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Originally published In Press as doi:10.1074/jbc.M605461200 on August 25, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31478-31485, October 20, 2006
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AS160 Regulates Insulin- and Contraction-stimulated Glucose Uptake in Mouse Skeletal Muscle*Formula

Henning F. Kramer{ddagger}, Carol A. Witczak{ddagger}, Eric B. Taylor{ddagger}, Nobuharu Fujii{ddagger}, Michael F. Hirshman{ddagger}, and Laurie J. Goodyear{ddagger}§1

From the {ddagger}Joslin Diabetes Center Research Division, Metabolism Section and the Department of Medicine, §Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02215

Received for publication, June 7, 2006 , and in revised form, August 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin and contraction are potent stimulators of GLUT4 translocation and increase skeletal muscle glucose uptake. We recently identified the Rab GTPase-activating protein (GAP) AS160 as a putative point of convergence linking distinct upstream signaling cascades induced by insulin and contraction in mouse skeletal muscle. Here, we studied the functional implications of these AS160 signaling events by using an in vivo electroporation technique to overexpress wild type and three AS160 mutants in mouse tibialis anterior muscles: 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate sites (4P); 2) AS160 mutated to abolish Rab GTPase activity (R/K); and 3) double mutant AS160 containing both 4P and R/K mutations (2M). One week following gene injection, protein expression for all AS160 isoforms was elevated over 7-fold. To determine the effects of AS160 on insulin- and contraction-stimulated glucose uptake in transfected muscles, we measured [3H]2-deoxyglucose uptake in vivo following intravenous glucose administration and in situ muscle contraction, respectively. Insulin-stimulated glucose uptake was significantly inhibited in muscles overexpressing 4P mutant AS160. However, this inhibition was completely prevented by concomitant disruption of AS160 Rab GAP activity. Transfection with 4P mutant AS160 also significantly impaired contraction-stimulated glucose uptake, as did overexpression of wild type AS160. In contrast, overexpressing mutant AS160 lacking Rab GAP activity resulted in increases in both sham and contraction-stimulated muscles. These data suggest that AS160 regulates both insulin- and contraction-stimulated glucose metabolism in mouse skeletal muscle in vivo and that the effects of mutant AS160 on the actions of insulin and contraction are not identical. Our findings directly implicate AS160 as a critical convergence factor for independent stimulators of skeletal muscle glucose uptake.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Skeletal muscle insulin resistance is a salient feature of type 2 diabetes. In humans and other mammals, skeletal muscle normally accounts for ~75% of whole body insulin-stimulated glucose transport (1–3). Impaired ability of the muscle to respond to insulin is therefore disruptive to systemic glucose homeostasis. Skeletal muscle also possesses contractile properties that can effectively restore glucose control in an insulin-independent manner, and this element is preserved in individuals with type 2 diabetes (4, 5). Although the precise mechanisms remain elusive, it is clear that both insulin and contraction signals converge upon GLUT4 vesicles and promote their appearance at the cell membrane (6, 7).

AS1602 (Akt substrate of 160 kDa) is a Rab GTPase-activating protein (GAP) shown to regulate GLUT4 translocation in insulin-sensitive 3T3-L1 adipocytes (8) and L6 myoblasts (9). In addition to its Rab GAP domain, AS160 also contains two phosphotyrosine-binding domains and multiple putative phosphorylation sites, including six phospho-Akt substrate (PAS) motifs (RXRXX(S*/T*) where asterisks denote phosphorylation residues) targeted by Akt, AMPK, and potentially other upstream kinases (10). Under basal conditions, AS160 has been shown to retain GLUT4 vesicles intracellularly through the activity of its GAP domain in 3T3-L1 cells (11, 12). GLUT4 vesicles are dynamic complexes that constantly migrate/recycle along cytoskeletal elements and are directed by GTP-bound Rab proteins and other molecular chaperones (9, 13). Thus, AS160 GAP activity could inactivate a critical, still unidentified Rab protein as part of the mechanism for controlling basal GLUT4 trafficking (13). When cells are treated with insulin, however, AS160 is rapidly phosphorylated at PAS motifs (14) and dissociates from GLUT4 vesicles (12). This is associated with accelerated rates of GLUT4 vesicular exocytosis, such that GLUT4 manifests predominantly at the cell surface and enhances glucose transport (15).

Both 3T3-L1 adipocytes and L6 GLUT4-Myc myoblasts transfected with a constitutively active AS160 incapable of being phosphorylated at four PAS regulatory motifs (4P mutant) exhibit significantly reduced insulin-induced GLUT4 translocation (8, 9). AS160 phosphorylation therefore appears to function in a permissive role by decreasing the GAP activity of the protein such that exocytosis of GLUT4 vesicles is allowed. Supporting this hypothesis, inactivation of the Rab GAP domain via point mutation of Arg973 to lysine appears to restore insulin-stimulated GLUT4 translocation in adipocytes coexpressing phosphorylation site-specific mutations in AS160 (2M) (8).

