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/ß, and Glycogen Synthase in Isolated Rat Soleus Muscle
School of Kinesiology and Health Science, York University, Toronto, Canada N3J 1P3
Address all correspondence and requests for reprints to: Rolando B. Ceddia, Department of Kinesiology and Health Science, York University, 4700 Keele Street, Toronto, Ontario, Canada N3J 1P3. E-mail: roceddia{at}yorku.ca.
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60%) insulin-stimulated glycogen synthesis and completely prevented the increase in glycogen content induced by insulin. AICAR did not affect either basal or insulin-stimulated glucose uptake but significantly increased insulin-stimulated (20%) lactate production in soleus muscles. Interestingly, basal glucose uptake was significantly increased (1.4-fold) in the epitrochlearis muscle, even though neither basal nor insulin-stimulated rates of glycogen synthesis, glycogen content, and lactate production were affected by AICAR. We also report the novel evidence that AICAR markedly reduced insulin-induced Akt-Thr308 phosphorylation after 15 and 30 min exposure to insulin, which coincided with a marked reduction in glycogen synthase kinase 3 (GSK)-3
/ß phosphorylation. Importantly, phosphorylation of glycogen synthase was increased by AICAR treatment 45 min after insulin stimulation. Our results indicate that AICAR-induced AMP-activated protein kinase activation caused a time-dependent reduction in Akt308 phosphorylation, activation of glycogen synthase kinase-3/ß, and the inactivation of glycogen synthase, which are compatible with the acute reduction in insulin-stimulated glycogen synthesis in oxidative but not glycolytic skeletal muscles. |
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) and two regulatory subunits (ß and 
). AMPK is activated by a rise in the AMP to ATP ratio of the cell and also via phosphorylation of the -subunit by upstream kinases. Once active, AMPK shuts down anabolic pathways and increases catabolic reactions in an attempt to restore the intracellular AMP to ATP ratio (1, 2). A commonly used pharmacological agent to induce activation of AMPK is the compound 5-aminoimidasole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), which has frequently been used to characterize the effects of AMPK activation on glucose homeostasis in a variety of tissues (1, 2). AICAR is rapidly taken up by cells and phosphorylated to form 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuronosil-5'-monophosphate, an AMP mimetic. 5-Aminoimidazole-4-carboxamide-1-ß-D-ribofuronosil-5'-monophosphate induces activation of AMPK without altering intracellular ATP and AMP levels (1). It has been repeatedly reported by in vitro and in vivo studies that AICAR-induced AMPK phosphorylation/activation causes an increase in glucose uptake in skeletal muscles (3, 4, 5), improves insulin sensitivity in obese ZDF rats (6) and human type 2 diabetics (7). To date, there seems to be a consensus that AMPK activation leads to an increase in glucose uptake in skeletal muscle. Based on these evidences, AMPK has emerged as a potential drug target for the treatment of metabolic diseases such as insulin resistance and type 2 diabetes mellitus (8). In recent years, the major research focus has been on the effects of AMPK activation on basal and insulin-stimulated glucose uptake with very little being published regarding the metabolic fate of glucose in muscle cells, especially regarding the effects of AMPK activation on muscle glycogen synthesis.
