Download Cardiac glycogen accumulation after dexamethasone is regulated

Am J Physiol Heart Circ Physiol 295: H1753–H1762, 2008;
doi:10.1152/ajpheart.518.2008.
Cardiac glycogen accumulation after dexamethasone is regulated by AMPK
Prasanth Puthanveetil,1 Fang Wang,1 Girish Kewalramani,1 Min Suk Kim,1 Elham Hosseini-Beheshti,1
Natalie Ng,1 William Lau,1 Thomas Pulinilkunnil,3 Michael Allard,2 Ashraf Abrahani,1
and Brian Rodrigues1
1
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, 2Department of Pathology and Laboratory
Medicine, The University of British Columbia, Vancouver, British Columbia, Canada; and 3Division of Endocrinology,
Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School,
Boston, Massachusetts
Submitted 15 May 2008; accepted in final form 20 August 2008
glucocorticoids; insulin resistance; glucose transporter 4; glycogen
synthase
ON METABOLIC DEMAND,
glycogen, a mobilized storage form of
glucose, is readily broken down to yield glucose moieties. The
major rate-limiting enzymes involved with the metabolism of
glycogen include glycogen synthase (GS) and phosphorylase.
For its synthesis, the core protein glycogenin acts as a primer
for the attachment of UDP-glucose moieties, promoting the
growth of this polymer (31). GS catalyzes the attachment of
UDP-glucose to the nonreducing end of already formed glycogen (36). Glycogen breakdown is mediated by glycogen
Address for reprint requests and other correspondence: B. Rodrigues, Div. of
Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The Univ.
of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3
(e-mail: [email protected]).
http://www.ajpheart.org
phosphorylase. The activity of these enzymes is regulated by
both phosphorylation and allosteric stimulation (24).
Glycogen is an immediate source of glucose for cardiac
tissue to maintain its metabolic homeostasis (3). However, its
excess has been suggested to bring about structural and physiological impairments including an ionic imbalance, a change
in pH, and stimulation of pathways leading to hypertrophic
signaling. Thus, glycogen accumulation that is associated with
mutation of 5⬘-AMP-activated protein kinase (AMPK) has
been reported to cause cardiac hypertrophy, conduction system
failure, and ventricular arrhythmias (1). In glycogen storage
diseases like Pompe disease, characterized by deficiency of
debranching enzyme function, an excessive accumulation of
cardiac glycogen leads to left ventricular hypertrophy and
subsequent failure (55).
Glucocorticoids have widespread use as anti-inflammatory
and immunosuppressive agents (47). However, both excess
endogenous and exogenous glucocorticoids are known to contribute toward cardiovascular complications (39, 48). These
cardiac abnormalities could be secondary to glucocorticoidinduced insulin resistance and type 2 diabetes and alterations in
cardiac metabolism. With the latter, we have previously demonstrated that in hearts from dexamethasone (Dex)-treated
animals, amplification of lipoprotein lipase provided the heart
with excessive fatty acids that are known to induce cardiomyopathy (43, 53, 57). Glycogen accumulation was also enhanced
in these hearts. We rationalized that this increase in glycogen
was a consequence of compromised glucose oxidation observed in Dex-treated hearts (43). In the present study, we
examined whether additional factors related to glycogen synthesis, such as glucose entry and GS, also play a role in the
accumulation of this stored polysaccharide. Our data suggest
that in the presence of intact insulin signaling, AMPK-mediated glucose entry combined with the activation of GS and the
previously reported reduction in glucose oxidation act together
to promote glycogen storage. Should these effects persist
chronically, they may explain the increased morbidity and
mortality observed with long-term excesses in endogenous or
exogenous glucocorticoids.
MATERIALS AND METHODS
Experimental animals. This investigation conformed with the Guide
for the Care and Use of Laboratory Animals (National Institutes of
Health) and the University of British Columbia and was approved by the
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.
0363-6135/08 $8.00 Copyright © 2008 the American Physiological Society
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Puthanveetil P, Wang F, Kewalramani G, Kim MS, HosseiniBeheshti E, Ng N, Lau W, Pulinilkunnil T, Allard M, Abrahani A,
Rodrigues B. Cardiac glycogen accumulation after dexamethasone is
regulated by AMPK. Am J Physiol Heart Circ Physiol 295: H1753–
H1762, 2008; doi:10.1152/ajpheart.518.2008.—Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic
homeostasis. However, its excess brings about cardiac structural and
physiological impairments. Previously, we have demonstrated that in
hearts from dexamethasone (Dex)-treated animals, glycogen accumulation was enhanced. We examined the influence of 5⬘-AMP-activated
protein kinase (AMPK) on glucose entry and glycogen synthase as a
means of regulating the accumulation of this stored polysaccharide.
