1 Department of Kinesiology, University of Wisconsin-Madison, Madison, Wisconsin
2 Division of Kinesiology, University of Michigan, Ann Arbor, Michigan
3 Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire
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ABSTRACT |
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The signaling pathways that mediate insulins many actions remain incompletely understood, but the following sequence has been well characterized: insulin binds to its receptor, leading to receptor autophosphorylation and activation of receptor tyrosine kinase, which in turn results in tyrosine phosphorylation of endogenous substrates including insulin receptor substrate proteins. These docking proteins engage downstream signaling molecules such as phosphatidylinositol (PI) 3-kinase (13). PI 3-kinase catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate on the D3 position of inositol, and the resultant PI 3,4,5-trisphosphate binds and activates more distal signaling proteins, including phosphoinositide-dependent kinase-1 and Akt, a serine/threonine kinase. Akt has been implicated as a key signaling protein for several of insulins actions, including activation of glycogen synthesis, protein synthesis, and GLUT4 translocation to the cell surface, thereby increasing glucose transport (15). Identification of the intermediate signaling steps linking Akt to insulins diverse actions remains incomplete.
Recent research using 3T3-L1 adipocytes demonstrated that insulin leads to the phosphorylation of multiple proteins that contain one or more consensus sequences for phosphorylation by Akt (6). Among these Akt substrates was a 160-kDa protein, named Akt substrate of 160 kDa (AS160), which contained six Akt consensus sequences that become phosphorylated in insulin-treated adipocytes. AS160 also includes a GTPase-activating domain for small G-proteins, known as Rabs, which participate in vesicular trafficking (7). A point mutation of two or more of the consensus phosphorylation sites for Akt resulted in a marked decline in insulin-stimulated GLUT4 redistribution to the cell surface. Thus, in 3T3-L1 adipocytes, phosphorylation of AS160 was strongly implicated as an intermediate step linking insulins activation of Akt to increased glucose transport. More recently, Zeigerer et al. (8) demonstrated that AS160 is important for insulins activation of GLUT4 vesicle exocytosis without altering insulin-mediated inhibition of GLUT4 internalization.
Insulin also activates Akt in skeletal muscle (9,10), and AS160 is expressed by this tissue (6), which is a major target for insulin action. An important question is this: Does insulin, in skeletal muscle, lead to increased phosphorylation of AS160 and/or other proteins containing the Akt phosphomotif? Accordingly, our first aim was to characterize, in skeletal muscle, the time course and dose response for insulin on phosphorylation of substrates of Akt.
Several studies have indicated that, in addition to its activation by insulin, Akt can be activated in skeletal muscle by in vitro contractile activity or in vivo exercise (9,11,12). Therefore, the second major aim of this study was to determine if AS160 or other proteins containing the Akt phosphomotif become phosphorylated in response to muscle contraction. For both the insulin and contraction experiments, we also evaluated the effects of these stimuli on Akt phosphorylation, so that we could assess the relationship between phosphorylation of Akt and its putative substrates, and determined if wortmannin influenced their effects to assess the possible involvement of PI 3-kinase.
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RESEARCH DESIGN AND METHODS |
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Animal treatment.
Research protocols were approved by the University of Wisconsin-Madison Research Animal Review Committee and the University Committee on the Use and Care of Animals at the University of Michigan. Male Wistar rats purchased from Harlan (St. Louis, MO) were housed in a 12 h:12 h/light:dark cycle (lights off at 1800) and allowed free access to HarlanTeklad rodent food (Madison, WI) until 1700 the night before the experiment, when they were restricted to 5 g of food. The next day between 1030 and 1330, rats (150190 g body wt) were anesthetized with an intraperitoneal injection of sodium pentobarbital (70 mg/kg wt). While rats were under deep anesthesia, both epitrochlearis muscles were rapidly dissected out.
Muscle incubations for insulin stimulation.