Because AMPK is activated under conditions of cellular stress to promote ATP synthesis and restore the AMP to ATP ratio, it is expected that energy consuming processes, such as glycogen synthesis, would be shut down by activation of AMPK in skeletal muscle (9). In support of this hypothesis, Carling and Hardie (10) provided evidence that AMPK phosphorylates isolated and purified glycogen synthase (GS), therefore decreasing its activity. However, subsequent in vitro and in vivo studies provide conflicting results regarding the role of AMPK activation in skeletal muscle glycogen metabolism. It has been demonstrated that incubation of isolated flexor digitorum brevis and epitrochlearis (fast twitch muscles) with AICAR did not alter either GS or glycogen phosphorylase activity (11). Glycogen phosphorylase, which catalyzes the degradation of glycogen to glucose-1-phosphate, is an important determinant of the rate of glycogenolysis. Activation of glycogen phosphorylase by AMPK is expected to lead to a reduction in glycogen content in skeletal muscle. In this context, Young et al. (12) demonstrated that either basal or insulin-stimulated glycogen synthesis were unaffected by AICAR treatment in soleus muscles (primarily slow twitch), even though glycogen phosphorylase activity and lactate production were increased. However, in this previous in vitro study reporting that acute AICAR treatment induced an increase in glycogen phosphorylase activity, glycogen content in skeletal muscle was not measured (12). To complicate this matter further, it has been demonstrated that rats chronically treated (5–28 d) with AICAR have increased (up to 2-fold) muscle glycogen content (13, 14, 15). Interestingly, white fast-twitch muscles elicit the most pronounced increases in glycogen content after chronic AICAR treatment (13, 14), suggesting important fiber type differences regarding the role of AMPK activation in muscle glycogen metabolism. Even though these in vivo studies provide relevant information regarding the metabolic responses to AICAR-induced AMPK activation, they do not allow for separation of direct from systemic effects of AICAR on skeletal muscle glycogen metabolism.
Another aspect that remains poorly explored is the role of AMPK activation in insulin-stimulated skeletal muscle glycogen synthesis and the impact on whole-body insulin-stimulated glucose homeostasis. This is particularly important, given the fact that insulin-stimulated muscle glycogen synthesis has been demonstrated to account for the majority of whole-body glucose uptake and virtually the entire nonoxidative glucose metabolism in both normal and diabetic subjects (16). In this context, it has been demonstrated that AICAR-induced AMPK activation causes phosphorylation of the insulin receptor substrate (IRS)-1 on Ser-789 residues in C2C12 myotubes, suggesting cross talk between AMPK and early steps of insulin signaling that could have important implications for glycogen metabolism (17). Importantly, there is evidence that serine-phosphorylated forms of IRS-1 fail to associate with an active phosphatidylinositol 3-kinase (PI3-kinase), resulting in decreased translocation of glucose transporters and other associated downstream events related to glucose metabolism (18). From a glycogen synthesis perspective, it is hypothesized that AMPK activation could cause IRS-1 phosphorylation on serine residues, impair downstream insulin signaling events that depend on PI3-kinase activation, reduce phosphorylation and inactivation of glycogen synthase kinase (GSK)-3, and cause inactivation of glycogen synthase in skeletal muscle. However, no data supporting this hypothesis have been published.
The experiments outlined in this investigation are designed to elucidate the effects of AICAR-induced AMPK activation on insulin-stimulated glycogen metabolism and major intracellular insulin-signaling events relevant to the regulation of glycogen synthesis in oxidative and glycolytic skeletal muscles. Here we show that AICAR-induced AMPK activation reduces insulin-stimulated glycogen synthesis in isolated soleus muscles. Additionally, we provide novel evidences that AMPK activation caused time-dependent alterations in insulin-induced Akt-Thr308, GSK-3/ß, and GS phosphorylation, which are compatible with the acute reduction in insulin-stimulated glycogen synthesis in oxidative skeletal muscles.
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Chemicals
AICAR was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Glycogen, fatty-acid-free albumin, amyloglucosidase, hexokinase, and glucose-6-phosphate dehydrogenase were obtained from Sigma (St. Louis, MO). Human insulin (Humulin R) was purchased from Eli Lilly Inc. (Toronto, Ontario, Canada). D-[U-14C]glucose was purchased from GE Healthcare Radiochemicals (Québec City, Québec, Canada). 2-[1,2-3H]deoxy-D-glucose and D-[1-14C]mannitol were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Lactate reagent and standards were purchased from Trinity Biotech (Berkeley Heights, NJ). ATP, nicotinamide adenine dinucleotide phosphate, mannitol, and 2-deoxy-D-glucose were obtained from BioShop Canada Inc. (Burlington, Ontario, Canada). All other reagents used for the experiments were of the highest grade available.
