Prior treatment with the AMPK activator AICAR induces subsequently enhanced glucose uptake in isolated skeletal muscles from 24-month-old rats

Abstract

5′ AMP-activated protein kinase (AMPK) activation may be part of the exercise-induced process that enhances insulin sensitivity. Independent of exercise, acute prior treatment of skeletal muscles isolated from young rats with a pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), causes subsequently improved insulin-stimulated glucose uptake (GU). However, efficacy of a single prior AICAR exposure on insulin-stimulated GU in muscles from old animals has not been studied. The purpose of this study was to determine whether brief, prior exposure to AICAR (3.5 hours before GU assessment) leads to subsequently increased GU in insulin-stimulated skeletal muscles from old rats. Epitrochlearis muscles from 24 month-old male rats were isolated and initially incubated ±AICAR (60 minutes), followed by incubation without AICAR (3 hours), then incubation ±insulin (50 minutes). Muscles were assessed for GU (via 3-O-methyl-[3H]-glucose accumulation) and site-specific phosphorylation of key proteins involved in enhanced GU, including AMPK, Akt, and Akt substrate of 160 kDa (AS160), via western blotting. Prior ex vivo AICAR treatment resulted in greater GU by insulin-stimulated muscles from 24 month-old rats. Prior AICAR treatment also resulted in greater phosphorylation of AMPK (T172) and AS160 (S588, T642 and S704). GLUT4 protein abundance was unaffected by prior AICAR and/or insulin treatment. These findings demonstrate that skeletal muscles from older rats are susceptible to enhanced insulin-stimulated GU after brief activation of AMPK by prior AICAR. Consistent with earlier research using muscles from young rodents, increased phosphorylation of AS160 is implicated in this effect that was not attributable to altered GLUT4 glucose transporter protein abundance.

Serum Is Not Necessary for Prior Pharmacological Activation of AMPK to Increase Insulin Sensitivity of Mouse Skeletal Muscle

“…The data presented herein oppose the findings by Fisher et al [ 14 ], though the muscles studied differed with regards to type, species, and gender. However, a recent study reported that prior AICAR stimulation improved insulin sensitivity of rat epitrochlearis muscle in the absence of serum [ 20 ], emphasizing that the discrepancies observed between the present study and that of Fisher et al [ 14 ] are not due to differences in muscle type or species. Since Oki et al [ 20 ] and Fisher et al [ 14 ] investigated muscle from old (24 months-old) and young (likely ~6 weeks-old) rats, respectively, the observed difference could be due to an effect of age somehow affecting the necessity of a serum factor to mediate improvements in muscle insulin sensitivity after prior AICAR stimulation.…”

“…However, a recent study reported that prior AICAR stimulation improved insulin sensitivity of rat epitrochlearis muscle in the absence of serum [ 20 ], emphasizing that the discrepancies observed between the present study and that of Fisher et al [ 14 ] are not due to differences in muscle type or species. Since Oki et al [ 20 ] and Fisher et al [ 14 ] investigated muscle from old (24 months-old) and young (likely ~6 weeks-old) rats, respectively, the observed difference could be due to an effect of age somehow affecting the necessity of a serum factor to mediate improvements in muscle insulin sensitivity after prior AICAR stimulation. However, the mice used in the present study were young, suggesting that, at least in mice, the ability for pharmacological AMPK activation to increase skeletal muscle insulin sensitivity in the absence of serum is not restricted to aged muscle.…”

“…Evidence implies that a serum factor is necessary for improving insulin sensitivity after contraction of isolated rodent skeletal muscle [ 13 , 14 , 15 , 16 , 19 ] as well as after AICAR stimulation of rat skeletal muscle [ 14 ]. One study recently questioned this requirement of a serum factor by demonstrating improved insulin sensitivity after prior AICAR stimulation of isolated skeletal muscle from old rats in the absence of serum [ 20 ]. Therefore, we investigated whether the effect of AICAR on insulin sensitivity for glucose uptake in isolated mouse extensor digitorum longus (EDL) was dependent on the presence of serum, as this is currently unknown.…”

AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase

“…We therefore determined the GTPase activity of fulllength TBC1D4 protein toward recombinant Rab10 in vitro. Purified GST-Rab10 was loaded with [γ- 32 P]GTP, incubated with purified full-length TBC1D4 protein, and the rate of [ 32 P] phosphate release resulting from hydrolysis of [γ- 32 P]GTP was measured as described previously (7). Figure 2, A and B illustrate that purified GST-Rab10 exhibited some endogenous GTPase activity, but the initial velocity of GTP hydrolysis was substantially increased in the presence of full-length TBC1D4.…”

Cooperative actions of Tbc1d1 and AS160/Tbc1d4 in GLUT4-trafficking activities

“…Although the difference was subtle, we observed statistically significant differences in the phosphorylation at Ser 704 of AS160 in some cases. Phosphorylation of this site has been reported to be modulated by exercise and its related signals (26,27). In fact, AS160 has several additional phosphorylation sites, and phosphorylation at some of these sites is reported to be sustained after several hours of exercise (28 -32), including Ser 341 as well as Ser 704 (26).…”