Epitrochlearis muscles used to study the effects of insulin were incubated in glass vials containing Krebs-Henseleit buffer (KHB) + 0.1% BSA supplemented with 8 mmol/l glucose (KHB + BSA + glucose) or were shaken 60 min in a water bath at 35°C with continuous gassing (95% O2/5% CO2). Temperature, shaking, and gassing remained constant throughout all subsequent incubations. After the initial incubation, some muscles were immediately blotted, rapidly trimmed of connective tissue, and freeze-clamped with liquid N2cooled aluminum tongs and then stored at 80°C until subsequent homogenization and analysis. These muscles were used for basal (no insulin) values. To determine the time course for insulin-stimulated phosphorylation of Akt and its putative substrates, other muscles were transferred to a vial containing KHB + BSA + glucose supplemented with 120 nmol/l insulin for 1, 2.5, 5, 10, 15, 30, or 60 min.
Other muscles, used to characterize insulins dose response for signaling, underwent an initial 60-min incubation before being transferred to vials containing KHB + BSA + glucose and varying insulin concentrations (0, 0.15, 0.3, 0.6, 12, or 120 nmol/l) for a 30-min incubation period. Subsequently, all muscles were rapidly blotted, trimmed of connective tissue, and freeze-clamped with liquid N2cooled aluminum tongs then and stored at 80°C until subsequent homogenization and analysis.
The PI 3-kinase inhibitor wortmannin was dissolved in DMSO at 1 mmol/l and stored at 20°C until used. Muscles were incubated in vials containing KHB + BSA + glucose supplemented with wortmannin (final concentration of 500 nmol/l) or an equal volume of vehicle (DMSO; 0.05% final concentration) for 30 min at 35°C and then transferred to another vial with the same solution as the preceding vial but with or without insulin (120 nmol/l) supplementation for 30 min. Muscles were then frozen and stored as described above.
In vitro muscle contractions.
Muscles dissected from other rats were used to study the effects of in vitro contractile activity. Both epitrochlearis muscles from each rat were mounted in a temperature-controlled bath. The distal end of the muscle was attached to a glass rod, and the proximal end was attached to a force transducer (Radnoti, Litchfield, CT) as previously described (13). The mounted muscles were preincubated for 30 min in KHB + 2 mmol/l Na pyruvate with continuous gassing of 95% O2/5% CO2 before the contraction protocol was begun. One muscle was then stimulated to contract (Grass S48 Stimulator; Grass Instruments, Quincy, MA) for 5 min in fresh KHB + 2 mmol/l Na pyruvate using a protocol previously described by Sakamoto et al. (9): pulse duration of 0.1 ms, pulse rate of 100 pulses/s, train duration of 10 s, train rate of 2/min, train duration of 10 s. The contralateral muscle remained attached in the bath for 5 min with passive tension set at 0.4 g but was not stimulated to contract. Immediately after the 5-min period of contraction or passive tension, the muscles were blotted, trimmed of connective tissue, frozen using liquid N2cooled tongs, and stored at 80°C until homogenization.
To determine the effect of wortmannin on contraction-stimulated phosphorylation of Akt and AS160, paired muscles were attached to the force transducer as described above and incubated for 30 min in KHB + 2 mmol/l Na pyruvate supplemented with wortmannin (100 or 500 nmol/l) or an equal volume of vehicle (DMSO). Muscles were then stimulated to contract as described above. Other muscles, serving as resting controls, were incubated with wortmannin or DMSO for the same duration as the contracting muscles before being blotted, trimmed, frozen, and stored at 80°C.
Muscle incubations for 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside stimulation.
Paired muscles were incubated in vials containing KHB and 8 mmol/l glucose for 30 min at 35°C. Muscles were then transferred to another vial containing KHB + glucose, with or without 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) (2 mmol/l) for 40 min at 35°C. Subsequently, muscles were blotted, trimmed of connective tissue, frozen using liquid N2cooled tongs, and stored at 80°C until homogenization.
Homogenization.
Frozen muscles were homogenized in 0.6 ml ice-cold homogenization buffer (20 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaCl, 1% NP40, 2 mmol/l Na3VO4, 10 mmol/l NaF, 2 mmol/l EDTA, 2 mmol/l EGTA, 2.5 mmol/l NaPP, 20 mmol/l ß-glycerophosphate, 2 mmol/l PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin) using glass-on-glass tubes (Kontes, Vineland, NJ). Homogenates were subsequently rotated at 4°C for 1 h before being centrifuged (12,000g for 10 min at 4°C). A portion of the resultant supernatant was used to determine protein concentration by the bicinchoninic acid assay (14), and the remaining supernatant was stored at 80°C until it was further analyzed.
Immunoprecipitation.
Homogenized muscle lysate (200250 µg protein at 1 mg/1 ml) was incubated with 1.52 µg of anti-AS160 at 4°C with gentle rotation overnight, and then 100 µl of a 50% slurry of prewashed protein A agarose beads (Upstate, Lake Placid, NY) was added to the lysate + antibody mix. The lysate + antibody + bead mix was rotated at 4°C for 2 h before centrifugation at 2,600g, and the supernatant was aspirated. After washing (nine times with 300 µl homogenization buffer), the protein bound to the protein A beads was eluted with 50 µl 2 x SDS loading buffer and boiled before loading on a polyacrylamide gel.
Immunoblotting.
After separation using SDS-PAGE, proteins were electrophoretically transferred to nitrocellulose, which was then rinsed with Tris-buffered saline plus Tween (TBST) (0.14 mol/l NaCl, 0.02 mol/l Tris base, pH 7.6, and 0.1% Tween), blocked with 5% nonfat dry milk in TBST for 1 h at room temperature, washed 3 x 5 min at room temperature, and treated with the primary antibody anti-PAS (1:1,000 in TBST + 5% BSA) overnight at 4°C. Blots were then washed 3 x 5 min with TBST; incubated with the secondary antibody, goat anti-rabbit IgG HRP conjugate (Upstate, Lake Placid, NY) (1:5,000 in TBST + 5% milk), for 1 h at room temperature; washed again 3 x 5 min with TBST; and developed with enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Densitometry.
Protein bands were quantified by the densitometric method (Bio-Rad GS-670; Bio-Rad, Hercules, CA). The amount of protein loaded in each lane for immunoblotting was within the linear range (i.e., for protein loaded vs. band density) using antibodies against P-Akt, PAS, and P-AMPK. Band densities were expressed relative to the respective basal values within each blot.
Statistical analysis.
Statistical analyses were done using Sigma Stat version 2.0 (San Rafael, CA). Data are expressed as means ±SE. One-way ANOVA was used to determine significant differences in protein phosphorylation in the insulin time course and dose-response experiments. When data failed the Levene Median test for equal variance, the Kruskal-Wallis nonparametric ANOVA on ranks was used. A P value 0.05 was considered statistically significant. As appropriate, parametric (Dunnett) and nonparametric (Dunn) post hoc methods were used to identify which insulin-treated groups were significantly different from basal. Two-way ANOVA was used to determine significant differences in the experiments that evaluated wortmannins effects on AS160 phosphorylation in insulin-stimulated or contraction-stimulated muscles, and the source of significant (P
0.05) variance was detected with Tukeys post hoc test. For insulin-stimulated phosphorylation of AS160, a t test was used to compare basal and insulin-treated muscles. For contraction-stimulated effects on phosphorylation of Akt and its substrates and for AICAR-stimulated effects on phosphorylation of AMPK, Akt, and AS160, a paired t test was used to compare muscles stimulated by contraction or AICAR with their paired nonstimulated muscles. A paired t test was also used for phosphorylation of Akt in muscles stimulated by contraction or insulin compared with their contralateral muscles stimulated identically, but in the presence of wortmannin.
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RESULTS |
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Insulin dose response.
Akt-Ser phosphorylation was not significantly increased above basal with 0.15 or 0.3 nmol/l insulin, although there was a trend to increase at these insulin concentrations (Fig. 2A). A significant increase, sevenfold above basal, was evident with 0.6 nmol/l insulin (P < 0.05). Akt-Ser phosphorylation was even more dramatically elevated above basal with 12 nmol/l (16.2-fold) and 120 nmol/l (20.8-fold) insulin (P < 0.05).
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Insulin-induced phosphorylation of AS160.
To confirm that the PAS immunoreactivity in the 160-kDa band was AS160, samples were immunoprecipitated using an antibody against AS160 and then immunoblotted with
PAS. With this approach, muscles that had been incubated with 0.6 nmol/l insulin had a 2.0-fold increase in AS160 phosphorylation over basal (P < 0.01; Fig. 3A). A similar increase above basal (1.9-fold) was found with 120 nmol/l insulin (P < 0.01; Fig. 3B).
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Wortmannin effect on contraction-stimulated muscles.
Akt-Ser phosphorylation in contraction-stimulated muscles (3.69 ± 0.84; relative to basal = 1.00) was eliminated (P < 0.05) in paired muscles (n = 6) that underwent contraction in the presence of 500 nmol/l wortmannin (1.13 ± 0.33). Wortmannin at either 100 or 500 nmol/l eliminated the 2.4-fold contraction-stimulated increase above basal in AS160 phosphorylation; therefore, pooled data from both concentrations are shown in Fig. 7 (contraction without wortmannin vs. all other groups; P < 0.001).
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DISCUSSION |
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The time course for insulins effects on Akt phosphorylation was slightly more rapid than the results reported by Song et al. (10), likely because of the higher incubation temperature in our experiments (35°C compared with 30°C). The significant increase in Akt-Ser phosphorylation with 0.6 nmol/l insulin is in agreement with results from Derave et al. (15), who found that Akt activity was also increased in perfused rat hindlimb with 0.6 nmol/l insulin. The current study appears to be the first published characterization of the insulin dose response for Akt-Ser phosphorylation in isolated skeletal muscle.
Immunoblotting with PAS revealed that two proteins,
250 kDa (PAS-250) and
160 kDa (PAS-160), became phosphorylated in response to insulin, with peak values at 10 and 15 min, respectively. Akt-Ser phosphorylation increased more rapidly (peaked at 5 min), consistent with a mechanism whereby insulin-stimulated Akt-Ser phosphorylation was required for subsequent phosphorylation of PAS-250 and PAS-160. The significantly increased phosphorylation of PAS-250 and PAS-160 with 0.6 nmol/l insulin indicates that these modifications may be physiologically relevant for insulin action in skeletal muscle.
Using the PAS antibody with 3T3-L1 adipocytes, Kane et al. (6) identified seven insulin-responsive putative Akt substrates, including proteins of 160 and 250 kDa. We confirmed that the 160-kDa protein that responded to insulin in skeletal muscle was AS160, consistent with the results in adipocytes. The 250-kDa protein in 3T3-L1 adipocytes was recently identified by mass spectrometry and found to include a predicted GTPase-activated domain for Rheb and Rap (16). Future research will be needed to determine if the insulin-responsive 250-kDa protein in skeletal muscle corresponds to the insulin-responsive 250-kDa protein found in 3T3-L1 adipocytes. Gridley et al. (16) identified a 105-kDa protein of unknown function that, in 3T3-L1 adipocytes, responded to insulin with elevated immunoreactivity against the PAS antibody, and they found the protein to be highly abundant in skeletal muscle, although they did not assess insulins effects in muscle. We consistently observed strong PAS immunoreactivity at
100 kDa that did not respond to any of our interventions (data not shown).
As expected, wortmannin inhibited the effect of insulin on Akt-Ser phosphorylation. Wortmannin also blocked insulin-stimulated phosphorylation of AS160, in support of the idea that AS160 was phosphorylated by Akt. Furthermore, taken together, our results are consistent with the hypothesis that AS160 phosphorylation is important for insulin-stimulated glucose transport in skeletal muscle, as it is in 3T3-L1 adipocytes.
Our data in epitrochlearis muscles that were stimulated to contract confirm the findings of Sakamoto et al. (9) for rat extensor digitorum longus muscles: in both muscles, in vitro contraction induced a rapid increase in Akt-Ser phosphorylation. While immunoblotting with PAS, we identified two putative Akt substrates (160- and 250-kDa proteins) that were significantly phosphorylated in response to in vitro contraction. Immunoprecipitation with
AS160 and subsequent immunodetection using
PAS demonstrated that the contraction-responsive protein of 160 kDa was AS160.
Wortmannin blocked contraction-stimulated Akt-Ser phosphorylation in agreement with observations of Sakamoto et al. (9), who found that wortmannin (100 or 500 nmol/l) eliminated the contraction-induced increase in Akt activity. Contractile activity does not increase class IA (9) or class II (17) PI 3-kinase activity, leading Sakamoto et al. to suggest that muscle contractions may activate class IB PI 3-kinase, leading ultimately to Akt phosphorylation (9). Regardless of the mechanism for activating Akt, we found that wortmannin also inhibited contraction-stimulated phosphorylation of AS160. This result is especially notable because wortmannin (1002,000 nmol/l) does not inhibit contraction-stimulated glucose transport by isolated skeletal muscle (1821). The phosphomotifs on AS160 identified by PAS immunoreactivity, which were responsive to contractile activity and inhibited by wortmannin, are evidently not essential for contraction-stimulated glucose transport. It remains possible that regulation of AS160 is relevant for contraction-stimulated glucose transport, e.g., by Akt-independent phosphorylation on sites not recognized by PAS.