Muscle extraction and incubation
Before muscle extraction, all animals were anesthetized with a single ip injection of ketamine/xylazine (0.2 ml per 100 g body weight). Subsequently the soleus (15–20 mg) and epitrochlearis (10–15 mg) muscles were quickly extracted and mounted onto thin, stainless steel wire clips to maintain optimal resting length. The incubation procedures were performed as previously described (19). Briefly, immediately after extraction, the muscles were placed in plastic scintillation vials containing 2 ml of gassed [30 min with O2:CO2-95:5% (vol/vol)] Krebs-Hanseleit bicarbonate (KHB) buffer with 4% fat-free BSA and 6 mM glucose. The scintillation vials were then sealed with rubber stoppers, and gasification was continued for the entire 1-h preincubation period. After preincubation, the muscles were transferred to a second set of vials with 1.5 ml of KHB buffer containing D-[U-14C]glucose (0.2 µCi/ml). All muscles were maintained in either the absence or presence of the following conditions: insulin (100 nM), AICAR (2 mM), and AICAR plus insulin for the entire 1-h incubation. For the AICAR plus insulin conditions, all muscles were exposed to AICAR for 30 min before the addition of insulin.
Measurement of glycogen synthesis in isolated muscle
Glycogen synthesis was assessed by measuring the incorporation of D-[U-14C]glucose into glycogen as previously described (19). Briefly, upon termination of the incubation experiment as outlined above, muscles were quickly washed in ice-cold PBS, blotted on filter paper, frozen (N2), and digested in 0.5 ml of 1 M KOH at 70 C for 1 h. Of the digested muscle solution, aliquots were taken for protein determination (Bradford method), determination of glycogen content, and glycogen synthesis. Formation of glycogen from labeled glucose was estimated by adding 10 mg of carrier glycogen to the hydrolysates. Subsequently glycogen was precipitated overnight with 100% ethanol. The precipitate was resuspended in 0.5 ml of water and its radioactivity was determined using a scintillation counter (19).
Measurement of glycogen content and lactate production
After incubation of muscles in the presence of the various conditions outlined above, the muscles were digested in 0.5 ml of 1 M KOH. For analysis of glycogen content, the pH of muscle digest was titrated to 4.8 before the addition acetate of buffer (pH 4.8) and 0.5 mg/ml amyloglucosidase. Subsequently glycogen was hydrolyzed at 40 C for 2 h and glucose was analyzed enzymatically (20) and the absorbance read in a spectrophotometer (Ultraspec 2100 pro; Biochrom Ltd., Cambridge, UK) at 340 nm wavelength. Lactate concentration was measured in deproteinized and neutralized muscle incubation medium using a commercially available kit.
Measurement of glucose transport into muscle
Soleus and epitrochlearis muscles were extracted as described above. Subsequently they were preincubated for 1 h in KHB buffer containing 8 mM glucose, 32 mM mannitol, and 0.1% BSA. After the preincubation, all muscles were exposed for 1 h to the following conditions: insulin (100 nM), AICAR (2 mM), and AICAR plus insulin. All AICAR plus insulin conditions received AICAR for 30 min before the 1-h incubation period. After the incubation, all muscles were washed for 10 min in KHB buffer containing 40 mM mannitol at 29 C and, if present during the previous incubation period, insulin (100 nM) and AICAR (2 mM). For measurement of glucose transport, the muscles were transferred to new flasks and incubated for 20 min at 29 C in 1.5 ml KHB buffer containing 0.5 µCi/ml 2-[1,2-3H]deoxy-D-glucose (2-DG, and 8 mM nonlabeled 2-DG) and 0.1 µCi/ml [U-14C]mannitol as an extracellular space marker (21). To terminate the experiment, immediately after the 20-min glucose uptake period, muscles were blotted (4 C) and quickly frozen in liquid N2. Muscles were then digested in 0.5 ml of 1 M KOH at 70 C and centrifuged (1000 x g). Aliquots (450 µl) of the muscle extract supernatant and the incubation medium were counted for radioactivity using a scintillation counter with channels preset for simultaneous 3H and 14C counting. The amounts of D-[U-14C]mannitol and 2-DG present in the samples were used to calculate extracellular space and glucose transport, respectively, as previously described (22). The intracellular water content of the muscles was calculated subtracting the measured extracellular space water from total muscle water. Total water content was assumed to be 77%, which is the average value for soleus and epitrochlearis muscles after drying the tissues to a constant weight in our laboratory.
