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Am J Physiol Endocrinol Metab 290: E789-E797, 2006. First published December 13, 2005; doi:10.1152/ajpendo.00564.2004
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Effects of chronic Akt activation on glucose uptake in the heart

Takashi Matsui,1 Tomohisa Nagoshi,1 Eun-Gyoung Hong,2 Ivan Luptak,3 Kirsten Hartil,4 Ling Li,1 Naira Gorovits,4 Maureen J. Charron,4 Jason K. Kim,2 Rong Tian,3 and Anthony Rosenzweig1

1Program in Cardiovascular Gene Therapy, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; 2Section of Endocrinology and Metabolism, National Institutes of Health-Yale Mouse Metabolic Phenotyping Center, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut; 3NMR Laboratory for Physiological Chemistry, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts; and 4Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York

Submitted 7 December 2004 ; accepted in final form 6 December 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake (P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo (P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin.

glucose transporters; transgenic mice; heart

IN THE NORMAL ADULT HEART, fatty acids are the preferential substrate for metabolism and ATP generation. However, glucose is an important alternative energy source in specific pathological conditions, such as ischemia (33), where the more favorable bioenergetics of glucose metabolism are thought to confer important benefits (reviewed in Ref. 9). Glucose utilization is initiated by glucose uptake and in some setting this appears to be the rate-limiting step in glucose utilization (26). Thus understanding control of glucose uptake in the heart may have implications for our approach to a variety of cardiac conditions.

In most tissues, insulin provides the dominant stimulus to enhance glucose transport, largely through activation of the downstream signaling molecules insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3 kinase (PI3K), and the serine/threonine kinase Akt (or protein kinase B). We (30) previously found that acute activation of Akt significantly increased glucose uptake in vitro in cardiomyocytes to a level comparable to that seen with pharmacological insulin stimulation, accompanied by increased sarcolemmal glucose transporter-4 (GLUT4) in vivo. This observation is consistent with prior studies in L6 skeletal myoblasts (15, 42) and adipocytes (5, 17, 22). However, less is known about the effects of chronic Akt activation in the heart.

Glucose uptake is controlled by GLUTs on the plasma membrane. GLUT1 and GLUT4 appear to be the major glucose transporters in cardiac and skeletal muscle as well as in adipose tissue. Although GLUT4 is more abundant than GLUT1 in both the heart and skeletal muscle, the amount of GLUT1 relative to GLUT4 is much higher in the heart than in skeletal muscle (12, 27). In addition, the distribution of these glucose transporters differs between cardiac and skeletal muscle. In skeletal muscle, GLUT4 is more abundant in the membranes of intracellular vesicles and is recruited to the plasma membrane after insulin stimulation, whereas GLUT1 is localized mainly to the plasma membrane at baseline and is not significantly affected by insulin stimulation (43). In contrast, in the heart, GLUT1 is also located in intracellular pool and is recruited to the plasma membrane in response to insulin (2). Thus both GLUT1 and GLUT4 may contribute to enhanced glucose uptake after insulin stimulation in the heart (35).

In addition to GLUTs, hexokinase II plays an important role in regulating insulin-stimulated glucose uptake (3, 14) by catalyzing the first committed step in glycolysis, phosphorylation of glucose to glucose 6-phosphate. Hexokinase II enhances glucose uptake and glycogen synthesis in the heart (25).

To investigate the effects of chronic Akt activation in the heart, we generated transgenic (TG) mice with cardiac-specific expression of a constitutively active form of Akt (myr-Akt) driven by the myosin heavy chain-{alpha} promoter and measured glucose uptake at baseline and in response to insulin with the ex vivo Langendorff perfusion system using 31P-NMR spectroscopy. We found an increase in basal glucose uptake but a blunted response to insulin stimulation. In addition to ex vivo perfused heart, we confirmed increased basal glucose uptake using in vivo awake mice. Although the level of total GLUT1 expression was similar in both TG and littermate nontransgenic (NTG) mice, GLUT4 in TG mice was substantially lower than in NTG mice. Sucrose gradient protein fractionation demonstrated that, in intracellular vesicular pools, GLUT4 in hearts from TG mice was dramatically decreased in addition to a slight reduction on GLUT1. These reductions may contribute to impaired insulin sensitivity in hearts of TG mice. These studies delineate the effects of chronic Akt activation on glucose uptake in the heart and may have relevance for heart failure or other conditions in which Akt activity is enhanced (16).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of TG mice. The Subcommittee on Research Animal Care in Massachusetts General Hospital, Harvard Medical School, approved all aspects of animal care and experimentation performed in this study. TG mice expressing the hemagglutinin (HA)-tagged Akt with src myristoylation (myr) signal under the direction of the murine {alpha}-myosin heavy-chain promoter have previously been described in detail (29). Two viable lines (TG20 and TG564) have been backcrossed >10 generations to C57BL/6 wild-type mice. These lines exhibit stable Mendelian transgene inheritance consistent with autosomal (TG564) and X-linked transmission (TG20). 10- to 18-wk-old age-matched mice were used in all experiments, and either NTG or C57BL/6 wild-type mice were used as control. Because of X-chromosome inactivation, hearts from female TG20 mice exhibit chimeric transgene expression (29). TG564 mice were used for the described experiments unless specified in the figure legend.

Ex vivo glucose uptake using 31P-NMR spectroscopy. After animals were deeply anesthetized with pentobarbital sodium (60 mg/kg ip) following 30 min of heparin (1,000 IU/kg ip) anticoagulation, hearts from overnight-fasted mice were subjected to the ex vivo Langendorff model perfused with phosphate-free Krebs-Henseleit buffer (in mM: 118 NaCl, 25 NaHCO3, 5.3 KCl, 2.5 CaCl2, 1.2 MgSO4, 0.5 EDTA, 5 pyruvate, and 5 glucose). 31P-NMR spectra were collected as previously described (40). Briefly, after a 20-min control perfusion, the phosphate-free Krebs-Henseleit buffer was switched to a buffer containing 3 mM of the glucose analog 2-deoxy-D-glucose (2-DG), instead of glucose, and 1.2 mM KH2PO4 to replenish the phosphate pool. All data were normalized to {gamma}-ATP peak area for quantification. The transport rate (mmol·l–1·min–1) of 2-DG was assessed by the time-dependent accumulation of 2-DG-phosphate (2-DG-P). The rates were measured both before and after the addition of insulin (2 mU/ml) to the perfusate (40).