There are currently no reports establishing a regulatory role for AS160 on skeletal muscle glucose metabolism. Data published by our lab (10) and others (16) indicate that AS160 phosphorylation at PAS motifs occurs following both insulin and contraction in skeletal muscle. Furthermore, these phosphorylation events are regulated in a distinct and potentially additive manner by insulin-stimulated Akt2 and contraction-stimulated AMPK{alpha}2 activities (10). It is plausible, then, that AS160 is a common, downstream point of convergence mediating the effects of both insulin and contraction on skeletal muscle glucose uptake. The purpose of this study was to determine the effects of wild type and mutant AS160 on basal and insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle in vivo. Specifically, we overexpressed wild type and three different mutant AS160 DNA constructs by direct injection into the tibialis anterior muscle of mice followed by in vivo electroporation. Mutant constructs included: 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate (PAS) sites (4P); 2) AS160 mutated to inhibit Rab GTPase activity (R/K), and 3) a double mutant AS160 containing both 4P and R/K mutations (2M). Measurements of glucose uptake in vivo using tracer methodology suggest that AS160 phosphorylation at PAS motifs is required for full insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle. The observed inhibition with 4P mutant overexpression was dependent upon the Rab GAP activity of AS160, because muscles transfected with mutant AS160 coexpressing 4P and Rab GAP mutations (2M) exhibited normal insulin-induced glucose uptake and increased glucose uptake following contraction. Basal skeletal muscle glucose uptake in sham-operated transfected muscles was also significantly increased by Rab GAP mutant overexpression (R/K and 2M) compared with empty vector and wild type AS160 controls. Thus, AS160 directly regulates insulin- and contraction-stimulated uptake in mouse skeletal muscle. Furthermore, the effects of wild type and mutant AS160 on the actions of insulin and contraction are not identical and suggest distinct modes of regulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Total AS160 was detected using an affinity-purified pan-AS160 antibody raised against the mouse C-terminal amino acid epitope (PTNDKAKAGNKP) (8, 14). Phosphorylation-specific antibodies included Akt Thr308 (Cell Signaling Technology), AMPK Thr172, and AS160 Thr642 (BIOSOURCE International). This latter antibody was affinity purified and targets the peptide fragment RRRAHpTFSHPPS on AS160 (10). Protein expression was assessed with anti-GLUT4, anti-GLUT1 (Chemicon International), anti-AMPK {alpha}2 (Upstate%20Biotechnology">Upstate Biotechnology, Inc.), anti-Akt1/2 and anti-hexokinase II (Santa Cruz Biotechnology). Horseradish peroxidase-conjugated antirabbit, anti-mouse, and anti-goat antibodies (Amersham Biosciences) were used to bind and detect all primary antibodies.

Animals—Protocols for animal use were reviewed and approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center and were in accordance with National Institutes of Health guidelines. Female ICR mice 8 weeks old (25–30 grams) were purchased from Taconic (Hudson, NY). All of the mice were housed with a 12 h:12 h light:dark cycle and fed standard laboratory chow and water ad libitum. The mice were fasted overnight (10 p.m. to 8 a.m.) prior to the morning of the experiment.

Plasmid DNA Constructs—Human wild type AS160 DNA and three distinct mutant AS160 DNA constructs have been characterized previously in 3T3-L1 cells (8). The expressed mutant AS160 isoforms include: 1) AS160 mutated at four PAS motifs (Ser318, Ser588, Thr642, and Ser751), rendering these sites incapable of being phosphorylated (4P); 2) AS160 mutated to lysine at Arg973 within its Rab GAP domain, effectively eliminating AS160 GAP activity (R/K); and 3) AS160 double mutant containing both the 4P and R/K mutations (2M). For the purposes of DNA injection and in vivo electroporation in adult mouse skeletal muscle, AS160 DNA was excised from the original CMV-10 vector, affixed to a Myc tag leader sequence via specific primers and PCR amplification (composite forward primer: AA CTC GAG GCC ACC ATG GAG CAA AAG CTT ATT TCT GAA GAG GAC TTG ATG GAG CCG CCC AGC TGC ATT CAG GAT GA; composite reverse primer: TT CTC GAG TTA TGG CTT ATT TCC TAT CTT GGC TTT GTT GTT) and subcloned into pCAGGS plasmids. This expression vector drives a target gene under the CAG (cytomegalovirus immediate-early enhancer chicken beta-actin hydrid) promoter and has been demonstrated to have high activity in skeletal muscle (17, 18). AS160 DNA constructs were confirmed for accuracy using the high throughput DNA sequencing service at Brigham and Women's Hospital (Boston, MA). Plasmid DNA was then amplified in Escherichia coli TOP10 cells (Invitrogen), extracted using an endotoxin-free Plasmid Mega Kit (Qiagen), and suspended in saline at 4 µg/µl.