Western blot determination of phosphorylated (p)-AMPK, p-acetyl-CoA carboxylase (ACC), p-Akt (Thr308, Ser473), p-GSK-3/ß, p-GS, AMPK1, AMPK2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Soleus muscles were incubated in the absence or presence of insulin (100 nM), AICAR (2 mM), and AICAR plus insulin. All muscles were preincubated in KHB buffer as previously described. To investigate time-dependent alterations in phosphorylation levels of Akt (Thr308 and Ser473), GSK-3/ß, and GS, we exposed the muscles to insulin for 15, 30, and 45 min. AICAR was added to the incubation medium 30 min before adding insulin and remained in the medium thereafter. Control muscles received neither AICAR nor insulin. To test the effectiveness of AICAR to induce AMPK activation, we measured the phosphorylation state of ACC, a downstream target of AMPK (23). In addition, we also examined the distribution of AMPK1 and AMPK2 in soleus and epitrochlearis muscles to determine the fiber type-specific distribution of the two AMPK catalytic isoform subunits. Immediately after all treatments, muscles were frozen in liquid N2 and stored at –80 C until analysis. For preparation of muscle lysates, the solei and epitrochlearis muscles were homogenized in buffer containing 135 mM NaCl, 1 mM MgCl2, 2.7 mM KCl, 20 mM Tris (pH 8.0), 1% Triton X-100, 10% glycerol, and protease and phosphatase inhibitors (0.5 mM Na3VO4, 10 mM NaF, 1 µM leupeptin, 1 µM pepstatin, 1 µM okadaic acid, and 0.2 mM phenylmethylsulfonyl fluoride), and heated (65 C, 5 min). An aliquot of the homogenate was used to determine the protein concentration in each sample by the Bradford method.
Before loading onto SDS-PAGE gels, the samples were diluted 1:1 (vol/vol) with 2x Laemmli sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% (wt/vol) sodium dodecyl sulfate, 50 mM dithiothreitol, and 0.01% (wt/vol) bromophenol blue]. Aliquots of muscle homogenates containing 75 µg of protein were run through SDS-PAGE gels [12% for p-AMPK, 7.5% for P-ACC, and 10% for P-Akt (Thr308 and Ser473), P-GSK-3/ß, P-GS, and GAPDH] and then transferred to polyvinyl difluoride membranes (Bio-Rad Laboratories, Burlington, Ontario, Canada). The phosphorylation of AMPK was determined using a P-AMPK (Thr172) antibody, which detects AMPK only when activated by phosphorylation at Thr-172. The phosphorylation of Akt was determined using P-Akt (Thr308) and P-Akt (Ser473) antibodies, which detect Akt only when phosphorylated at Thr-308 or Ser-473. The phosphorylation of GSK was determined using a P-GSK-3/ß (Ser21/9) antibody, which detects GSK only when phosphorylated at Ser-21 and Ser-9. GS and ACC phosphorylation was detected using P-GS and P-ACC-specific antibodies, which recognize GS and ACC when phosphorylated at serine 641 (Ser641) and serine 79 (Ser79), respectively. Equal loading of all gels was confirmed by Coomassie staining of all gels and use of GAPDH as a loading control. Specific antibodies against P-Akt, P-GSK-3/ß, P-GS, and P-AMPK were purchased Cell Signaling Technology Inc. (Beverly, MA). Specific antibodies against the -1 and -2 subunits of AMPK were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). P-ACC was obtained from Upstate Biotechnology (Charlottesville, VA), and GAPDH was from Abcam, Inc. (Cambridge, MA). All antibodies were applied in a 1:1000 dilution, except GAPDH, which was used in a 1:5000 dilution.