Glycogen content. Sections were prepared as previously described (29). Periodic Acid-Schiff (PAS) staining was performed with the PAS Staining System (Sigma), according to the manufacturer's instructions. Some samples were pretreated with {alpha}-amylase before PAS staining to confirm the specificity of positive staining. Biochemical assay demonstrated myocardial glycogen content with the amount of glucose released from glycogen after alkaline extraction as previously described (40).

Basal glucose uptake and glycogen synthesis in vivo. Basal rates of glucose uptake and glycogen synthesis in heart were assessed in awake TG and littermate NTG mice (n = 4–5 for both groups) as previously described (20). After an overnight fast, [3-3H]glucose (0.1 µCi/min; PerkinElmer Life and Analytical Sciences, Boston, MA) was continuously infused for 2 h, and 2-deoxy-D-[1-14C]glucose (2-[14C]DG) was administered as a bolus (10 µCi) at 30 min before the end of experiments. Plasma samples were taken at 5-min intervals during the last 30 min of experiments to measure the concentrations of 2-[14C]DG and [3H]glucose. At the end of study, mice were anesthetized with pentobarbital sodium injection, and heart samples were rapidly taken for biochemical assays. Because 2-DG is a glucose analog that is phosphorylated but not further metabolized, glucose uptake in heart can be estimated by determining the myocardial content of 2-[14C]DG-6-P and plasma 2-[14C]DG profile, which was fitted with a double exponential or linear curve using MLAB (Civilized Software, Bethesda, MD). Cardiac glycogen synthesis in vivo was estimated by determining the incorporation of 3H into myocardial glycogen as previously described (20).

Cardiac muscle fractionation. Sucrose gradient purification was used to isolate cytosolic, intracellular vesicle pools and plasma membrane fractions from cardiac muscle, as previously described (43). In brief, hearts from 12- to 16-wk-old male mice were removed from deeply anesthetized animals by means of pentobarbital sodium (60 mg/kg ip), washed with cold PBS, and homogenized in cold lysis buffer (20 mM HEPES, 250 mM sucrose, 1 mM EDTA, 5 mM benzamidine, 1 µM aprotinin, 1 µM pepstatin, 1 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4). The homogenate was then centrifuged at 2,000 g for 10 min, and supernatants removed and centrifuged at 9,000 g for 20 min. Pellets were resuspended in 500 µl of PBS with protease inhibitors, as in the lysis buffer, and kept as a part of plasma membrane fraction (P1). Supernatants were then centrifuged at 180,000 g for 90 min, and the supernatants from this centrifugation were removed and kept as the cytosolic fraction. The remaining pellets were resuspended in 500 µl of PBS with protease inhibitors. Protein concentration was determined, and the same amount of protein from each sample was loaded on a 10–30% (wt/wt) continuous sucrose gradient and centrifuged at 48,000 rpm for 55 min in an SW-50 rotor. Gradients were separated into 26 fractions, starting from the bottom of the tube (heavy fraction). The pellet was resuspended in PBS and named P2 (plasma membrane fraction 2). Each experiment was done with a pair of littermate TG and NTG mice. Fractions isolated from TG and NTG hearts were subjected in parallel to SDS-PAGE (12%) and transferred to a single nitrocellulose membrane for immunoblotting.

Western blotting. Frozen hearts were crushed in liquid nitrogen and homogenized in cold lysis buffer, as previously described (29). After the samples were lysed, SDS-PAGE was performed under reducing conditions on 10 or 12% separation gels with a 4% stacking gel. Proteins were transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies to GLUT1 (FabGennix), GLUT4 (FabGennix), GSK-3beta (Cell Signaling), phospho-Ser9 GSK-3beta (Cell Signaling), beta1-integrin (Santa Cruz Biotechnology), SCAMP2 (Abcam), HA (12CA5, Roche), or hexokinase type II (IREAGQR, Chemicon) and subsequently with horaseradish peroxidase-conjugated secondary antibodies. Signal was detected using enhanced chemiluminescence and band intensity quantified using NIH Image.

Quantitative RT-PCR. Total RNA was isolated from TG564 and NTG mice and quantitative RT-PCR (QRT-PCR) performed using the Brilliant One-Step QRT-PCR kit (Stratagene, La Jolla, Ca) containing SYBR Green I (Sigma), as described previously (6). Accumulation of PCR product was monitored in real time and the crossing threshold (CT) was determined with the Mx4000 system (Stratagene). Fold change in gene expression was determined using the {Delta}CT method with normalization to GAPDH (6, 32). QRT-PCRs were performed with the following sets of primers: for GLUT4, forward 5'-ATGGCTGTCGCTGGTTTCTC and reverse 5'-TAAGGACCCATAGCATCCGC; for GAPDH, forward 5'-TGGTGAAGCAGGCATCTGAG and reverse 5'-TGCTGTTGAAGTCGCAGGAG.