In Vivo Gene Transfer in Mouse Skeletal Muscle—Enriched yields of AS160 plasmid clones were directly injected into mouse tibialis anterior muscles by a modified protocol (17), originally described by Aihara and Miyazaki (19). The mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (90 mg/kg), and 100 µg of DNA (empty pCAGGS or AS160 constructs) was injected longitudinally into the tibialis anterior muscle with a 29-guage needle. Stainless steel electrode needles fixed 4 mm apart were then inserted into the transverse muscle belly and square wave electrical pulses (200 V/cm) were applied eight times at a rate of one pulse/s (duration, 20 ms) using a Grass S88 pulse generator (Grass Instruments, Quincy, MA). Our lab has demonstrated that transfection efficiency with the LacZ gene under this protocol is ~85% and manifests relatively homogenously throughout the muscle (17).

Glycogen Determination—Glycogen content of frozen, pulverized muscle was determined by HCl hydrolysis followed by NaOH neutralization (20). Resultant free glucosyl concentration was determined spectrophotometrically using a hexokinase-based assay kit (Sigma).

In Situ Muscle Contraction—The mice were transfected with identical DNA constructs in each tibialis anterior muscle. Seven days after DNA injection, the mice were anesthetized with intraperitoneal administration of pentobarbital sodium (90 mg/kg of body weight). Peroneal nerves from both legs were surgically exposed for electrode placement. Although one leg was left unstimulated (basal/sham control), the other leg was subjected to electrical stimulation using a Grass S88 pulse generator for 15 min of contractions (train rate, 1/s; train duration, 500 ms; pulse rate, 100 Hz, duration, 0.1 ms at 2–7 V).

Intravenous Glucose Injections—The mice were transfected with different DNA constructs in each tibialis anterior muscle. Seven days after DNA injection, the mice were anesthetized with pentobarbital sodium (90 mg/kg) and administered a saline or 20% glucose bolus (1.0 g of glucose/kg of body weight) through the retroorbital sinus. This dosage stimulates a physiologic insulin response (from ~0.82 to ~3.4 ng/ml spike within 5 min, followed by persistent but declining concentrations at 15 min post-injection, see supplementary data obtained from the Diabetes Endocrinology and Research Center Specialized Assay Core Insulin enzyme-linked immunosorbent assay) and does not induce significant hypoglycemia (21, 22).

In Vivo Skeletal Muscle Glucose Uptake—Base-line blood samples were collected from the tail vein prior to intravenous delivery of [3H]2-deoxyglucose through the retroorbital vein. This tracer bolus was combined with the unlabeled glucose or saline solution in glucose injection experiments and occurred simultaneous with the onset of in situ peroneal nerve stimulation in contraction studies. For all treatments, the blood samples were taken from the tail vein at 5, 10, 15, 25, 35, and 45 min post-injection for the determination of blood glucose and [3H]2-deoxyglucose specific activity. After collection of the final blood sample, the animals were euthanized, and tibialis anterior muscles were removed and frozen in liquid nitrogen. Accumulation of [3H]2-deoxyglucose in pulverized tissue was determined via a precipitation protocol adapted from Ferre et al. (23) using barium hydroxide/zinc sulfate and perchloric acid. Small amounts of the muscle were set aside and used for immunoblots.

Tissue Processing and Immunoblotting—Frozen muscle tissue was homogenized with a Polytron (Brinkman Instruments) in chilled lysis buffer (20 mM Tris, pH 7.5, 5 mM EDTA, 10 mM Na3P2O4, 100 mM NaF, 2 mM Na3VO4, 10 µg/ml leupeptin, 3 mM benzamidine, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Three-fourths of this homogenate was aliquoted for measurement of glucose uptake (see above). The remaining one-fourth was additionally processed with detergent (1% Nonidet P-40), rotated end over end at 4 °C for 1 h and then centrifuged at 14,000 x g for 15 min at 4 °C. The supernatants were isolated, and protein concentrations were determined via Bradford assay as described. Equal amounts of skeletal muscle proteins (40–50 µg) were resolved by SDS-PAGE (24) for Western blot analysis (25). Antibody-bound proteins were visualized using enhanced chemiluminescence (Amersham Biosciences). The protein bands were scanned by ImageScanner (Amersham Biosciences) and quantitated by densitometry (Fluorchem 2.0; Alpha Innotech, San Leandro, CA).


