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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

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.
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pone.0127388.g003: 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.

Mentions: In search for additional factors responsible for the decrease in AMPK phosphorylation caused by a high glucose concentration, we assessed cellular energy state. In keeping with previous observations [15], we found no differences in tissue concentrations of ATP, ADP, AMP or CrP in muscles incubated with 25 vs. 5.5mM glucose for 30min, 1h, or 2h (Table 2). The abundance of CAMKKβ, a known upstream kinase of AMPK, was unchanged at all timepoints measured (data not shown). Activation of another known regulator, GSK3β, which has been shown to inhibit catabolic activity of AMPK by associating with the β-subunit and phosphorylating it at Thr479 of the α-subunit [21], was also unchanged (S1B Fig) at all timepoints measured. Since glycogen has been shown to inhibit AMPK by associating with the CBD of the β-subunit [12], we measured muscle glycogen content following a 30 or 60 min incubation in 25 vs. 5.5mM glucose. We found that muscle glycogen was significantly increased at both timepoints, suggesting that glycogen may be responsible for the early and sustained inhibition of AMPK (Fig 3A).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127388.g003: 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.
Mentions: In search for additional factors responsible for the decrease in AMPK phosphorylation caused by a high glucose concentration, we assessed cellular energy state. In keeping with previous observations [15], we found no differences in tissue concentrations of ATP, ADP, AMP or CrP in muscles incubated with 25 vs. 5.5mM glucose for 30min, 1h, or 2h (Table 2). The abundance of CAMKKβ, a known upstream kinase of AMPK, was unchanged at all timepoints measured (data not shown). Activation of another known regulator, GSK3β, which has been shown to inhibit catabolic activity of AMPK by associating with the β-subunit and phosphorylating it at Thr479 of the α-subunit [21], was also unchanged (S1B Fig) at all timepoints measured. Since glycogen has been shown to inhibit AMPK by associating with the CBD of the β-subunit [12], we measured muscle glycogen content following a 30 or 60 min incubation in 25 vs. 5.5mM glucose. We found that muscle glycogen was significantly increased at both timepoints, suggesting that glycogen may be responsible for the early and sustained inhibition of AMPK (Fig 3A).

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