Statistical analysis. All data are presented as means ± SE from at least three independent experiments and were compared using a two-tailed Student's t-test. The null hypothesis was rejected at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Glucose uptake in ex vivo myr-Akt transgenic mice. To minimize variability due to endogenous insulin release, we measured basal and insulin-stimulated glucose uptake in hearts from overnight-fasted TG and NTG mice. Cardiac glucose uptake was assessed in an ex vivo Langendorff perfusion system using 31P-NMR spectroscopy and glucose analog 2-DG. At baseline, the rate of 2-DG-P accumulation was significantly higher in TG than in NTG mice (Fig. 1A, top). Cumulative data demonstrated that the basal glucose uptake in TG mice was greater than that seen in NTG mice (0.87 ± 0.18 vs. 0.18 ± 0.10 mmol·l–1·min–1, P < 0.05, Fig. 1B). After baseline 2-DG-P accumulation was measured for 40 min, insulin (final concentration = 2 mU/ml) was added to the perfusate and glucose uptake assessed in the same way. As we expected, glucose uptake in NTG hearts was enhanced by insulin more than 18-fold. In contrast, insulin-stimulating glucose uptake in the heart of TG mice demonstrated much less response than that seen in NTG mice (1.9 vs. 18.1-fold, Fig. 1, A and B). Although glucose uptake after insulin perfusion in TG mice is almost twice bigger than that in baseline, there is no statistical difference (P = 0.06, Fig. 1B).


Figure 1
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  		Fig. 1. Glucose uptake in isolated perfused hearts from transgenic (TG) and non-TG (NTG) mice. A: representative NMR spectra. PCr, phosphocreatine. After 20-min control perfusion with 2-deoxyglucose (2-DG)-free buffer supplemented with glucose and pyruvate, heart from TG564 or NTG mice was perfused with glucose-free buffer containing 2-DG for 40 min before stimulation with insulin for 20 min. Top: spectra before insulin stimulation (Baseline); bottom: spectra during 2 mU/ml insulin stimulation (Insulin stim.). Five hearts from each group were examined. Representative data of TG564 and NTG mice are shown. B: rates of glucose uptake. Rates of 2-DG-phosphate (2-DG-P) accumulation in NTG (n = 5) and TG564 (n = 5) were measured as described in EXPERIMENTAL PROCEDURES.

 
Glycogen content in myr-Akt TG mice. To examine whether enhanced glucose uptake resulted in increased storage of glycogen, we performed histochemical staining and biochemical analyses. PAS staining of hearts from fasted NTG mice did not reveal significant glycogen stores (Fig. 2A). In contrast, PAS staining of hearts from both male and female TG20 mice revealed substantial glycogen accumulation. Treatment with {alpha}-amylase completely abrogated this staining, confirming its specificity (Fig. 2A). As noted above, the X-linked transgene in TG20 mice results in chimeric transgene expression in hearts from female mice (29). Consistent with this, fewer cardiomyocytes in female TG20 hearts stained positive with PAS compared with those in male TG20 mice, although staining in male TG20 mice was also somewhat heterogenous (Fig. 2A). These data suggest that the effect of myr-Akt on glycogen deposition is likely a cell autonomous effect of the transgene rather than a secondary effect of the hypertrophy seen in both male and female TG20 mice. PAS staining in TG564 mice appeared similar to that seen in TG20 male mice (data not shown). To quantitate glycogen content, we measured the amount of glucose released by alkaline extraction (40). Glycogen content in Akt TG mice was more than sixfold higher than that seen in NTG littermates (37.1 ± 1.2 vs. 5.5 ± 2.6 µmol glucose/g wet wt, P < 0.001; Fig. 2B). We next examined expression and phosphorylation (Ser9) of GSK-3beta, which causes inhibition of GSK-3 and consequently increases the activity of glycogen synthase. To minimize the contribution of leukocytes and other circulating factors, we studied GSK-3beta phosphorylation in the heart after a 20-min ex vivo perfusion in the Langendorff system. The level of phosphorylation in TG564 mice was substantially higher than that in NTG littermates (Fig. 2C).


Figure 2
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  		Fig. 2. Glycogen accumulation. A: representative Periodic Acid-Schiff (PAS) staining. Hearts from overnight-fasted male and female TG20 mice and negative female littermates were harvested and snap-frozen for 10-µm sections. Pretreatment with {alpha}-amylase confirmed the specificity of staining (bottom). B: quantification of glycogen content. Glycogen content in hearts from overnight-fasted TG564 (n = 3) and NTG (n = 3) mice was measured as described in EXPERIMENTAL PROCEDURES. C: phosphorylation of GSK-3beta. Total cell lysates (25 µg) were subjected to Western Blotting with anti-phospho (P-)(Ser9) GSK-3beta and total GSK-3beta. Blots are representative of 3 independent experiments.

 
Basal glucose uptake and glycogen synthesis in vivo. Akt TG mice show cardiac hypertrophy (2.3-fold greater than NTG in TG564) (29), which could cause an overestimation of glucose uptake in the ex vivo perfused heart system of NMR. Therefore, we also evaluated cardiac glucose uptake in vivo using a combination of continuous [3-3H]glucose infusion and bolus 2-[14C]DG injection in awake mice. In vivo, the basal rate of cardiac glucose uptake in Akt TG mice was substantially higher than that in NTG mice (320.6 ± 56.3 vs. 49.1 ± 10.6 nmol·g–1·min–1, P < 0.005; Fig. 3A). This change in basal glucose uptake in vivo agrees well with that measured by NMR in ex vivo perfused hearts (4.8-fold increase ex vivo vs. 6.5-fold in vivo). By use of these samples, basal glycogen synthesis in vivo was estimated as the rate of 3H incorporation into myocardial glycogen. The results indicate that basal glycogen synthesis in hearts from Akt TG mice was markedly elevated compared with the NTG mice (20.7 ± 4.0 vs. 4.2 ± 2.4 nmol·g–1·min–1, P < 0.01; Fig. 3B). Increased glycogen synthesis in vivo was also consistent with the result of accumulated glycogen content in the hearts of Akt TG mice in histological analysis and biochemical assay.


Figure 3
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  		Fig. 3. Basal glucose uptake and glycogen synthesis in heart from TG and NTG mice in vivo. A: basal glucose uptake. Cardiac glucose uptake was measured in awake TG564 (n = 4) and NTG (n = 5) mice. B: basal glycogen synthesis. Cardiac glycogen synthesis was measured in awake TG564 (n = 4) and NTG (n = 5) mice.

 
GLUT1 and GLUT4 expression in the myr-Akt TG mice. To identify potential mechanisms contributing to glucose uptake in the heart, we examined the expression of the two main cardiac glucose transporters, GLUT1 and GLUT4. Total expression of GLUT4 was significantly reduced in whole cell lysates from TG mice compared with NTG mice (~32% reduced in TG, P < 0.05; Fig. 4A). In contrast, overall GLUT1 expression appeared comparable between TG and NTG (Fig. 4A). Because GLUTs enhance glucose uptake functionally at the plasma membrane, we analyzed the distribution of both GLUT1 and -4 in hearts by sucrose gradient fractionation. This method distinguishes proteins associated with intracellular membrane structures such as intracellular vesicles from those associated with the plasma membrane (23, 43). Immunoblotting for the sarcolemmal protein beta1-integrin confirmed localization of plasma membrane proteins to the P2 (and to a lesser extent P1) fractions without cross-contamination in the intracellular or cytosolic fractions (Fig. 4B). Secretory carrier-associated membrane proteins (SCAMPs) are located in cell surface recycling vesicles that contain GLUT4 (43). Immunoblotting for SCAMP2 confirmed the distribution of these small vesicles containing GLUT1 and GLUT4 in the heart (Fig. 4B). Immunoblotting for the HA epitope encoded by the Akt transgene revealed the presence of myr-Akt in both the plasma membrane and the cytosolic fractions (Fig. 4B), consistent with prior reports (4). In NTG mice, immunoreactive GLUT4 was evident in both the plasma membrane and intracellular vesicular fractions but not in the cytosol (Fig. 4B). Cumulative densitometric quantitation demonstrated that, at baseline, GLUT4 was predominantly in the intracellular vesicular fractions rather than in the plasma membrane fractions (3.8-fold higher in intracellular vesicular fractions; Fig. 4C), whereas GLUT1 protein was comparable in both sites (Fig. 4C). Sucrose gradient showed that in Akt TG hearts GLUT4 was substantially reduced in both the plasma membrane (21.6 ± 5.8 vs. 100, P < 0.01) and the intracellular vesicular fractions (137.6 ± 122.8 vs. 379.5 ± 156.5, P < 0.05; Fig. 4, B and C). The level of GLUT1 protein in Akt TG hearts was slightly lower in both the plasma membrane and the intracellular vesicular fractions than that in NTG hearts, although these differences did not achieve statistical significance (Fig. 4, B and C). To address the mechanism of increased basal glucose uptake in Akt TG hearts, we evaluated the distribution of GLUT1 protein in TG hearts between the plasma membrane (PM) and intracellular vesicular (IV) compartments. No statistically significant difference was seen in the PM/IV ratio of immunoreactive GLUT1 [1.21 ± 0.25 vs. 1.59 ± 0.47, NTG vs. TG, P = not significant (ns)]. To study a regulation of GLUT4 expression in the heart of myr-Akt TG mice, we examined the mRNA level of GLUT4 by QRT-PCR (Fig. 4D). By use of {Delta}CT, quantitative analysis was done with normalization to GAPDH. Cumulative data demonstrated that the mRNA level of GLUT4 in TG564 mice was lower than that in NTG mice (0.73 ± 0.05 vs. 1.02 ± 0.11, P < 0.05; Fig. 4D).


Figure 4
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  		Fig. 4. Expression of GLUT4 and GLUT1 in hearts from TG and NTG mice. A: total expression of GLUT4 and -1. Proteins (25 µg) from whole cell lysate were loaded and subjected to Western blotting. Representative immunoblot of GLUT4 and -1 from cardiac lysates of TG564 and NTG is shown from 3 independent experiments. GAPDH immunoblotting is shown as a loading control. Densitometric quantitation of GLUT4 and -1 from TG564 (n = 7) and NTG (n = 7) mice is shown in each bar graph. B: sucrose gradient fractionation of cardiac muscle proteins. Cardiac muscle protein fractions from TG564 and littermate NTG mice were prepared as described in EXPERIMENTAL PROCEDURES (Cardiac muscle fractionation). In each experiment, the same amount of protein was loaded in pellet 1 (P1), P2, and cytosolic fraction (Cyt). For intracellular (vesicular) membrane fractions (lanes 3–24), the same volume of sample from each fraction was loaded. Arrows indicate size corresponding to Akt with src myristoylation (myr-Akt; bottom). IB, immunoblot; SCAMP, secretory carrier-associated membrane protein; HA, hemagglutinin. Representative data from 5 independent experiments are shown. C: densitometric quantitation of GLUT4 and GLUT1 in cardiac protein fractions from TG564 and NTG mice. Expression levels of GLUT4 and GLUT1 in plasma membrane (PM) and total intracellular vesicular (IV) fractions from TG564 (n = 3) and NTG (n = 3) mice were measured. D: Quantitative RT-PCR of GLUT4. Total RNA was isolated from TG564 (n = 5) and NTG (n = 5) mice. Fold change of mRNA was calculated as described in EXPERIMENTAL PROCEDURES.

 
At baseline, GLUT4 expression in total lysate and sucrose gradient fractionation from the hearts of TG mice was significantly lower than that in NTG mice (Fig. 4, A–C). The reduction in intracellular or vesicular pools of GLUT4 likely contributes to the blunted insulin response seen in these mice. To confirm the response to insulin on the distribution of GLUT4 expression, we performed sucrose gradient fractionation in the ex vivo heart stimulated with a 20-min insulin perfusion, which was done with the same condition to 31P-NMR spectroscopy (Fig. 1). In NTG mice, the GLUT4 expression in the intracellular vesicular fraction was significantly reduced by insulin exposure (387.6 ± 153.4 vs. 172.2 ± 110.4, P < 0.05; Fig. 5). In addition, the PM/IV ratio was dramatically increased after 20-min insulin exposure (0.25 to 0.79), suggesting the translocation of GLUT4 from the intracellular vesicular fraction to the plasma membrane, although an increase of GLUT4 expression in the plasma membrane was not statistically significant (100 vs. 135.1 ± 11.2, unstimulated vs. insulin-stimulated hearts). In contrast, the PM/IV ratio in TG mice by insulin stimulation was comparable to that in unstimulated hearts (0.62 vs. 0.51, unstimulated vs. insulin-stimulated hearts) in addition to there being no change in the GLUT4 expression in the intracellular vesicular fraction (151.7 ± 71.0 vs. 120.1 ± 55.6, unstimulated vs. insulin-stimulated hearts) and in the plasma membrane (94.8 ± 14.2 vs. 68.0 ± 13.7, unstimulated vs. insulin-stimulated hearts). These data demonstrated less capacity of GLUT4 recruitment to the plasma membrane by acute insulin stimulation in TG mice.


