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

Coughlan KA, Balon TW, Valentine RJ, Petrocelli R, Schultz V, Brandon A, Cooney GJ, Kraegen EW, Ruderman NB, Saha AK - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Endocrinology and Diabetes, Boston University Medical Center, Boston, Massachusetts, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Glucose infusion decreases AMPK Thr172 phosphorylation and α2 AMPK activity, and increases AMPK Ser485/491 phosphorylation in red gastrocnemius muscle.AMPK Thr172 (A) and AMPK Ser485/491 (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.
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pone.0127388.g007: Glucose infusion decreases AMPK Thr172 phosphorylation and α2 AMPK activity, and increases AMPK Ser485/491 phosphorylation in red gastrocnemius muscle.AMPK Thr172 (A) and AMPK Ser485/491 (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.

Mentions: To determine whether the effects of glucose observed in the incubated EDL also occurred in vivo and in the presence of insulin, rats were infused with 50% glucose for 0, 3, 5 or 8h at a rate adjusted to maintain plasma glucose concentration at 16–17mM. The plasma insulin level was 250μU/L during the infusion vs. 50μU/L prior to its start. In the red gastrocnemius (RG) muscle, the glucose infusion decreased both AMPK Thr172 phosphorylation (Fig 7A) and AMPKα2 activity (by the SAMS peptide assay, Fig 7C) with a significant change observed at 5h. Interestingly, this was followed by a secondary increase in AMPK activity, something we have not observed in previous studies [25, 26]. In keeping with these findings, the phosphorylation of ACC also was only significantly decreased at 5h (Fig 7D), although it is somewhat diminished at 3 and 8h of infusion. In contrast, the increase in phosphorylation of AMPK at Ser485/491 (Fig 7B) and the decreases in SIRT1 protein (Fig 8A), NAD/NADH (Fig 8B), NAMPT activity (Fig 8C) and lactate/pyruvate ratio (Fig 8D), which occurred after the decrease in AMPK-Thr172 in the incubated EDL, took place at the same time (5h) and were maintained at 8h. Only the increase in PP2A activity (Fig 4B) was a later event (observed at 8h), as it was in the EDL incubated with a high glucose medium (Fig 4A).


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

Coughlan KA, Balon TW, Valentine RJ, Petrocelli R, Schultz V, Brandon A, Cooney GJ, Kraegen EW, Ruderman NB, Saha AK - PLoS ONE (2015)

Glucose infusion decreases AMPK Thr172 phosphorylation and α2 AMPK activity, and increases AMPK Ser485/491 phosphorylation in red gastrocnemius muscle.AMPK Thr172 (A) and AMPK Ser485/491 (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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440828&req=5

pone.0127388.g007: Glucose infusion decreases AMPK Thr172 phosphorylation and α2 AMPK activity, and increases AMPK Ser485/491 phosphorylation in red gastrocnemius muscle.AMPK Thr172 (A) and AMPK Ser485/491 (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.
Mentions: To determine whether the effects of glucose observed in the incubated EDL also occurred in vivo and in the presence of insulin, rats were infused with 50% glucose for 0, 3, 5 or 8h at a rate adjusted to maintain plasma glucose concentration at 16–17mM. The plasma insulin level was 250μU/L during the infusion vs. 50μU/L prior to its start. In the red gastrocnemius (RG) muscle, the glucose infusion decreased both AMPK Thr172 phosphorylation (Fig 7A) and AMPKα2 activity (by the SAMS peptide assay, Fig 7C) with a significant change observed at 5h. Interestingly, this was followed by a secondary increase in AMPK activity, something we have not observed in previous studies [25, 26]. In keeping with these findings, the phosphorylation of ACC also was only significantly decreased at 5h (Fig 7D), although it is somewhat diminished at 3 and 8h of infusion. In contrast, the increase in phosphorylation of AMPK at Ser485/491 (Fig 7B) and the decreases in SIRT1 protein (Fig 8A), NAD/NADH (Fig 8B), NAMPT activity (Fig 8C) and lactate/pyruvate ratio (Fig 8D), which occurred after the decrease in AMPK-Thr172 in the incubated EDL, took place at the same time (5h) and were maintained at 8h. Only the increase in PP2A activity (Fig 4B) was a later event (observed at 8h), as it was in the EDL incubated with a high glucose medium (Fig 4A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Endocrinology and Diabetes, Boston University Medical Center, Boston, Massachusetts, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus