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. 2010 Sep 1;299(5):C1171–C1179. doi: 10.1152/ajpcell.00514.2009

A role for AMPK in increased insulin action after serum starvation

James Kain Ching 1, Pooja Rajguru 1, Nandhini Marupudi 1, Sankha Banerjee 1, Jonathan S Fisher 1,✉
PMCID: PMC2980310  PMID: 20810907

Abstract

Serum starvation is a common cell culture procedure for increasing cellular response to insulin, though the mechanism for the serum starvation effect is not understood. We hypothesized that factors known to potentiate insulin action [e.g., AMP-activated protein kinase (AMPK) and p38] or to be involved in insulin signaling leading to glucose transport [e.g., Akt, PKCζ, AS160, and ataxia telangiectasia mutated (ATM)] would be phosphorylated during serum starvation and would be responsible for increased insulin action after serum starvation. L6 myotubes were incubated in serum-containing or serum-free medium for 3 h. Levels of phosphorylated AMPK, Akt, and ATM were greater in serum-starved cells than in control cells. Serum starvation did not affect p38, PKCζ, or AS160 phosphorylation or insulin-stimulated Akt or AS160 phosphorylation. Insulin had no effect on glucose transport in control cells but caused an increase in glucose uptake for serum-starved cells that was preventable by compound C (an AMPK inhibitor), by expression of dominant negative AMPK (AMPK-DN), and by KU55933 (an ATM inhibitor). ATM protein levels increased during serum starvation, and this increase in ATM was prevented by compound C and AMPK-DN. Thus, it appears that AMPK is required for the serum starvation-related increase in insulin-stimulated glucose transport, with ATM as a possible downstream effector.

Keywords: glucose transport, ataxia telangiectasia mutated phosphorylation, compound C, AMP-activated protein kinase

the practice of serum starvation of differentiated myotubes as a means of increasing insulin-stimulated glucose uptake was established by the work of Klip and colleagues (21) in the 1980s, and serum starvation is now widely implemented in experimental settings for which increased insulin action is desired. However, the underlying mechanisms that link serum starvation to increased insulin-stimulated glucose transport have not been defined. The critical role of insulin in the maintenance of blood glucose homeostasis by stimulating glucose uptake, mainly into skeletal muscle and fat tissues (17), emphasizes the need to fully understand the mechanisms of models reportedly involved in increasing insulin sensitivity.

Stimulation of glucose transport into skeletal muscle by insulin involves phosphorylation and activation of Akt and/or protein kinase C-ζ (PKCζ) (33). Akt activation by insulin likely increases cell surface localization of the glucose transport protein GLUT4 via phosphorylation and inactivation of the Akt substrate of 160 kDa (AS160) (23). Recently, ataxia telangiectasia mutated (ATM) has been directly implicated as a participant in insulin signaling leading to GLUT4 translocation and glucose transport (13) in myotubes, and animals or cell lines lacking ATM are resistant to insulin (29) or have reduced glucose tolerance (24). Thus, any one of these proteins (i.e., Akt, PKCζ, AS160, or ATM) could potentially play roles in the serum starvation-related increase in insulin action.

Activation of the AMP-activated protein kinase (AMPK) (8, 9, 16, 20) or p38 MAP kinase (p38) (11) reportedly enhances insulin-stimulated glucose transport. The growing realization that AMPK is a potential target for many antidiabetic drugs such as metformin and rosiglitazone (10, 40) further strengthens the implication of AMPK in increasing insulin sensitivity. AMPK plays an important role in regulating cellular energy homeostasis (14) and thus is an attractive candidate for mediation of the serum starvation effect on insulin action.

We hypothesized that factors known to potentiate insulin action [e.g., AMPK (9, 20) or p38 (11)] or known to be involved in insulin signaling leading to glucose transport [e.g., Akt, PKCζ, AS160, and ATM (13, 34)] would be phosphorylated during serum starvation and would be responsible for increased insulin action after serum starvation. The aim of the present study was to provide a mechanistic link between serum starvation and increased insulin action in L6 myotubes.

MATERIALS AND METHODS

Materials.

Dulbecco's modified Eagle's medium (DMEM), solution A, and trypsin were purchased from Washington University Tissue Culture Center (St. Louis, MO). Humalog insulin was obtained from Lilley (Indianapolis, IN). Cell culture dishes were obtained from Becton Dickinson Labware (Franklin Lakes, NJ). Reagents for enhanced chemiluminescence were obtained from PerkinElmer Life Sciences (Boston, MA). Pre-cast 4–20% Tris-HEPES polyacrylamide gels were obtained from NuSep (Austell, GA), while NuPAGE 3-8% Tris acetate minigels were obtained from Invitrogen (Carlsbad, CA). Antibodies recognizing AMPK, phosphorylated AMPK, Akt, phosphorylated Akt S473, phosphorylated Akt T308, p38, phosphorylated p38, PKCζ, phosphorylated PKCζ, phosphorylated acetyl-CoA carboxylase (p-ACC), phosphorylated ATM (S1981) and phosphorylated substrate of Akt (P-AS) were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against the Akt substrate of 160 kDa (AS160) were obtained from Upstate (Lake Placid, NY). An antibody against GAPDH was obtained from Novus Biologicals (Littleton, CO). Cytochalasin B, horseradish peroxidase-linked streptavidin, and antibodies against ATM were from Sigma (St. Louis, MO). Compound C and KU55933 were generous gifts from Merck and KUDOS, respectively.

Cell culture.

L6 cells were obtained from the American Type Culture Collection (Manassas, VA). This cell line has been demonstrated to serve as a suitable cell culture system in which to study insulin-stimulated glucose transport (21). L6 myoblasts were cultured in 10-cm-diameter dishes in DMEM containing 10% FetalPlex (Gemini Bio-Products, Woodland, CA) and 1% antibiotic-antimycotic solution. The cells were incubated at 37°C in 5% CO2 and were passaged every 48 h by trypsinization using 0.05% trypsin in 0.02% EDTA and were supplied fresh medium. To induce differentiation of myoblasts into myotubes, cells were seeded in 24-well plates (for determination of glucose transport) or six-well plates (for Western blot experiments). After growth, cells were supplied with fresh DMEM containing 2% horse serum and 1% antibiotic-antimycotic mixture every 48 h for 5–7 days.

Serum starvation.

To determine the effects of serum starvation, cells were incubated in serum-free medium after being rinsed twice with phosphate-buffered saline (PBS), for 3 h before experiments, as is common in myotube glucose transport studies (e.g., Refs. 2 and 39). To determine whether serum starvation altered insulin-stimulated phosphorylation of Akt and AS160, myotubes were incubated with serum-free or serum-replete medium for 3 h followed by a 20-min incubation in the absence or presence of 100 nM insulin and subsequent Western blots.

Western blot analysis.

Differentiated L6 myotubes were serum starved for 3 h, washed twice with ice-cold PBS, and then scraped in lysis buffer (50 mM HEPES with pH 7.4, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mm Na3PO4, 100 mM NaF, 2 mm Na3VO4, 10 μg/ml leupeptin, 0.5 μg/ml pepstatin, 10 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Cell lysates were centrifuged at 14,000 rpm for 10 min, and protein concentration was determined using the bicinchoninic acid (BCA, Pierce Biotechnology) method with BSA as standard. Equal aliquots of proteins, solubilized in Laemmli sample buffer, were separated in SDS-PAGE 4–20% pre cast polyacrylamide electrophoresis gels for low molecular weight proteins and 3–8% pre cast Tris acetate gels for high molecular weight proteins, transferred to nitrocellulose membrane, and blocked with 5% nonfat milk in Tris-buffered saline. Membranes were exposed to primary antibodies for 1 h at room temperature or overnight at 4° C, washed, and probed with the appropriate horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were detected using enhanced chemiluminescence.

Streptavidin conjugated to horseradish peroxidase was used to probe for ACC, the only high molecular weight biotin-containing protein in myotube lysates.

Glucose transport.

During the 3-h serum starvation, cells were incubated with or without a protein kinase inhibitor: 20 μM compound C (AMPK inhibitor) or 1 μM KU55933 (ATM inhibitor). Following serum starvation, cells were incubated in HEPES-buffered saline (HBS: 20 mM HEPES-Na, 140 mm NaCl, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2) containing 5 mM d-glucose and in the presence or absence of 100 nM insulin at 37°C for 20 min. The glucose transport assay was performed based on the method as previously described (20). Prior to the glucose transport assay, cells were rinsed twice with glucose-free HBS. Cells were then incubated in 200 μl HBS containing 2-[3H] deoxyglucose (3 μCi/ml) and unlabeled 10 μM 2-deoxyglucose at room temperature for 10 min. Insulin was included in the transport medium for cells that were previously treated with insulin during preincubation. Immediately after 10 min, the transport medium was removed and glucose transport terminated by rapidly washing the cells 3 times with ice-cold 0.9% NaCl. To lyse the cells, 200 μL of 0.2% SDS in 0.2N NaOH were added to each well. Radioactivity was counted by a liquid scintillation spectrophotometer. The protein concentration in this experiment was measured by the BCA method.

Adenovirus experiment.

To further investigate the involvement of AMPK in the serum starvation-related increase in insulin-stimulated glucose transport, L6 myotubes were infected with adenoviral vectors for expression of green fluorescent protein (Ad-GFP) or myc-tagged dominant negative AMPKα2 (Ad-AMPK-DN) and incubated for 48 h before experiments (20). The GFP adenoviral vector was a generous gift from Dr. Kenneth Walsh (Boston University School of Medicine, Boston, MA). The myc-tagged AMPK-DN adenoviral vector (constructed by Morris Birnbaum, University of Pennsylvania, Philadelphia, PA) was constructed from AMPKα2 bearing a mutation of Lys-45 to arginine (K45R), as described previously (25, 26, 41). AMPK-DN when expressed in cells depletes endogenous AMPK (20, 25). Western blot analysis was performed using antibodies specific to AMPKα1, AMPKα2, AMPK, and myc-tag.

Effects of AICAR.

To determine whether activation of AMPK is sufficient to increase ATM protein levels, myotubes were incubated in the absence or presence of 2 mM AICAR, an AMPK activator (6), as previously described (20, 30), before Western blots for ATM.

Positive and negative controls for ATM in Western blots.

To validate the ATM antibody, we anesthetized an ATM-deficient (1) mouse (ATM−/−) and a wild-type littermate (ATM+/+) with 50 mg/kg pentobarbital sodium and collected liver samples to run as positive (ATM+/+) and negative (ATM−/−) controls on Western blots. The mice were raised from ATM+/− breeders obtained from The Jackson Laboratory (Bar Harbor, ME). All animal procedures were approved by the Institutional Animal Care and Use Committee of Saint Louis University.

Statistical analysis.

Data were analyzed using ANOVA, followed by Fisher's least significant difference post hoc comparisons when P < 0.05 was obtained. All values are expressed as means ± SE.

RESULTS

Phosphorylation of potential mediators of increased insulin action.

As a first step to assess the possible link between serum starvation and increased insulin sensitivity, we determined whether serum starvation enhances phosphorylation of potential mediators of increased insulin action. As shown in Fig. 1, 3 h of serum starvation caused significant increases in levels of phosphorylated AMPK (∼40%, P < 0.05, Fig. 1A) and the AMPK target ACC (∼70%, P < 0.05, Fig. 1B). Levels of phosphorylated ATM S1981 (∼50%, P < 0.05, Fig. 1C), Akt S473 (∼80%, P < 0.01, Fig. 1D), and Akt Thr308 (∼50%, P < 0.05, Fig. 1E) were also increased by serum starvation. Phosphorylation of AS160 (Fig. 1F), p38 (Fig. 1G), and PKCζ/λ (Fig. 1H) was not affected. Total AMPK and ATM were found to increase in parallel with levels of phosphorylated AMPK and ATM, respectively, under serum-starved conditions; thus data for phosphorylated forms of AMPK and ATM were not normalized to the respective total AMPK and ATM but instead were normalized to milligram cellular protein. The increases in total AMPK and ATM were unlikely to have been the result of a global increase in protein abundance, because levels of other proteins (e.g., Akt, AS160, p38, and ACC) were not altered by serum starvation.

Fig. 1.

Fig. 1.

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Effects of serum starvation on the phosphorylation status of potential insulin sensitivity factors. Shown are representative Western blots from cells incubated at 37°C in serum-containing medium (C, control) or serum-free medium (SS, serum starved) for 3 h. A: phosphorylated AMP-activated protein kinase (P-AMPK) and AMPK (n = 6/group). B: phosphorylated acetyl-CoA carboxylase (P-ACC) and ACC (probed with streptavidin) (n = 8–9/group). C: phosphorylated ataxia telangiectasia mutated (P-ATM)-S1981 and ATM (n = 8/group). D: P-Akt S473 and Akt (n = 15/group). E: P-Akt T308 and Akt (n = 6/group). F: phosphorylated Akt substrate (P-AS) and AS160 (n = 6/group). G: P-p38 and p38 (n = 6/group). H: P-PKCζ/λ and Akt (n = 3/group). I: Western blotting using lysates from control and serum-starved myotubes in parallel with lysates from livers of wild-type mice (ATM+/+) and transgenic mice (ATM−/−) lacking functional ATM. The ATM bands from myotubes correspond with the positive control ATM band from ATM+/+ mouse liver and are at the appropriate molecular weight. Values are means ± SE. *Significantly different corresponding value from non-serum-starved control (P < 0.05).

To demonstrate the validity of the serum starvation effect on ATM, control and serum-starved myotube samples were run on the same gel as positive and negative ATM control samples from livers of ATM+/+ and ATM−/− mice. The ATM band in serum-starved myotubes corresponds with the ATM band from livers of an ATM+/+ mouse, and there is no ATM band in the sample from an ATM−/− mouse (Fig. 1I).

Serum starvation does not affect insulin-stimulated Akt phosphorylation.

Akt is a central mediator of insulin signaling (12). To determine the effects of serum starvation on insulin-stimulated Akt phosphorylation, serum-starved myotubes were incubated in the presence or absence of 100 nM insulin at 37°C for 20 min before extraction in ice-cold lysis buffer. Despite a serum starvation-related increase in Akt phosphorylation at S473 (Fig. 2A, P < 0.05) and T308 (Fig. 2B, P < 0.05) in the absence of insulin, serum starvation did not affect insulin-stimulated Akt phosphorylation at either Akt phosphorylation site. Likewise, insulin-stimulated phosphorylation of the Akt target AS160 was unaffected by serum starvation (Fig. 2C).

Fig. 2.

Fig. 2.

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Serum starvation does not affect insulin-stimulated Akt or AS160 phosphorylation. Differentiated L6 myotubes were incubated 3 h in serum-replete (control) or serum-free medium (serum-starved) and further incubated at 37°C for 20 min in the presence or absence of 100 nM insulin before extraction with ice-cold lysis buffer. Cell lysates were subjected to SDS/PAGE and Western blot analysis. A: P-Akt S473 and Akt (n = 3/group). B: P-Akt T308 and Akt (n = 3/group). C: phosphorylated Akt substrate and AS160 (n = 3/group). Data represent means ± SE. *Significant difference due to serum starvation (P < 0.05). For A–C, there is a main effect for insulin (P < 0.05).

Insulin-stimulated glucose transport in serum-starved myotubes was prevented by compound C and KU55933.

To investigate whether AMPK and ATM were necessary for increased insulin-stimulated glucose transport after serum starvation, glucose transport assays were performed with cells incubated with or without compound C (an AMPK inhibitor) or KU55933 (an ATP-competitive inhibitor of ATM) during the serum starvation. Insulin had no effect on glucose transport in control cells but caused a ∼40% increase in glucose uptake in serum-starved myotubes (P < 0.05). This insulin action in serum-starved myotubes was prevented by compound C (Fig. 3A, P < 0.05) and KU55933 (Fig. 3B, P < 0.05).

Fig. 3.

Fig. 3.

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Inhibition of AMPK or ATM prevents serum starvation-related increase in insulin-stimulated glucose transport. A: differentiated cells were incubated in serum-free medium in the presence or absence of 20 μM compound C (comp C) for 3 h. They were further incubated in HEPES-buffered saline (HBS) containing 5 mM nonradiolabeled d-glucose in the presence or absence of 100 nM insulin for 20 min before glucose transport assay (n = 18/group). B: differentiated cells were incubated in serum-free medium in the presence or absence of 1 μM KU55933 for 3 h. They were further incubated in HBS containing 5 mM nonradiolabeled d-glucose in the presence or absence of 100 nM insulin for 20 min before glucose transport assay (n = 12/group). 2DG, 2-deoxy-d-glucose. Data represent means ± SE. *Significant difference from corresponding control not treated with insulin (P < 0.05); †significant inhibition of the insulin effects by the inhibitors.

Expression of Ad-AMPK-DN prevents the serum starvation-related increase in insulin action.

As shown in Fig. 4A, adenoviral vectors were effective for expression of AMPK-DN, as evidenced by increased AMPK (caused by expression of AMPK-DN), increased AMPKα2 (the AMPK isoform of which AMPK-DN is a mutant), and presence of the myc tag from the AMPK-DN protein. In cells expressing GFP, insulin increased glucose transport after serum starvation but not without serum starvation (P < 0.01). The serum starvation-related increase in insulin action did not occur in cells expressing AMPK-DN (Fig. 4B; P < 0.05).

Fig. 4.

Fig. 4.

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Expression of AMPK-dominant negative (DN) prevents the insulin-sensitizing effect of serum starvation. Uninfected (control) myotubes and myotubes that were adenovirally transduced with green fluorescent protein (GFP) or AMPK-DN were incubated in serum-containing or serum-free medium for 3 h. A: Western blot was performed using antibodies specific to AMPKα1, AMPKα2, AMPK, and myc-tag, and P-ACC. The use of myc-tag here is to demonstrate that the AMPK-DN (which is AMPKα2 containing a mutation of a single amino acid) was effectively expressed. B: representation of a glucose transport assay with cells expressing AMPK-DN or GFP. Differentiated cells were incubated in serum-free medium for 3 h. They were further incubated in HBS containing 5 mM nonradiolabeled d-glucose in the presence or absence of 100 nM insulin for 20 min before glucose transport assay (n = 12/group). *Significant difference from corresponding control not treated with insulin (P < 0.05); †significant inhibition of the insulin effects by expression of AMPK-DN. C: ATM and ACC (n = 9/group). D: P-ACC and ACC (n = 9/group). *Serum-starvation-related increase (P < 0.05), which does not occur (†P < 0.05 vs. serum-starved cells expressing GFP) in cells expressing AMPK-DN (C and D). E: myotubes were incubated in the absence (C, control) or presence (A, AICAR) of 2 mM AICAR (an AMPK activator) for 1 h and assessed for ATM (n = 5/group) or P-AMPK and AMPK (n = 5–6/group). *Effect of AICAR (P < 0.05). Data represent means ± SE.

Higher basal (i.e., in the absence of insulin) glucose transport in myotubes in control (serum-containing) medium compared with myotubes in serum-free medium (as is shown in Fig. 3, A and B) has been previously reported (21). We cannot explain why basal (non-insulin-stimulated) glucose transport is similar in non-serum-starved and serum-starved cells in Fig. 4B, except to say that these assays are under different conditions (i.e., myotubes transduced with GFP or AMPK-DN), and it is possible that the transduction altered basal glucose transport levels.

Because inhibition of ATM prevented the serum starvation-related increase in insulin action (Fig. 3B), and it also appears that AMPK plays a role in the serum starvation effect (Figs. 3A and 4B), we investigated the possibility that AMPK might influence ATM levels in myotubes. As shown in Fig. 4C, in myotubes expressing GFP, serum starvation resulted in an increase of ATM (P < 0.05). However, serum starvation had no effect on ATM protein in cells expressing AMPK-DN. As shown in Fig. 4D, expression of AMPK-DN prevents the serum starvation-related increase in phosphorylation of the AMPK target ACC. The potential role of AMPK in the serum starvation effect of increased ATM levels raised the question of whether or not activation of AMPK affects ATM. As shown in Fig. 4E, incubation of myotubes with the AMPK activator AICAR was sufficient to increase ATM protein levels (P < 0.05).

Inhibition of AMPK does not prevent serum starvation-stimulated Akt phosphorylation but prevents an increase in ATM phosphorylation and protein.

To determine whether increased Akt phosphorylation during serum starvation is dependent on AMPK, myotubes were incubated in either serum-free or serum-containing medium in the presence of 20 μM compound C (an AMPK inhibitor) or DMSO (vehicle) for 3 h. Compound C did not prevent the effects of serum starvation on Akt phosphorylation (Fig. 5, A and B).

Fig. 5.

Fig. 5.

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Inhibition of AMPK does not prevent phosphorylation of Akt by serum starvation but blocks an increase in ATM. Differentiated cells were incubated in serum-containing or serum-free medium in the presence of 20 μM compound C or DMSO (vehicle) for 3 h before extraction in ice-cold lysis buffer. Cell lysates were subjected to SDS/PAGE and then Western blot analysis. A: P-Akt S473 and Akt (n = 3/group). B: P-Akt T308 and Akt (n = 3/group). C and D: P-ATM S1981 (n = 9/group) and ATM (n = 6/group). Data represent means ± SE. *Significant difference due to serum starvation (P < 0.05). †The serum starvation effect is prevented by compound C (P < 0.05).

To further examine whether the increase in ATM during serum starvation was caused by the increase in AMPK, myotubes were incubated in serum-free medium containing 20 μM compound C or DMSO (vehicle) for 3 h. Compound C prevented the serum starvation-related increase of ATM phosphorylation (Fig. 5C, P < 0.05). Similarly, the increase in ATM under serum-starved conditions was prevented by compound C (Fig. 5D, P < 0.05). Prevention of the serum starvation-related increase in ATM by AMPK-DN (Fig. 4C) and compound C (Fig. 5, C and D) suggests that AMPK activity is required for the increase of ATM and ATM phosphorylation that occurs during serum starvation.

Inhibition of ATM does not prevent serum starvation-stimulated AMPK and Akt phosphorylation.

ATM has previously been implicated in activation of AMPK (31, 32) and Akt (13, 36). To investigate whether ATM is involved in the increased levels of phosphorylated AMPK or Akt in serum-starved cells, differentiated cells were incubated in either serum-free or serum-containing medium in the presence of 1 μM KU55933 or DMSO (vehicle) for 3 h. The increased level of phosphorylated AMPK in serum-starved cells was not prevented by KU55933 (Fig. 6A). Similarly, KU55933 did not prevent serum starvation-stimulated Akt phosphorylation at either S473 (Fig. 5B) or Akt T308 (Fig. 5C), though KU55933 reduced basal P-Akt T308 (P < 0.05).

Fig. 6.

Fig. 6.

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Inhibition of ATM does not prevent phosphorylation of AMPK and Akt by serum starvation. Differentiated cells were incubated in serum-containing or serum-free medium in the presence of 1 μM KU55933 or DMSO (vehicle) for 3 h before extraction in ice-cold lysis buffer. Cell lysates were subjected to SDS/PAGE and then Western blot analysis. A: P-AMPK and AMPK (n = 6/group). B: P-Akt S473 and Akt (n = 3/group). C: P-Akt T308 and Akt (n = 6/group). D: effects of serum starvation and KU55933 on p53 protein level (n = 12/group). *Significant difference due to serum starvation (P < 0.05); †effect of KU55933 (P < 0.05). Data represent means ± SE.

As shown earlier (Fig. 3B), KU55933 prevents the serum starvation-related increase in insulin-stimulated glucose transport. However, KU55933 does not reduce P-AMPK, it does not decrease insulin-stimulated Akt phosphorylation, and if anything, it tends to increase the increment in P-Akt caused by insulin. Thus, if there is an ATM effect to promote increased insulin action after serum starvation, it seems unlikely to be mediated by alterations in P-AMPK or P-Akt. In fact, it seems more likely (given that inhibition of AMPK or expression of AMPK-DN prevents serum starvation-related increases in ATM and insulin-stimulated glucose transport) that AMPK lies upstream of ATM in the effect of serum starvation on insulin action.

The p53 tumor-suppressor protein is a key ATM substrate and its stabilization has been commonly used as a read-out of ATM activity (3). As shown in Fig. 6D, p53 significantly accumulated in response to serum starvation (P < 0.05), and this increase in p53 was prevented by 1 μM KU55933 (P < 0.05).

DISCUSSION

In the current study, we present new information about the role of serum starvation in increasing insulin-stimulated glucose transport. For example, we have shown that serum starvation increases the abundance of phosphorylated Akt, AMPK, and ATM but not p38, PKCζ, or AS160. Furthermore, inhibition of either AMPK or ATM prevents the serum starvation-related increase in insulin action.

Although Akt, a major effector in the insulin signaling pathway, was significantly phosphorylated in serum-starved myotubes compared with non-serum myotubes, it appears that increased basal Akt phosphorylation during serum starvation is not sufficient to increase subsequent insulin action. For example, basal Akt phosphorylation was not affected by inhibition of AMPK or inhibition of ATM, both of which prevented the serum starvation effect on insulin action. Furthermore, insulin-stimulated Akt phosphorylation was not affected by serum starvation. Thus, it seems unlikely that increased insulin-stimulated glucose transport after serum starvation is mediated by an alteration of insulin signaling through Akt. In this regard, the increased insulin-stimulated glucose transport after serum starvation is similar to the increase in insulin stimulated glucose transport in exercised muscle in humans and in rat muscle exposed to the AMPK activator AICAR, both of which occur without a change in insulin-stimulated Akt phosphorylation (9, 38).

Serum starvation appears to be unusual in its ability to potentiate insulin-stimulated glucose transport without increasing AS160 phosphorylation. AS160 is a well-defined effector of Akt, and the bulk of data so far reported (4, 7, 22, 23, 35) identify AS160 as a convergent point at the crossroads of distinct signaling pathways geared toward GLUT4 trafficking. Our data suggest that phosphorylation of AS160 may not be the only mechanism that enhances glucose transport in muscle cells. The inferences from these findings are not far from those of a previous study by Eguez et al. (7) in which silencing of AS160 using short hairpin RNA in 3T3 adipocytes resulted in only a partial redistribution of GLUT4 from intracellular compartments to the plasma membrane, thereby suggesting the existence of both AS160-dependent as well as AS160-independent mechanisms for insulin-mediated GLUT4 trafficking. Recently, Randhawa et al. (27) have also suggested an AS160-independent GLUT4 trafficking pathway. Potentially, phosphorylation of AS160 at sites that might not be reactive with the P-AS antibody could play a role in increased insulin action after serum starvation. Likewise, it is possible that altered phosphorylation of TBC1D1, a Rab GAP that like AS160 influences membrane localization of GLUT4 (5, 28), could also play a role in the serum starvation effect.

Our results demonstrate that AMPK is required for enhancement of insulin-stimulated glucose transport induced by serum starvation. For example, expression of dominant inhibitory AMPK or incubation of myotubes with compound C (an AMPK inhibitor) prevented the serum starvation-related increase in insulin-stimulated glucose transport. It is known that insulin-stimulated glucose transport (in response to maximal insulin concentrations) is normal in muscle of mice lacking AMPKα2 and in mice expressing dominant inhibitory AMPK (the same as AMPK-DN used in the current study) (25, 37). On the other hand, oral glucose tolerance is decreased in AMPKα2 knockout mice (19), suggesting a role for AMPK in regulation of insulin sensitivity. However, experiments designed to determine whether there is increased insulin sensitivity in muscle of AMPK-deficient animals after an AMPK-activating stimulus (e.g., exercise or treatment with AICAR) have not yet been done.

The data from the current study suggest that ATM might play a role downstream of AMPK in mediation of the serum starvation effect on insulin action. Interestingly, the serum starvation-related increase in ATM protein was prevented by dominant inhibitory AMPK or compound C, and activation of AMPK (with AICAR) was sufficient to increase ATM protein. In contrast, inhibition of ATM did not affect AMPK. However, because inhibition of ATM prevented the serum starvation-related increase in insulin-stimulated glucose transport, it seems possible that ATM plays a role in the serum starvation effect, perhaps downstream of AMPK. It must be acknowledged that there is no genetic evidence for a role of ATM in the serum starvation effect in the present study, only the data obtained from the use of an inhibitor (with the potential for off-target effects). On the other hand, the ATM inhibitor KU55933 was used at a concentration of 1 μM. Even at 10 μM, the ATM inhibitor reportedly does not interfere with any of a panel of 60 kinases tested (15). The 1 μM concentration of KU55933 is substantially lower than the IC50 values for other members of the phosphatidylinositol 3-kinase (PI3K) family kinases, including PI3K, mammalian target of rapamycin, and ATM- and Rad3-related protein (15). At 1 μM, KU55933 does not affect insulin-stimulated phosphorylation of Akt in L6 myotubes (18). Thus, it seems possible that the KU55933 effects in the current study are effects of inhibition of ATM as opposed to off-target effects.

In contrast to previous reports that implicate ATM as essential for insulin-stimulated activation of Akt (13, 36) and ATM as an AMPK activator (31, 32), our data suggest that ATM is not required for increased phosphorylation of Akt and AMPK during serum starvation. Indeed, in our model, ATM appears to be downstream of AMPK. Taken together with previous findings by other groups (13, 31, 32, 36), our data suggest that directional interactions among AMPK, Akt, and ATM are context-specific.

In conclusion, the present study suggests that the effect of serum starvation to enhance insulin action requires AMPK and that ATM might play a role in the serum starvation effect.

GRANTS

This work was supported by Public Health Service Grants K01 DK066330, R15 DK080437, and R15 DK080437–01S2 (American Recovery and Reinvestment Act supplement to provide research experiences for science teachers) from the National Institute of Diabetes and Digestive and Kidney Diseases. Some support was provided by the Pfizer-Solutia Students and Teachers as Research Scientists (STARS) Program (University of Missouri-St. Louis, Dr. Kenneth Mares, Director).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

ACKNOWLEDGMENTS

Rona Robinson-Hill, Torris Caston, and Jill Anderson provided technical support. Compound C and KU55933 were generous gifts from Merck and KUDOS, respectively.

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