Figure 5
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  		Fig. 5. GLUT4 expression in ex vivo perfused hearts with or without insulin stimulation. A: sucrose gradient fractionation of unstimulated and insulin-stimulated hearts. Hearts from TG564 and NTG mice were perfused in ex vivo Langendorff model. After 20-min control perfusion, hearts were stimulated with (+) or without (–) 2 mU/ml insulin in perfusion buffer for 20 min. Sucrose gradient fractionation was performed as described in EXPERIMENTAL PROCEDURES. Representative immunoblot of GLUT4 from TG564 and NTG with or without insulin stimulation is shown from 3 independent experiments. B: densitometric quantitation of GLUT4 in sucrose gradient fractionations from TG564 and NTG mice. Expression levels of GLUT4 in PM and total IV fractions from hearts of TG564 and NTG mice were measured. Graphs show cumulative data from 3 hearts in each setting (total 12): unstimulated NTG, insulin-stimulated NTG, unstimulated TG564, and insulin-stimulated TG564.

 
Hexokinase II, relevant to glucose uptake in the myr-Akt TG mice. To identify potential contributors to the increased basal glucose uptake seen in Akt TG mice, we examined hexokinase, which controls the first committed step in glycolysis (26). We observed no difference in hexokinae II expression between fasted TG and NTG mice (Fig. 6). Similarly, hexokinase was not different in fed TG and NTG mice (data not shown).


Figure 6
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  		Fig. 6. Expression of hexokinase II (HKII) in hearts from TG and NTG mice. Representative immunoblot of HKII from cardiac lysates of TG564 and NTG is shown from 3 independent experiments. GAPDH immunoblotting is shown as loading control. Densitometric quantitation of HKII from TG564 (n = 9) and NTG (n = 8) is shown in each bar.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the heart. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake but a blunted response to insulin. These changes were associated with an increase in glycogen deposition (>6-fold) and synthesis (5-fold). Studies in chimeric hearts of female X-linked transgenic Akt mice suggest that increased glycogen deposition occurs as a cell autonomous effect of transgene expression. Although acute Akt activation appears to increase glucose uptake predominantly through enhanced sarcolemmal GLUT4 (30), chronic Akt activation actually led to a significant decrease in total expression of GLUT4. Sucrose gradient showed that intracellular GLUT4 was substantially reduced in addition to a modest reduction of intracellular pools of GLUT1 in chronic Akt activation. Because translocation of these pools to the plasma membrane mediates the acute increase in glucose uptake seen in the heart after insulin stimulation (2, 12), it seems likely that this reduction contributes to the loss of insulin responsiveness that we observed. In fact, the maximal insulin-stimulated 2-DG uptake in hearts of TG mice was only one-half of that in NTG mice, reflecting a decrease in capacity of GLUT4 (32% in whole lysates and 64% in the intracellular vesicular fractions).

The observed increase in glycogen deposition in Akt TG hearts likely reflects the persistently increased glucose uptake in these hearts, which in other settings appears sufficient to increase glycogen content in the heart (40). However, other signaling effects could also contribute. For example, the activation of Akt by insulin inactivates GSK-3beta through Ser9 phosphorylation, thereby enhancing activation of glycogen synthase (7, 8). In fact, after ex vivo perfusion, we did observe an increase in GSK-3beta phosphorylation (Fig. 2C), suggesting that this mechanism could contribute to the increased glycogen in the heart of TG mice. Interestingly, previous studies (29, 37) with nonperfused, freshly isolated hearts did not detect this difference, perhaps reflecting a higher background contribution of noncardiomyocyte population, e.g., leukocytes, to GSK-3beta phosphorylation. Recently, it has been reported that muscle-specific deletion of GLU4 in mice increases glycogen content despite reduced glucose transport (21). In that case, they demonstrated an increase of hexokinase II expression and protein phosphatase-1 (PP1) activity with dominating over GSK-3 effects. In the present study, GSK-3 effects seem more dominant on regulation of glycogen content, because we did see significant phosphorylation of GSK-3beta with enhanced baseline glucose uptake but no increase of hexokinase II. We examined glycogen content and synthesis in Akt TG mice to see the effect of enhanced basal glucose uptake in chronic Akt activation. Although a series of ex vivo and in vivo experiments demonstrating an increase of basal glucose uptake and glycogen deposition supports the hypothesis that glycogen is a dominant fate of the increased glucose uptake in this setting, future studies will be directed at investigating other potential metabolic fates of glucose.

The dramatic reduction in GLUT4 expression that we observed is consistent with prior studies documenting decreased GLUT4 after prolonged insulin exposure in other tissues (36, 39). Interestingly, a similar phenomenon is also seen in pressure overload hypertrophy in the heart (41), a condition in which Akt is activated (11). Using QRT-PCR, we showed a decrease of the mRNA level of GLUT4 in a model of chronic Akt activation, which is consistent with a prior report demonstrating that chronic insulin treatment decreases GLUT4 mRNA in an in vitro cell line (13). In addition to transcriptional regulation, other mechanisms, such as modification at translation, might be involved in this setting, as reported previously (39). To examine insulin-induced GLUT4 translocation, hearts stimulated with insulin were subjected to sucrose gradient protein purification. In NTG mice, acute insulin stimulation dramatically reduced the expression of GLUT4 in the intracellular vesicular fractions and increased the PM/IV ratio, although an increase of GLUT4 expression in the plasma membrane did not achieve statistical significance. On the other hand, the expression level of GLUT4 in the intracellular vesicular fractions in myr-Akt TG mice was not changed by insulin. These data suggest that chronic Akt activation reduces the expression level of GLUT4, especially in insulin-responsive compartments.