As expected, AICAR treatment resulted in a robust increase in AMPK phosphorylation, concomitant with no detectable change in Akt-Ser phosphorylation. Therefore, the effect of AICAR on AS160 phosphorylation was apparently attributable to AMPK activation and independent of Akt activation. In support of this interpretation, purified AMPK can phosphorylate purified AS160 in vitro, as detected with the PAS antibody (H. Sano, G.E.L., unpublished data). Both AMPK and Akt have been demonstrated to phosphorylate Ser466 of the cardiac isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (22), providing a precedent for the idea that these kinases also share a common phosphorylation site on AS160. However, it is also possible that AMPK and Akt phosphorylate different AS160 sites that are immunoreactive with the PAS antibody.
Wortmannin does not inhibit the AMPK activity of purified enzymes (23) or the AMPK activity from H-2Kbcells stimulated by hyperosmotic stress (24). Thus, the wortmannin-induced inhibition of AS160 phosphorylation in contraction-stimulated muscles was likely the result of blocking the PI-3 kinasephosphoinositide-dependent kinase-1Akt signaling pathway and not inhibition of AMPK. However, it is unclear why a residual effect of AMPK on AS160 phosphorylation was not detected in the muscles incubated with wortmannin during contractile activity, which is known to activate AMPK (18,25).
An 180-kDa protein tended to have a contraction-induced increase in immunoreactivity against PAS, but there was no detectable response to insulin in this protein, even at supraphysiological concentrations of insulin, either in isolated skeletal muscle or 3T3-L1 adipocytes (6,7), indicating that increased Akt-Ser phosphorylation was insufficient for increasing PAS immunoreactivity of this protein. It seems worthwhile to pursue the identity of PAS-180. One candidate is tuberin, a protein (molecular weight of 180200 kDa) that is a substrate for Akt in HEK-293 and HeLa cells (26,27). Tuberin has multiple roles, including inhibition of cell growth and tumorigenesis, and phosphorylation of tuberin by Akt is thought to relieve tuberin-mediated cell growth inhibition (28). Additional studies will be needed to determine if PAS-180 is tuberin and, if so, to understand why insulin did not cause a detectable increase in its PAS immunoreactivity in isolated skeletal muscle.
In conclusion, two putative Akt substrates, PAS-250 and AS160, were phosphorylated in insulin-stimulated skeletal muscle with time courses and dose responses that are consistent with insulins rapid physiological actions. Although the specific bioeffects influenced by the phosphorylation of these proteins in skeletal muscle have not been established, in light of results with 3T3-L1 adipocytes (68), it seems reasonable to suspect that phosphorylation of AS160 in skeletal muscle is part of the insulin signaling pathway leading to stimulation of GLUT4 translocation and glucose transport. AS160 phosphorylation was also increased by in vitro contractile activity or incubation with AICAR, although only contraction led to elevated Akt-Ser phosphorylation. The contraction-induced increase in AS160 phosphorylation was inhibited by wortmannin, indicating that contractions effect on phosphorylation of AS160 was likely attributable to PI 3-kinasemediated activation of Akt. Future research will be needed to fully understand these results, e.g., to determine if insulin, contractile activity, and AICAR act on distinct or overlapping pools of AS160; to identify the specific phosphomotifs on AS160, as detected by PAS immunoreactivity, that are modulated by each stimulus; and, ultimately, to determine the bioeffects influenced by AS160 and other novel Akt substrates in skeletal muscle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address correspondence and reprint requests to Gregory D. Cartee, PhD, University of Michigan Kinesiology, Rm. 3040E, 401 Washtenaw Ave., Ann Arbor, MI 48109-2214. E-mail: gcartee{at}umich.edu
Received for publication March 23, 2004 and accepted in revised form October 4, 2004
AICAR, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; AS160, Akt substrate of 160 kDa; KHB, Krebs-Henseleit buffer; PAS, phospho-(Ser/Thr) Akt substrate; PI, phosphatidylinositol; TBST, Tris-buffered saline plus Tween
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REFERENCES |
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