5.5 mM glucose (without leucine).

Fig 7. Glucose infusion decreases AMPK Thr¹⁷² phosphorylation and α2 AMPK activity, and increases AMPK Ser^485/491 phosphorylation in red gastrocnemius muscle.

AMPK Thr¹⁷² (A) and AMPK Ser^485/491 (B) and ACC Ser⁷⁹ (D) phosphorylation were analyzed by western blot. α2 AMPK activity (C) was measured using the SAMS peptide assay as described in the methods section. Data are means ± SEM. n = 4–6 rats per group, P < 0.05 vs. 0h group.

Fig 8. Glucose infusion diminishes SIRT1 abundance (A), NAD/NADH ratio (B), and NAMPT activity (C), and increases lactate/pyruvate ratio (D) in RG muscles.

Red gastrocnemius muscles from rats infused with glucose for 0, 3, 5, or 8h were analyzed for SIRT1 protein expression (A) NAD/NADH ratio (B) NAMPT activity (C) and lactate/pyruvate (D). Data are means ± SEM. N = 4–6 rats per group, *P < 0.05 vs. 0h group.