We did not observe an increase of GLUT1 expression previously noted in these settings (24, 36, 39, 41). Moreover, sucrose gradient fractionation demonstrated that GLUT1 in Akt TG hearts was slightly lower in both the plasma membrane and the intracellular vesicular fractions than that in NTG hearts, although these differences did not achieve statistical significance. Because GLUT1 in the heart, as with GLUT4, is recruited to the plasma membrane in response to insulin (2, 35), a reduction of both GLUT4 and GLUT1 in intracellular pools likely accounts for the blunted insulin response seen with chronic Akt activation.

Although GLUT4 is more abundant than GLUT1 in both the heart and skeletal muscle, the ratio of GLUT1 to GLUT4 is much higher in the heart than in skeletal muscle (12, 27). Furthermore, the localization of GLUT1 at baseline, seen predominantly in the plasma membrane, suggests that GLUT1 plays an important role in basal glucose uptake in the heart (35). Hearts from NTG showed that GLUT1, but not GLUT4, was predominantly localized in the plasma membrane, consistent with previous reports (12, 35). To address the mechanism of increased glucose uptake in Akt TG mice, we compared the distribution of GLUT1 in TG mice with that in NTG with the ratio of PM/IV immunoreactive protein. No difference was seen in this ratio, suggesting that GLUT1 did not account for the increased basal glucose uptake in TG mice. The present studies cannot exclude the possibility that the intrinsic activity of GLUT1 and GLUT4 might be altered and thereby contribute to the enhanced basal glucose uptake (18).

Although acute Akt activation shows beneficial effects on the ischemic heart (28), our findings raise the possibility that chronic activation of these pathways may cause adverse effects on cardiac function in some settings such as ischemia-reperfusion injury. Indeed, glycogen deposition and cardiac insulin resistance can be associated with cardiomyopathy in different settings (1, 31). Tissue insulin resistance has previously been associated with a variety of signaling abnormalities, including IRS-1/2 (see review in Ref. 38). In fact, we (32) have recently reported that chronic Akt activation impairs cardiac functional recovery and increases injury in an ex vivo model of ischemia-reperfusion injury through negative feedback inhibition of IRS-1/PI3K signaling. Reductions in the levels of both IRS-1 and IRS-2 culminate in impaired activation of PI3K in response to ligands such as insulin or IGF-I and reperfusion (32). Thus we infer that the combination of impaired upstream insulin signaling together with the alterations in GLUT1 and GLUT4 levels is likely to account for the diminished ability of Akt TG hearts to increase glucose uptake after insulin stimulation. This "cardiac insulin resistance" may have relevance for heart failure (16) or other conditions associated with chronic activation of Akt signaling. In these settings, Akt activation may reflect increased expression of growth factors such as IGF-I induced as an initially compensatory response (19, 34).

Baseline function in hearts from Akt TG mice manifested moderate increases in both positive and negative dP/dt measured ex vivo, whereas other major cardiac functions, such as the mean left ventricular systolic pressure (LVSP) and end-diastolic pressure (LVEDP) were comparable to those in NTG (32). However, functional recovery and injury after ischemia-reperfusion were dramatically worse in hearts from Akt TG mice compared with NTG (32). Because glucose is utilized as a critical energy source during cardiac ischemia, the inability of Akt TG hearts to enhance glucose uptake may well contribute to the observed deleterious effects of chronic Akt activation in this model. The current study demonstrating less expression of GLUT4 in Akt TG mice suggests an additional mechanism contributing to this cardiac deterioration after ischemia-reperfusion injury in addition to Akt-driven feedback inhibition of IRS-1(32).

Although the lower levels of GLUT1 and GLUT4 in the intracellular vesicular fractions likely contribute to the loss of insulin responsiveness, the molecular explanation for enhanced basal glucose uptake in Akt TG hearts has not been identified in the current study. Hexokinase II is known to increase glucose uptake in the heart (3, 14), but it was not significantly different between Akt TG and NTG hearts. A variety of other glucose transporters and enzymes contribute to glucose uptake in skeletal and heart muscle. For example, although GLUT8 expression in the heart is less than in other tissue such as testis, the high efficiency of this glucose transporter suggests that it may play a role in glucose uptake in the heart (10). However, GLUT8 was not increased in hearts from Akt TG mice (data not shown). It is possible that enhanced expression of novel cardiac glucose transporters contributes to the observed increase in basal glucose uptake. In this context, the Akt TG mice could provide a valuable substrate for investigating the possibility of novel glucose transportors and/or modulatory pathways by facilitating subtractive screens to identify such mechanisms.

In summary, the present data demonstrate that chronic Akt activation in the heart led to a substantial increase in the basal glucose uptake and glycogen deposition but blunted the response to insulin. Decreases in the insulin-responsive intracellular pools of GLUT1 and GLUT4 could represent an additional mechanism of insulin resistance on chronic Akt activation in the heart, in addition to IRS-1/PI3K inhibition (32). Additional studies will be necessary to define the contribution of these mechanisms to pathophysiological conditions in the heart such as cardiomyopathy or heart failure.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by grants from the National Institutes of Health (HL-04250 to T. Matsui; HL-58119 and DK-47425 to M. J. Charron; U24 DK-59635 to J. K. Kim; HL-59246 and HL-67970 to R. Tian; and HL-59521 and HL-61557 to A. Rosenzweig), American Diabetes Association (1-04-RA-47 to J. K. Kim), and The Robert Leet and Clara Guthrie Patterson Trust Award to J. K. Kim. T. Matsui is a recipient of a Grant-in-Aid from the American Heart Association Northeast Research Consortium. I. Luptak is a recipient of the American Heart Association Northeast Consortium Fellowship. A. Rosenzweig and R. Tian are Established Investigators of the American Heart Association.


   ACKNOWLEDGMENTS
 
We thank Drs. Konstantin V. Kandror and Tatyana A. Kupriyanova for their helpful advice.

Present address of E.-G. Hong and J. K. Kim: Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA.

Present address of T. Matsui, L. Li, and A. Rosenzweig: Cardiovascular Division, Beth Israel Deaconess Medical Center, Boston, MA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Matsui, Program in Cardiovascular Gene Therapy, Cardiovascular Research Center, Massachusetts General Hospital–EAST, 114 16th St., Rm. 2625, Charlestown, MA 02129 (e-mail: tmatsui{at}partners.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Arad M, Maron BJ, Gorham JM, Johnson WH Jr, Saul JP, Perez-Atayde AR, Spirito P, Wright GB, Kanter RJ, Seidman CE, and Seidman JG. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N Engl J Med 352: 362–372, 2005.[Abstract/Free Full Text]
  2. Becker C, Sevilla L, Tomas E, Palacin M, Zorzano A, and Fischer Y. The endosomal compartment is an insulin-sensitive recruitment site for GLUT4 and GLUT1 glucose transporters in cardiac myocytes. Endocrinology 142: 5267–5276, 2001.[Abstract/Free Full Text]
  3. Chang PY, Jensen J, Printz RL, Granner DK, Ivy JL, and Moller DE. Overexpression of hexokinase II in transgenic mice. Evidence that increased phosphorylation augments muscle glucose uptake. J Biol Chem 271: 14834–14839, 1996.[Abstract/Free Full Text]
  4. Coffer PJ, Jin J, and Woodgett JR. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 335: 1–13, 1998.
  5. Cong LN, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, and Quon MJ. Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11: 1881–1890, 1997.[Abstract/Free Full Text]
  6. Cook SA, Matsui T, Li L, and Rosenzweig A. Transcriptional effects of chronic Akt activation in the heart. J Biol Chem 277: 22528–22533, 2002.[Abstract/Free Full Text]
  7. Cross DA, Alessi DR, Cohen P, Andjelkovich M, and Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785–789, 1995.[CrossRef][Medline]
  8. Cross DA, Watt PW, Shaw M, van der Kaay J, Downes CP, Holder JC, and Cohen P. Insulin activates protein kinase B, inhibits glycogen synthase kinase-3 and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue. FEBS Lett 406: 211–215, 1997.[CrossRef][ISI][Medline]
  9. Depre C, Vanoverschelde JL, and Taegtmeyer H. Glucose for the heart. Circulation 99: 578–588, 1999.[Free Full Text]
  10. Doege H, Schurmann A, Bahrenberg G, Brauers A, and Joost HG. GLUT8, a novel member of the sugar transport facilitator family with glucose transport activity. J Biol Chem 275: 16275–16280, 2000.[Abstract/Free Full Text]
  11. Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, and Rockman HA. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation 105: 85–92, 2002.[Abstract/Free Full Text]
  12. Fischer Y, Thomas J, Sevilla L, Munoz P, Becker C, Holman G, Kozka IJ, Palacin M, Testar X, Kammermeier H, and Zorzano A. Insulin-induced recruitment of glucose transporter 4 (GLUT4) and GLUT1 in isolated rat cardiac myocytes. Evidence of the existence of different intracellular GLUT4 vesicle populations. J Biol Chem 272: 7085–7092, 1997.[Abstract/Free Full Text]
  13. Flores-Riveros JR, McLenithan JC, Ezaki O, and Lane MD. Insulin down-regulates expression of the insulin-responsive glucose transporter (GLUT4) gene: effects on transcription and mRNA turnover. Proc Natl Acad Sci USA 90: 512–516, 1993.[Abstract/Free Full Text]
  14. Fueger PT, Heikkinen S, Bracy DP, Malabanan CM, Pencek RR, Laakso M, and Wasserman DH. Hexokinase II partial knockout impairs exercise-stimulated glucose uptake in oxidative muscles of mice. Am J Physiol Endocrinol Metab 285: E958–E963, 2003.[Abstract/Free Full Text]
  15. Hajduch E, Alessi DR, Hemmings BA, and Hundal HS. Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells. Diabetes 47: 1006–1013, 1998.[Abstract]
  16. Haq S, Choukroun G, Lim H, Tymitz KM, del Monte F, Gwathmey J, Grazette L, Michael A, Hajjar R, Force T, and Molkentin JD. Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation 103: 670–677, 2001.[Abstract/Free Full Text]
  17. Hill MM, Clark SF, Tucker DF, Birnbaum MJ, James DE, and Macaulay SL. A role for protein kinase Bbeta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes. Mol Cell Biol 19: 7771–7781, 1999.[Abstract/Free Full Text]
  18. Huang C, Somwar R, Patel N, Niu W, Torok D, and Klip A. Sustained exposure of L6 myotubes to high glucose and insulin decreases insulin-stimulated GLUT4 translocation but upregulates GLUT4 activity. Diabetes 51: 2090–2098, 2002.[Abstract/Free Full Text]
  19. Isgaard J, Wahlander H, Adams MA, and Friberg P. Increased expression of growth hormone receptor mRNA and insulin-like growth factor-I mRNA in volume-overloaded hearts. Hypertension 23: 884–888, 1994.[Abstract/Free Full Text]
  20. Kim HJ, Higashimori T, Park SY, Choi H, Dong J, Kim YJ, Noh HL, Cho YR, Cline G, Kim YB, and Kim JK. Differential effects of interleukin-6 and -10 on skeletal muscle and liver insulin action in vivo. Diabetes 53: 1060–1067, 2004.[Abstract/Free Full Text]