Statistical analyses
All statistical analyses were performed by one-way ANOVAs followed by Fisher post hoc tests. The level of significance was set at P < 0.05.
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). Interestingly, whereas treatment with AICAR alone did not alter the basal rate of glycogen synthesis, it resulted in a approximately 60% reduction of glycogen synthesis in the presence of insulin. Treatment of epitrochlearis muscles with insulin resulted in a 1.8-fold increase in glycogen synthesis relative to control (Fig. 1B). AICAR treatment did not affect either basal or insulin-stimulated conditions in epitrochlearis muscles.
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27%) in glycogen content relative to control (Fig. 2A). Interestingly, AICAR completely reversed the increase in glycogen content induced by insulin in soleus muscles. Incubation of epitrochlearis muscles (Fig. 2B) with insulin, AICAR, and AICAR plus insulin did not have any effect on glycogen content.
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), incubation with insulin resulted in a significant, approximately 1.6-fold, increase in lactate production relative to control. The effect of AICAR on the basal condition showed a trend toward increased lactate production but did not reach statistical significance (Fig. 3A). However, incubation of the soleus with AICAR and insulin caused a 2-fold increase in lactate production relative to control, which was approximately 21% higher than that of insulin alone (Fig. 3A). The epitrochlearis muscles (Fig. 3B) also showed a similar increase in lactate production (1.7-fold) when treated with insulin. However, AICAR did not affect insulin-stimulated lactate production in these muscles (Fig. 3B).
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). The addition of AICAR to the medium did not affect either the basal or insulin-stimulated rate of glucose transport in the soleus (Table 1). As expected, epitrochlearis muscles elicited an increase in glucose uptake (1.7-fold) in the presence of insulin. AICAR treatment also caused a significant increase (1.4-fold) in glucose uptake in epitrochlearis muscles (Table 1). The addition of AICAR to the insulin-stimulated epitrochlearis muscles resulted in approximately 2.0-fold increase in glucose uptake. However, this increase was not statistically different from the insulin or AICAR only conditions.
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/ß, and GS in soleus muscles). In addition, we also examined the differential expression of the AMPK1 and AMPK2 catalytic subunits in both, soleus and epitrochlearis muscles. The AMPK1 content was markedly higher (1.6-fold) in epitrochlearis than soleus muscles, whereas AMPK2 appeared similar in both muscles (Fig. 4B). To determine whether the effects of AICAR-induced AMPK activation affected major intracellular pathways of insulin signaling, we examined the phosphorylation states of Akt Thr308, Akt Ser473, GSK-3/ß, and GS. As anticipated, exposure of the soleus muscle to insulin for 15 min resulted in an approximately 9-fold increase in Akt Thr308 phosphorylation, compared with control. Even though this effect decreased as exposure time to insulin increased, Akt Thr308 phosphorylation remained markedly elevated after 45 min of insulin exposure, compared with control (Fig. 4C). AICAR on its own did not cause any changes in the phosphorylation state of Akt Thr308. However, pretreatment of soleus muscles with AICAR resulted in approximately 33 and 55% reductions in Akt Thr308 phosphorylation after 15 and 30 min of insulin exposure, respectively (Fig. 4C). Insulin also increased Akt Ser473 phosphorylation (1.6-fold), but this was unaffected by AICAR. To further investigate the effects of AICAR on enzymes involved in glycogen metabolism, we examined the phosphorylation state of GSK-3/ß (Fig. 4C). This enzyme is a downstream target of Akt and is active when in its dephosphorylated state. Under control conditions, GSK-3/ß phosphorylation was virtually undetectable. A pronounced increase in the phosphorylation state of GSK-3 was observed in the presence of insulin (Fig. 4C). Interestingly, AICAR markedly reduced the 15-, 30-, and 45-min insulin-stimulated phosphorylation of GSK-3. The phosphorylation of GSK-3ß under insulin stimulation was not as high as GSK-3; however, the effect of AICAR on the insulin-stimulated condition followed a similar pattern to that of GSK-3 (Fig. 4C). These data indicate that AICAR prevented the insulin-induced suppression of GSK-3/ß activity in soleus muscles.