Figure 1
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  		FIGURE 1.
Gene injection and in vivo electroporation of AS160 in mouse tibialis anterior muscles results in significant overexpression after 7 days. Empty pCAGGS vector (E) or Myc-tagged AS160 DNA constructs (wild type and 4P, R/K, and 2M mutants) were injected into the tibialis anterior muscles of anesthetized mice, followed by in vivo electroporation. The animals were allowed to recover, and recombinant protein expression was assessed 7 days post-injection. The muscle proteins were resolved by SDS-PAGE and immunoblotted with anti-pan-AS160 antibody. The data are expressed as the means ± S.E.; n = 12–16/group. *, p < 0.05 (versus empty pCAGGS controls).

 
Statistical Analysis—The data are expressed as the means ± S.E. Statistical analyses were performed using one-way analysis of variance. When analysis of variance revealed significant differences, Tukey's post hoc test for multiple comparisons was performed. p values less than 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression of AS160 in Transfected Mouse Skeletal Muscle—We initially determined the expression of recombinant wild type (WT) AS160 and 4P, R/K, and 2M mutant AS160 isoforms using our gene transfer approach. AS160 DNA injection and in vivo electroporation induced consistent 6–8-fold increases in AS160 protein after 7 days in mouse tibialis anterior muscles (Fig. 1). As expected, the magnitude of overexpression was comparable across all constructs, because each AS160 variant was subcloned into the same pCAGGS vehicle. Note that the pan-AS160 antibody detected both the recombinant Myc-tagged AS160 (upper band) and endogenous mouse AS160 (lower band). Both AS160 protein bands were used in densitometry quantifications. Overexpressed AS160 did not silence or enhance expression of endogenous AS160 in experimental muscles. Furthermore, AS160 overexpression was only detected in tibialis anterior muscles, because the adjacent extensor digitorum longus muscles exhibited no increases in AS160 (data not shown). These results validate the efficacy of localized DNA injections as a means of overexpressing wild type or altered gene products into skeletal muscle tissue.

Phosphorylation of Overexpressed AS160—We next examined the phosphorylation of overexpressed AS160 at Thr642, a critical PAS motif targeted by Akt and other kinases (Fig. 2A), as well as collective phosphorylation events at all PAS sites via the phospho-Akt substrate antibody (Fig. 2B). Basal phosphorylation of overexpressed WT and R/K AS160 at Thr642 was significantly increased compared with empty vector (lanes E) controls. However, 4P and 2M mutant AS160, which express point mutations at Thr642 and three other PAS motifs, exhibited no discernible increases in basal Thr642 phosphorylation compared with empty vector controls. Both contraction and insulin stimulated endogenous AS160 Thr642 phosphorylation and further increased Thr642 phosphorylation of overexpressed WT and R/K AS160. These trends were similarly observed in immunoblots with the PAS antibody (2B). Here, electrophoretic optimization (8% gel; 50:1 ratio acrylamide:bisacrylamide) enabled clear discernment of upper (exogenous) and lower (endogenous) AS160 protein phosphorylation. This demonstrates that recombinant Myc-tagged AS160 is phosphorylated in vivo and that the integrity of the AS160 point mutations (4P and 2M) is preserved following gene expression.


Figure 2
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  		FIGURE 2.
Phosphorylation of overexpressed AS160 in transfected muscles following basal or contraction- or insulin-stimulated conditions. Empty pCAGGS vector (E) or Myc-tagged AS160 DNA constructs (wild type and 4P, R/K, and 2M mutants) were injected into the tibialis anterior muscles of anesthetized mice, followed by in vivo electroporation. The animals recovered for 1 week and then were stimulated to contract in situ (15 min) or received intravenous glucose (1 g/kg). The mice were immediately sacrificed following the experimental protocol, and tibialis anterior muscles were harvested and processed for signaling. A, proteins were resolved by 10% SDS-PAGE (37.5:1 acrylamide:bisacrylamide) and immunoblotted (IB) with an anti-phospho-AS160 Thr642 antibody. B, proteins were resolved by 8% SDS-PAGE (50:1 acrylamide:bisacrylamide) to increase electrophoretic separation of high molecular weights, and immunoblotted with an anti-PAS antibody. This strategy enabled crisp discernment of upper (exogenous) and lower (endogenous) AS160 protein bands. Basal and contraction- and insulin-stimulated samples were run together in the same gels; however, because of the 15 different treatment conditions, it was not possible to load all of the treatments in a single gel. Therefore, internal loading controls were placed in all of the gels to enable comparisons from blot-to-blot. The images are cropped to elicit clarity, and the quantitations represent the normalized aggregate of six pairs of gels. The data are expressed as the means ± S.E. (n = 6–12/group). *, p < 0.05 (versus empty pCAGGS basal). {dagger}, p < 0.05 (versus empty pCAGGS for respective condition).

 
AS160 Regulates Insulin-stimulated Glucose Uptake—Although AS160 appears to be involved in insulin-mediated GLUT4 translocation in cellular models (8, 9, 11, 15), there is currently no data concerning the role of AS160 on glucose metabolism in adult skeletal muscle tissue. Intravenous glucose injections were conducted in fasted mice to induce hyperglycemia (Fig. 3A) and an associated physiologic insulin response (~4-fold increase in circulating concentrations within 5 min) (supplementary data; Refs. 21 and 22), whereas glucose uptake into injected muscles was measured in vivo using radiolabeled tracer. Blood glucose curves reflect aggregate data from all electroporated mice and are graphed as a single line because there were no significant differences between transfected animals. The regulatory effects of overexpressed WT and mutant AS160 on insulin-stimulated glucose uptake are shown in Fig. 3B. Muscles overexpressing 4P mutant AS160 exhibited significant decreases (~33%) in insulin-stimulated glucose uptake compared with both empty vector and overexpressed WT controls. Interestingly, AS160 coexpressing a mutated Rab GAP domain (2M) prevented the inhibitory effects of the 4P mutations alone and fully restored insulin-stimulated glucose uptake. These changes occurred concomitantly with normal insulin-stimulated signaling to Akt Thr308 across all muscles (Fig. 3C). No significant differences in insulin-stimulated glucose uptake were observed between empty vector controls and overexpressed WT, R/K, and 2M AS160. In addition, there were no significant alterations in glucose uptake compared with injected muscles in mice administered saline as a control. These data suggest that phosphorylation of AS160 at PAS motifs is required for full insulin-stimulated glucose uptake in vivo in adult skeletal muscle.


Figure 3
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  		FIGURE 3.
AS160 overexpression regulates insulin-stimulated glucose uptake without affecting Akt Thr308 phosphorylation in transfected mouse skeletal muscles. Empty pCAGGS vector (E) or Myc-tagged AS160 DNA constructs (wild type and 4P, R/K, and 2M mutants) were injected into the tibialis anterior muscles of anesthetized mice, followed by in vivo electroporation. The animals were allowed to recover, and basal and insulin-stimulated glucose uptake were assessed in vivo 7 days post-injection. The blood glucoses were evaluated in all electroporated mice at 0, 5, 10, 15, 25, 35, and 45 min following [3H]2-deoxyglucose injection combined with either saline or glucose bolus (1.0 grams glucose/kg of body weight) (A). Absolute skeletal muscle glucose uptake was determined 45 min after saline/glucose injections (B). The data are expressed as the means ± S.E. (n = 6–24/group). *, p < 0.05 (versus insulin-stimulated empty pCAGGS controls). In addition, muscles from basal (–) and insulin-stimulated conditions (+) were immunoblotted for antiphospho-Akt Thr308 (C). The data are expressed as the means ± S.E. (n = 5–12/group). *, p < 0.05 (versus basal).

 
AS160 Regulates Contraction-stimulated Glucose Uptake—Exercise and muscle contractions stimulate AS160 phosphorylation at PAS motifs through a wortmannin-insensitive pathway involving AMPK (10). However, it is not known whether these AS160 phosphorylation events, in turn, regulate contraction-stimulated glucose uptake. Therefore, we explored the effects of overexpressed WT AS160 and mutant AS160 on glucose uptake following 15 min of in situ contractions in transfected mouse tibialis anterior muscles (Fig. 4A). Muscles overexpressing 4P mutant AS160 exhibited significant impairments (~44%) in contraction-stimulated glucose uptake compared with empty vector controls. Contraction-stimulated glucose uptake in vivo was also inhibited (~24%) in muscles overexpressing wild type AS160, although it was still significantly higher (20%) than in 4P-overexpressing muscles. In contrast, in vivo glucose uptake after contraction was significantly increased in muscle overexpressing mutant AS160 devoid of Rab GAP activity (21 and 40% for R/K and 2M, respectively). It is important to note that contraction-stimulated phosphorylation of Akt Thr308 (Fig. 4B) and AMPK Thr172 (Fig. 4C) was normal and not significantly different between muscles. These results suggest that AS160 phosphorylation at PAS motifs is essential for full contraction-stimulated glucose uptake in skeletal muscle. Overexpression of constitutively active (4P) AS160 exerted a dominant inhibitory effect on glucose uptake following muscle contractions in vivo, whereas overexpression of AS160 lacking Rab GAP activity surprisingly enhanced skeletal muscle glucose uptake.