  21. Kim YB, Peroni OD, Aschenbach WG, Minokoshi Y, Kotani K, Zisman A, Kahn CR, Goodyear LJ, and Kahn BB. Muscle-specific deletion of the glut4 glucose transporter alters multiple regulatory steps in glycogen metabolism. Mol Cell Biol 25: 9713–9723, 2005.[Abstract/Free Full Text]
  22. Kohn AD, Summers SA, Birnbaum MJ, and Roth RA. Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271: 31372–31378, 1996.[Abstract/Free Full Text]
  23. Kupriyanova TA and Kandror KV. Cellugyrin is a marker for a distinct population of intracellular Glut4-containing vesicles. J Biol Chem 275: 36263–36268, 2000.[Abstract/Free Full Text]
  24. Laybutt DR, Thompson AL, Cooney GJ, and Kraegen EW. Selective chronic regulation of GLUT1 and GLUT4 content by insulin, glucose, and lipid in rat cardiac muscle in vivo. Am J Physiol Heart Circ Physiol 273: H1309–H1316, 1997.[Abstract/Free Full Text]
  25. Liang Q, Donthi RV, Kralik PM, and Epstein PN. Elevated hexokinase increases cardiac glycolysis in transgenic mice. Cardiovasc Res 53: 423–430, 2002.[Abstract/Free Full Text]
  26. Manchester J, Kong X, Nerbonne J, Lowry OH, and Lawrence JC Jr. Glucose transport and phosphorylation in single cardiac myocytes: rate-limiting steps in glucose metabolism. Am J Physiol Endocrinol Metab 266: E326–E333, 1994.[Abstract/Free Full Text]
  27. Marette A, Richardson JM, Ramlal T, Balon TW, Vranic M, Pessin JE, and Klip A. Abundance, localization, and insulin-induced translocation of glucose transporters in red and white muscle. Am J Physiol Cell Physiol 263: C443–C452, 1992.[Abstract/Free Full Text]
  28. Matsui T, Li L, del Monte F, Fukui Y, Franke TF, Hajjar RJ, and Rosenzweig A. Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro. Circulation 100: 2373–2379, 1999.[Abstract/Free Full Text]
  29. Matsui T, Li L, Wu JC, Cook SA, Nagoshi T, Picard MH, Liao R, and Rosenzweig A. Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J Biol Chem 277: 22896–22901, 2002.[Abstract/Free Full Text]
  30. Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, Force TL, Franke TF, Hajjar RJ, and Rosenzweig A. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation 104: 330–335, 2001.[Abstract/Free Full Text]
  31. McFalls EO, Murad B, Liow JS, Gannon MC, Haspel HC, Lange A, Marx D, Sikora J, and Ward HB. Glucose uptake and glycogen levels are increased in pig heart after repetitive ischemia. Am J Physiol Heart Circ Physiol 282: H205–H211, 2002.[Abstract/Free Full Text]
  32. Nagoshi T, Matsui T, Aoyama T, Leri A, Anversa P, Li L, Ogawa W, Del Monte F, Gwathmey JK, Grazette L, Hemmings B, Kass DA, Champion HC, and Rosenzweig A. PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury. J Clin Invest 115: 2128–2138, 2005.[CrossRef][ISI][Medline]
  33. Owen P, Dennis S, and Opie LH. Glucose flux rate regulates onset of ischemic contracture in globally underperfused rat hearts. Circ Res 66: 344–354, 1990.[Abstract/Free Full Text]
  34. Reiss K, Kajstura J, Zhang X, Li P, Szoke E, Olivetti G, and Anversa P. Acute myocardial infarction leads to upregulation of the IGF-1 autocrine system, DNA replication, and nuclear mitotic division in the remaining viable cardiac myocytes. Exp Cell Res 213: 463–472, 1994.[CrossRef][ISI][Medline]
  35. Russell RR, 3rd Yin R, Caplan MJ, Hu X, Ren J, Shulman GI, Sinusas AJ, and Young LH. Additive effects of hyperinsulinemia and ischemia on myocardial GLUT1 and GLUT4 translocation in vivo. Circulation 98: 2180–2186, 1998.[Abstract/Free Full Text]
  36. Sargeant RJ and Paquet MR. Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes. Biochem J 290: 913–919, 1993.
  37. Shioi T, McMullen JR, Kang PM, Douglas PS, Obata T, Franke TF, Cantley LC, and Izumo S. Akt/Protein kinase B promotes organ growth in transgenic mice. Mol Cell Biol 22: 2799–2809, 2002.[Abstract/Free Full Text]
  38. Shulman GI. Cellular mechanisms of insulin resistance. J Clin Invest 106: 171–176, 2000.[ISI][Medline]
  39. Taha C, Liu Z, Jin J, Al-Hasani H, Sonenberg N, and Klip A. Opposite translational control of GLUT1 and GLUT4 glucose transporter mRNAs in response to insulin. Role of mammalian target of rapamycin, protein kinase b, and phosphatidylinositol 3-kinase in GLUT1 mRNA translation. J Biol Chem 274: 33085–33091, 1999.[Abstract/Free Full Text]
  40. Tian R and Abel ED. Responses of GLUT4-deficient hearts to ischemia underscore the importance of glycolysis. Circulation 103: 2961–2966, 2001.[Abstract/Free Full Text]
  41. Tian R, Musi N, D'Agostino J, Hirshman MF, and Goodyear LJ. Increased adenosine monophosphate-activated protein kinase activity in rat hearts with pressure-overload hypertrophy. Circulation 104: 1664–1669, 2001.[Abstract/Free Full Text]
  42. Wang Q, Somwar R, Bilan PJ, Liu Z, Jin J, Woodgett JR, and Klip A. Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. Mol Cell Biol 19: 4008–4018, 1999.[Abstract/Free Full Text]
  43. Zhou M, Sevilla L, Vallega G, Chen P, Palacin M, Zorzano A, Pilch PF, and Kandror KV. Insulin-dependent protein trafficking in skeletal muscle cells. Am J Physiol Endocrinol Metab 275: E187–E196, 1998.[Abstract/Free Full Text]




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