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). Interestingly, AICAR treatment initially decreased the GS phosphorylation after 15 and 30 min of insulin exposure. However, GS phosphorylation markedly increased after 45 min of exposure to insulin (Fig. 4C). |
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60%) the insulin-induced increase in glycogen synthesis. AICAR treatment also prevented the insulin-induced increase in glycogen content in soleus muscles. Interestingly, neither basal nor insulin-stimulated glucose uptake was affected by AICAR in soleus, whereas epitrochlearis muscles elicited a significant increase in this variable. This differential response to AICAR in isolated soleus and epitrochlearis muscles has also been reported in previous studies (24, 25); however, no data regarding the effects of AICAR on other glucose pathways have been reported in these studies. It has been argued that the lack of a stimulatory effect of AICAR on glucose transport in slow-twitch skeletal muscles could be due to fiber type-specific differences in the expression of AMPK subunits (24, 26). In fact, it has been demonstrated that the presence of all three AMPK subunits is required for AICAR-induced increases in glucose transport in mouse skeletal muscle (26). Here we report clear differences in AMPK subunit content in slow-twitch and fast-twitch muscles (Fig. 4B). Whereas AMPK2 was equally present in both muscles investigated, AMPK1 was faintly detected in soleus, which could justify the different responses in glucose uptake to AICAR observed here.
In our system, we also observed that insulin-stimulated lactate production was increased by AICAR treatment, and because AICAR did not affect insulin-stimulated glucose uptake, it indicates that AICAR-induced AMPK activation shifted insulin-stimulated glucose metabolism toward lactate production instead of accumulation as glycogen in isolated soleus muscles. However, the AICAR-induced reduction in glycogen synthesis cannot be completely accounted for by the increase in lactate production, suggesting that other pathways may also participate in this metabolic shift. Previous investigations have demonstrated that glucose oxidation increases in isolated soleus muscles exposed to AICAR (27), which may also have been the case in the present study. In addition, our data are in partial agreement with a previous in vitro study showing that AICAR increased lactate production in soleus muscles exposed to low concentrations of insulin (12). However, in their study neither basal nor insulin-stimulated rates of glycogen synthesis were affected by AICAR, even though an increase in glycogen phosphorylase activity was reported (12). This is intriguing because increased glycogen phosphorylase activity is expected to decrease glycogen synthesis. In our investigation, the marked AICAR-induced decrease in insulin-stimulated glycogen synthesis as well as suppression of the increase in glycogen content in solei is compatible with the increased glycogen phosphorylase activity previously reported by Young et al. (12). There are important methodological differences between our study and the one by Young et al. (12) that may justify the differences regarding the effect of AICAR on insulin-stimulated glycogen synthesis. We used intact soleus muscle (15–20 mg) from 40- to 60-g rats, whereas Young et al. (12) used muscle strips from 130- to 140-g rats. Splitting the muscles may have influenced the response of the tissue to AICAR and insulin treatments. Also, the differences in AICAR concentrations (2 vs. 1 mM) may account for the differences in glycogen synthesis reported in these two independent studies.