Significant differences in basal glucose uptake were observed between sham-operated muscles. Under these conditions, muscles overexpressing Rab GAP mutant AS160 exhibited significant increases in basal glucose uptake compared with empty vector controls (71 and 49% for R/K and 2M AS160, respectively), without detectable changes in fasting glycogen content (data not shown). There were no differences between empty vector, WT, and 4P mutant injected muscles on basal glucose uptake. Taken together, overexpression of mutant AS160 devoid of Rab GAP activity appears to regulate both basal and contraction-stimulated glucose uptake in vivo. The combined regulatory effects that AS160 exerts on both insulin- and contraction-stimulated glucose uptake with in vivo transfections are consistent with a role for AS160 on molecular localization or trafficking.

AS160 Overexpression Does Not Alter Expression of Proteins Involved in Skeletal Muscle Glucose Uptake—To determine whether changes in skeletal muscle glucose uptake observed with AS160 overexpression might be due to adaptive effects on regulatory upstream and downstream proteins, we performed immunoblots for AMPK{alpha}2, Akt1/2, GLUT1, GLUT4, and hexokinase II (Table 1). There were no significant differences between muscles injected with empty vector or any of the AS160 gene constructs. Thus, gene delivery by localized AS160 plasmid injections does not cause genomic disruption or alterations in the expression of key signaling proteins that regulate glucose metabolism in skeletal muscle.


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  		TABLE 1
Immunoblot quantitations of AMPK{alpha}2, Akt1/2, GLUT1, GLUT4, and hexokinase II expression and AS160 expression and phosphorylation (Thr642) in tibialis anterior muscles 1 week following intramuscular injection of empty vector control (E), WT AS160, 4P mutant AS160, R/K mutant AS160, or double mutant (2M) AS160 DNA constructs

 


Figure 4
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  		FIGURE 4.
AS160 overexpression regulates basal (sham) and in situ contraction-stimulated glucose uptake without alterations in Akt Thr308 and AMPK Thr172 phosphorylation in transfected mouse skeletal muscles. Empty pCAGGS vector (E) or Myc-tagged AS160 DNA constructs (wild type and 4P, R/K, and 2M mutants) were injected into the tibialis anterior muscles of anesthetized mice, followed by in vivo electroporation. The animals were allowed to recover, and basal and contraction-stimulated glucose uptake were assessed in vivo 7 days post-injection. Absolute skeletal muscle glucose uptake was determined 45 min after [3H]2-deoxyglucose injection combined with either sham operations or 15 min in situ tibialis anterior muscle contractions (A). The data are expressed as the means ± S.E. (n = 6–24/group). *, p < 0.05 (versus contraction-stimulated empty pCAGGS controls); **, p < 0.05 (versus contraction-stimulated WT AS160 and empty vector controls); {dagger}, p < 0.05 (versus basal empty pCAGGS controls); {dagger}{dagger}, p < 0.05 (versus contraction-stimulated empty pCAGGS, wild type AS160, and 4P-AS160). In addition, muscles from basal/sham (–) and contraction-stimulated conditions (+) were immunoblotted with anti-phospho-Akt Thr308 (B) and anti-phospho-AMPK Thr172 antibodies (C). The data are expressed as the means ± S.E. (n = 5–12/group). *, p < 0.05 (versus sham).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Skeletal muscle is unique in its ability to promote glucose homeostasis through both insulin-dependent and insulin-independent pathways leading to GLUT4 translocation. Successful isolation of a point of signaling convergence just prior to GLUT4 would provide a promising target for therapeutic diabetes intervention. Our lab previously determined that insulin- and contraction-stimulated signaling cascades phosphorylate AS160 PAS motifs in a distinct and additive manner in mouse skeletal muscle (10). The purpose of this study was to characterize a functional role for AS160 on skeletal muscle glucose uptake. Using a local model of protein overexpression, we provide direct evidence that AS160 regulates insulin- and contraction-stimulated glucose uptake in vivo. Phosphorylation of AS160 at PAS motifs is essential for full insulin- and contraction-stimulated glucose uptake in adult skeletal muscle. To our knowledge, this is the first report of a single protein upstream of GLUT4 capable of orchestrating independent insulin and contraction effects on skeletal muscle glucose uptake.

