Limits...
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

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 Ser485/491 (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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4440828&req=5

pone.0127388.g005: 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 Ser485/491 (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.

Mentions: We next assessed whether the high-glucose induced inhibition of AMPK was dependent on the activation of mTOR signaling. In our previous study, we found that the mTORC1 inhibitor rapamycin had no effect on a leucine-induced decrease in AMPK Thr172 phosphorylation. Here, we found that rapamycin (100μM) also did not prevent the reduction in P-AMPK Thr172 caused by incubation with 25mM glucose for 1h (Fig 5A), although the increase in mTOR Ser2448 phosphorylation was prevented (Fig 5B). Since mTOR/p70S6K signaling was shown to stimulate phosphorylation of AMPK at Ser485/491 in the hypothalamus in response to leptin, we evaluated whether rapamycin could prevent the high-glucose induced phosphorylation of this site. As shown in Fig 5C, rapamycin did not affect the increase in AMPK Ser485/491 phosphorylation, suggesting that mTOR/p70S6K is not responsible for the inhibitory phosphorylation in this setting.


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)

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 Ser485/491 (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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127388.g005: 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 Ser485/491 (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.
Mentions: We next assessed whether the high-glucose induced inhibition of AMPK was dependent on the activation of mTOR signaling. In our previous study, we found that the mTORC1 inhibitor rapamycin had no effect on a leucine-induced decrease in AMPK Thr172 phosphorylation. Here, we found that rapamycin (100μM) also did not prevent the reduction in P-AMPK Thr172 caused by incubation with 25mM glucose for 1h (Fig 5A), although the increase in mTOR Ser2448 phosphorylation was prevented (Fig 5B). Since mTOR/p70S6K signaling was shown to stimulate phosphorylation of AMPK at Ser485/491 in the hypothalamus in response to leptin, we evaluated whether rapamycin could prevent the high-glucose induced phosphorylation of this site. As shown in Fig 5C, rapamycin did not affect the increase in AMPK Ser485/491 phosphorylation, suggesting that mTOR/p70S6K is not responsible for the inhibitory phosphorylation in this setting.

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