Previous in vivo studies also reported that glycogen content is not affected by AICAR treatment in red and white gastrocnemius muscles, even though major time-dependent fiber type differences have been reported regarding the activity of GS and glycogen phosphorylase (11). Additionally, in the same study, in vitro incubations of two fast-twitch muscles (flexor digitorum brevis and epitrochlearis) with AICAR affected neither GS nor glycogen phosphorylase activities (11). This is in line with our in vitro data showing that basal glycogen content was not affected by acute AICAR treatment in isolated soleus and epitrochlearis muscles. However, in another in vivo study, it was actually reported that glycogen synthesis was increased in white but not red quadricep muscles after AICAR treatment (5). The reasons for these discrepant results are not clear but may be attributed to the use of different muscle groups in various studies. In fact, it has been reported that major differences in the expression of AMPK isoforms in skeletal muscles with distinct fiber type compositions exist (28, 29, 30). For instance, Ai et al. (30) showed that the epitrochlearis, composed predominantly of type II fibers, expresses more AMPK1 subunits than type I fibers, which is in agreement with data presented in this manuscript (Fig. 4B). On the other hand, type II fibers exhibit a 5-fold greater expression of the -3 subunit than type I fibers (31). Even though specific muscle groups have been classified according to fiber type composition, current data on the expression and activity of AMPK isoforms in different muscle fibers, and their respective functional implications, still lack clarity. Therefore, the hypothesis that differential expression/activity of AMPK isoforms is responsible for the dissimilar results regarding the effects of AICAR treatment on glycogen metabolism requires additional investigation.
To examine whether the effects of AICAR on insulin-induced glycogen synthesis in soleus muscles are fiber type specific, we also used epitrochlearis muscles in our experiments. We found that AMPK activation does not alter either the rate of glycogen synthesis or glycogen content in fast-twitch muscles under basal and insulin-stimulated conditions (Figs. 1B and 2B). Additionally, lactate production was also unaffected by AICAR, indicating that the metabolic shift toward lactate production does not seem to occur in epitrochlearis muscle (Fig. 3B). Previous investigations have shown that either ip (11) or sc (5) injections of AICAR increase blood lactate levels in rats. However, because these were in vivo studies, we do not know how much of this lactate was derived from muscles vs. other tissues that might also have been affected by AICAR treatment.
Here we show novel evidence that AICAR interferes with the intracellular insulin signaling steps that are crucial to regulate glycogen metabolism. As expected, after soleus muscles were exposed to insulin for 30 min, GS phosphorylation was decreased relative to control, therefore increasing its activity. Interestingly, insulin-induced reduction in phosphorylation of GS was even more accentuated after 15 and 30 min of AICAR treatment, suggesting an initial increase in GS activity in soleus muscles. However, this was not accompanied by an increase in glycogen synthesis (Fig. 1A) after 1 h incubation with AICAR. This suggests that the AICAR-induced increase in GS phosphorylation in soleus after 45 min of exposure to insulin overcame the initial reduction in phosphorylation at 15 and 30 min. Furthermore, the time-course incubation of soleus muscles for the purpose of insulin signaling determination was performed up to 45 min, whereas the rate of glycogen synthesis and glycogen content were assessed after 1 h incubation. This leaves an additional 15 min during which GS may have become even more deactivated. These data are in agreement with previous in vitro experiments showing that AMPK from rat liver phosphorylates GS purified from rabbit skeletal muscle (10). However, a time-course analysis of the effects of AMPK on GS phosphorylation was not performed in those experiments.
Because AMPK activation has also been shown to effect insulin signaling enzymes upstream of GS (17, 32, 33), we examined whether AICAR had similar effects on GSK-3 and -3ß phosphorylation. When activated (dephosphorylated), GSK-3 and -3ß phosphorylate/deactivate GS and hence lead to decreased glycogen synthesis. As expected, insulin elicited a powerful effect on GSK-3 phosphorylation, and remarkably, treatment of the insulin-stimulated condition with AICAR led to a pronounced decrease in phosphorylation of this enzyme. Phosphorylation of GSK-3ß followed a pattern similar to GSK-3; however, the former did not appear to be affected to the same extent by insulin. The decreased phosphorylation of GSK-3 and -3ß by AICAR in insulin-stimulated soleus muscles may have been due to a direct effect of AICAR on this enzyme (32). Data from Jurkat T cells demonstrates that AICAR can have a direct inhibitory effect on GSK-3 phosphorylation, thus increasing its activity. In addition, treatment of differentiated hippocampal neurons with AICAR led to dephosphorylation/activation of GSK-3 and -3ß (33). However, these findings have not been shown in skeletal muscle cells.