We investigated AS160 function on glucose metabolism by transfecting mouse tibialis anterior muscles with empty vector, wild type AS160, and three mutant AS160 DNA constructs. Utilization of direct intramuscular DNA injection in combination with in vivo electroporation allows for local delivery and expression of the target gene without an immune response. Furthermore, this approach temporarily bypasses developmental compensations inherent to other genetic manipulations such as transgenic or knock-out models (26). Because it is limited to the superficial tibialis anterior muscle, isolated skeletal muscle preparations cannot be performed in a complementary manner. Nonetheless, the prevailing advantage of in vivo electroporation lies in its physiologic nature. The efficacy of this strategy has been validated previously by our lab (17, 18) and others (19) and very clearly allowed for consistent overexpression of wild type and all of the mutant AS160 isoforms in the current study. There were no changes in the expression or activity of known signaling proteins upstream of AS160, nor downstream effects on expression of GLUT1, GLUT4, and hexokinase II, which might otherwise confound interpretation of the results of AS160 on skeletal muscle glucose uptake.

AS160 regulation of insulin-stimulated glucose uptake in mouse skeletal muscles was determined following intravenous administration of glucose. Thus, instead of a supramaximal insulin stimulus, our experiments utilizing a glucose bolus were designed to induce a physiological insulin response (supplementary data) (21, 22). We found that 4P mutant AS160 acted in a dominant interfering capacity, inhibiting insulin-stimulated glucose uptake by over 33% compared with empty vector and WT AS160 controls. This result is consistent with impaired insulin-stimulated exocytosis of GLUT4 in 4P-transfected adipocytes (15). Furthermore, overexpression of the double mutant (2M) AS160, which coexpresses the 4P and Rab GAP domain mutations, effectively restored full insulin-stimulated glucose uptake relative to empty vector and WT AS160 transfected skeletal muscle. These data suggest that activity of the AS160 Rab GAP domain acts to restrain or inhibit glucose uptake, and is reflected in initial studies by Sano et al. (8), which explore the effects of each mutant on GLUT4 translocation in 3T3-L1 cells. However, we did not observe uncontrolled basal glucose transport following overexpression of Rab GAP mutant AS160, which is consistent with studies reporting AS160-independent mechanisms for basal GLUT4 retention (11). Residual activity of endogenous AS160 protein may also be sufficient to ameliorate basal dysregulations in glucose transport. Collectively, our findings implicate a requirement for AS160 phosphorylation to suppress intrinsic Rab GAP activity and thereby facilitate the full effects of insulin on glucose uptake. These are the first data directly determining a functional role for AS160 on insulin-stimulated glucose metabolism in adult skeletal muscle tissue.

The observed effects of AS160 on contraction-stimulated glucose uptake are also entirely novel, albeit more complex. Phosphorylation of AS160 on PAS motifs is regulated by Akt2 in insulin-dependent signaling to glucose uptake; however, these same motifs are also regulated by AMPK{alpha}2 and potentially other kinases involved in insulin-independent signaling to glucose uptake (10). In transgenic mice lacking AMPK{alpha}2 activity, both AICAR-stimulated AS160 phosphorylation (10) and glucose transport (27) are abolished, which suggests that AMPK{alpha}2-associated AS160 phosphorylation is necessary for AICAR-stimulated glucose uptake. However, these mice also exhibit significantly blunted contraction-stimulated AS160 phosphorylation (although they do produce significant contraction-stimulated increases compared with basal) (10), whereas glucose uptake following contraction appears normal after controlling for intrinsic differences in maximum contraction force (27). Developmental compensation could account for this apparent "mismatch" in transgenic animals (26), so we directly tested the function of contraction-stimulated AS160 phosphorylation at PAS motifs using our protein overexpression model in this study. Our data indicate that AS160 phosphorylation at four PAS motifs is necessary for the full effects of contraction on skeletal muscle glucose uptake in vivo. As in the insulin-stimulated condition, overexpression of 4P mutant AS160 exerted a significant dominant inhibitory effect, reducing contraction-stimulated glucose uptake by over 44% compared with empty vector control muscles and by 20% compared with muscles overexpressing WT AS160. These data provide evidence that contraction-mediated phosphorylation events on AS160 are functionally permissive for full stimulation of glucose uptake.

We were surprised to find that overexpression of wild type AS160 also decreased glucose uptake (24%) following contraction, although this value was still significantly higher than 4P-injected muscles. There are at least two possible interpretations of these data. The magnitude of overexpressed wild type and endogenous AS160 was too great for normal contraction-stimulated AS160 phosphorylation, and thus the remaining pool of unphosphorylated or partially phosphorylated AS160 exerted a dominant inhibitory effect. Our findings indirectly support this because we found substantial differences between AS160 overexpression (8-fold versus empty vector controls) and enhanced contraction-stimulated AS160 phosphorylation (~2-fold versus empty vector controls). These differences suggest that contraction-mediated kinase activity does not phosphorylate the exogenous AS160 in proportion to its overexpression, which may therefore account for the partial inhibition of contraction-stimulated glucose uptake by wild type AS160 overexpression. Alternatively, overexpression of wild type AS160 might suppress a molecule specifically activated by contraction but not insulin or perturb native stoichiometries that disrupt contraction-induced glucose uptake.

Overexpression of mutant AS160 devoid of Rab GAP activity (both R/K and 2M isoforms) generated increases in both unstimulated sham and contraction-stimulated glucose uptake compared with controls. These elevations in basal glucose uptake in transfected muscles are intriguing because utilization of siRNA to specifically knock down endogenous AS160 in 3T3-L1 adipocytes also resulted in increased basal glucose transport (11, 12). Re-expression of human wild type AS160 restored normal basal glucose transport in this model, whereas expression of R/K mutant AS160 did not. The interpretation of these data concluded that AS160 GAP activity is required for full intracellular retention of GLUT4 in the basal state (11). Overexpression of GAP-inactive AS160 in our skeletal muscle transfections resulted in similar dysregulation of basal glucose uptake, perhaps mediated by the dominant interfering capacity of these mutants on normal GLUT4 retention. To clarify, the basal glucose uptake values recorded in these experiments were from sham-operated muscles in the opposing leg of muscles stimulated to contract in situ. We did not detect significant changes in basal glucose uptake in animals administered saline as a control during IV glucose protocols. These latter saline basal glucose uptake values were approximately two-thirds lower than the sham basal uptake values obtained in contraction experiments. We have previously determined that sham operation by itself does not significantly increase AS160 phosphorylation above untreated or saline-injected control animals in vivo (10). Muscle contractions are well known to stimulate increases in cardiac output while reducing resistance of the peripheral vasculature. Thus, differences in blood flow and/or secretion of humoral factors likely account for the variation between saline and sham controls in our study, as has been shown in previous investigations (28, 29). However, the relative magnitude of sham basal uptake values afforded an increased sensitivity to detect significant differences in basal glucose uptake between the various AS160 isoforms.

Transfection of GAP-inactive AS160 also resulted in supraphysiological increases in contraction-stimulated glucose uptake in mouse skeletal muscle. In this scenario, we overexpressed a considerable pool of AS160 lacking normal braking (GAP) function on GLUT4 trafficking. AS160 is a relatively large protein with multiple domains, phosphorylation motifs, and docking sites conceivably designed for protein-protein interactions (30). It is possible that abundant AS160 without its inhibitory activity could act as a scaffold encouraging contraction-specific interactions that enhance glucose uptake. The role of AS160 as a scaffold protein will be pursued in future experiments.

To summarize, we used an in vivo electroporation technique to overexpress wild type and mutant AS160 in adult skeletal muscle and subsequently evaluated AS160 function on glucose uptake. Our findings reveal a regulatory role for AS160 on insulin- and contraction-stimulated glucose uptake. Overexpression of 4P mutant AS160, incapable of being phosphorylated at key PAS motifs, significantly inhibited glucose uptake following insulin or contractile stimuli in transfected muscles. Wild type AS160 overexpression also decreased contraction-induced uptake, although the mechanisms for this remain unclear. In contrast, both sham and contraction-stimulated glucose uptake were significantly increased by overexpressing mutant AS160 lacking normal Rab GAP activity. These collective changes occurred in the absence of significant alterations in the activity or expression of putative upstream AS160 kinases or downstream expression of GLUT4. Therefore, AS160 directly regulates insulin- and contraction-stimulated glucose transport in mouse skeletal muscle.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AR42238 and AR45670 (to L. J. G.), Individual Kirschstein National Research Service Awards F32 DK075851 (to E. B. T.) and AR051663 (to C. A. W.), Institutional Predoctoral Fellowship T32 (Penn State University Graduate Program in Physiology, to H. F. K.), and funds from the Joslin Diabetes Endocrinology and Research Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplementary data. Back

1 To whom correspondence should be addressed: Section Head of Metabolism, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2573; Fax: 617-732-2650; E-mail: laurie.goodyear{at}joslin.harvard.edu.

2 The abbreviations used are: AS160, Akt substrate of 160 kDa; GAP, GTPase-activating protein; PAS, phospho-Akt substrate; WT, wild type; 4P, AS160 mutated at four PAS motifs; R/K, mutation of arginine to lysine in AS160 GAP domain; 2M, double mutant AS160 containing both 4P and R/K mutations; AMPK, AMP-activated protein kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Gustav Lienhard (Dartmouth Medical School) for the pan-AS160 antibody and AS160 DNA constructs, as well as extensive helpful discussion. We also thank Dr. Eric Schaefer (Biosource) for the custom lot of anti-phospho-AS160 (Thr642) and Dr. Richard Ho for expertise on gene transfer and technical assistance on in vivo glucose uptake measurements.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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