The suppressive effects of AICAR on GSK-3 phosphorylation could also be linked to alterations in activity of upstream kinases such as Akt. Therefore, we also examined the effects of AICAR on insulin-stimulated phosphorylation of Akt-Ser473 and Akt-Thr308. Once again we observed a transient high degree of phosphorylation of Akt-Thr308 with insulin after 15 min of incubation, which appeared to fade at 30 and 45 min. However, AICAR reduced the insulin-induced phosphorylation of Akt-Thr308 at both 15 and 30 min of incubation. Phosphorylation of Akt-Ser473 was not affected by AICAR at any time points. Our data are in partial agreement with a recent study (33) showing that AICAR treatment reduces phosphorylation of Akt-Thr308 and Akt-Ser473. However, it is important to point out that whereas we examined the effects of AICAR on insulin-stimulated muscle tissue, the investigation by King et al. (33) tested the effects of AICAR on basal Akt phosphorylation in hippocampal cells. The use of different tissues and administration of AICAR to basal or insulin-stimulated states may account for the differences between our investigation and that by King et al. (33).
Another plausible mechanism by which AICAR exerts its effects on insulin-stimulated glycogen synthesis is mediated by events that take place upstream of Akt. In fact, it has previously been shown that AMPK directly affects early insulin-signaling events by phosphorylating IRS-1 on the Ser789 residue (17). Even though phosphorylation of IRS-1 on Ser789 residue did not impair insulin signaling in C2C12 myotubes (17), other investigations have shown that serine phosphorylation of IRS-1 is linked to reduced capacity of insulin to activate PI3-kinase (34, 35). Additionally, AICAR has also been shown to have a direct effect on inhibiting GSK-3 phosphorylation, thus activating it (32, 33). In turn, activated GSK-3 phosphorylates IRS-1 on serine residues, and this has also been demonstrated to attenuate insulin signaling via its phosphorylation of IRS-1 in CHO cells (34). In our system, AICAR treatment elicited a marked reduction in GSK-3/ß phosphorylation, which may have also influenced early steps of insulin signaling in isolated soleus muscle and downstream events associated with glycogen synthesis.
In conclusion, we demonstrate that acute AMPK activation by AICAR leads to suppression of insulin-stimulated glycogen synthesis in soleus but not epitrochlearis muscles. Additionally, we provide novel evidence that AICAR-induced AMPK activation affects major steps of the insulin signaling cascade in skeletal muscle. The suppression of glycogen synthesis and decrease in glycogen content by AICAR in isolated rat soleus muscles is compatible with the transient and time-dependent reduction in Akt308 phosphorylation, activation of GSK-3 /ß, and the inactivation of GS reported here.
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Footnotes |
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Author disclosure summary: S.F., M.P.G., and R.B.C. have nothing to declare.
First Published Online July 27, 2006
Abbreviations: ACC, Acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuronosil; AMPK, AMP-activated protein kinase; 2-DG, 2-[1,2-3H]deoxy-D-glucose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GS, glycogen synthase; GSK, glycogen synthase kinase; IRS, insulin receptor substrate; KHB, Krebs-Hanseleit bicarbonate; p, phosphorylated; PI3-kinase, phosphatidylinositol 3-kinase.
Received April 12, 2006.
Accepted for publication July 18, 2006.
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References |
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3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle. J Biol Chem 279:38441–38447-subunit isoforms of AMP-activated protein kinase suggests a major role for 3 in white skeletal muscle. Am J Physiol Endocrinol Metab 286:E194–E200
phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin. J Biol Chem 276:3543–3549This article has been cited by other articles:
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  P. G. Cammisotto, I. Londono, D. Gingras, and M. Bendayan Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats Am J Physiol Renal Physiol, April 1, 2008; 294(4): F881 - F889. [Abstract] [Full Text] [PDF]
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 K. D. Folmes, L. A. Witters, M. F. Allard, M. E. Young, and J. R. B. Dyck The AMPK {gamma}1 R70Q mutant regulates multiple metabolic and growth pathways in neonatal cardiac myocytes Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3456 - H3464. [Abstract] [Full Text] [PDF]
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