After Dex, cardiac tissue had a limited contribution toward the
development of whole body insulin resistance. Measurement of glucose transporter 4 (GLUT4) at the plasma membrane revealed an
excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated with
increased GLUT4 mRNA. Both total and phosphorylated AMPK
increased after Dex. Immunoprecipitation of Akt substrate of 160 kDa
(AS160) followed by Western blot analysis demonstrated no change
in Akt phosphorylation at Ser473 and Thr308 in Dex-treated hearts.
However, there was a significant increase in AMPK phosphorylation
at Thr172, which correlated well with AS160 phosphorylation. In
Dex-treated hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas glycogen synthase kinase3-␤ phosphorylation was augmented. Our data suggest that AMPKmediated glucose entry combined with the activation of glycogen
synthase and a reduction in glucose oxidation (Qi et al., Diabetes 53:
1790 –1797, 2004) act together to promote glycogen storage. Should
these effects persist chronically in the heart, they may explain the
increased morbidity and mortality observed with long-term excesses
in endogenous or exogenous glucocorticoids.
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Fig. 1. Dexamethasone (Dex) effects on whole body and tissue-specific insulin resistance. A: after an injection of vehicle or Dex for 4 h, whole body insulin
resistance was assessed using a euglycemic-hyperinsulinemic clamp. Insulin [HumulinR (3 mU 䡠 min⫺1 䡠 kg⫺1)] and D-glucose (50%) were continuously delivered
(by a cannula inserted into the left jugular vein) for 3 h. At regular intervals, blood samples taken from the tail vein were analyzed for glucose using a glucometer.
The glucose infusion rate (GIR) was adjusted accordingly to maintain euglycemia. CON, control. B–E: to determine tissue-specific insulin resistance, the skeletal
muscle (gastrocnemius and soleus muscles from the hind leg; B and C) and heart (D and E) from control (C) and 4-h Dex (D)-treated animals were evaluated
for phospho-insulin receptor substrate-1 [P-IRS-1 (Tyr989); C and E] and phospho-Akt [P-Akt (Ser473); B and D] and total IRS-1 (T-IRS-1) and total Akt (T-Akt)
before and after 15 min of injection of rapid acting insulin (I) into the tail vein (8 U HumulinR) using Western blot analysis. Results [in arbitrary units (AU)]
are means ⫾ SE of 3–5 rats/group. *Significantly different from control; ⫹significantly different from all other groups; #significantly different from control
animals given insulin (P ⬍ 0.05).
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performed as previously described (2). Measurement of the phosphorylated form of AMPK, ACC, GS, and GS kinase (GSK)-3-␤ is a
surrogate for the estimation of their activities. This was done using
Western blot analysis. Reaction products were visualized using an
ECL detection kit and quantified by densitometry.
Measurement of mRNA. mRNA levels were measured using quantitative real-time PCR. cDNA was synthesized from 1 ␮g RNA and
purified using a sample purification kit (QIAGEN). RNA levels were
determined from standard curves generated for each primer. The
sample run was carried out for 40 cycles. The oligonucleotide primers
were as follows: GLUT4 mRNA, forward 5⬘-GGGCAAAGGAACACAACAGT-3⬘ and reverse 5⬘-TGGAGGGGAACAAGAAAGTG-3⬘;
AMPK-␣1 mRNA, forward 5⬘-GCAGAGAGATCCAGAACCTG-3⬘
and reverse 5⬘-CTCCTTTTCGTCCAACCTTCC-3⬘; and AMPK-␣2
mRNA, forward 5⬘-GCTGTGGATCGCCAAATTAT-3⬘ and reverse
5⬘-GCATCAGCAGAGTGGCAATA-3⬘. Sample amplifications were
done with the help of a fluorescent SYBR green dye (Roche Applied
Science) in a Roche Applied Science Light Cycler system. ␤-Actin
was used as an internal reference.
Fig. 2. Cardiac glycogen accumulation after Dex. Cardiac glycogen was
determined as glucose residues using a glucokinase method after acid hydrolysis (A) and by histochemical analysis of cross sections of ventricular tissue
using periodic acid-Schiff staining (B). Results are means ⫾ SE of 5 rats/
group. *Significantly different from control (P ⬍ 0.05).
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Animal Care and Use Committee (Protocol No. A07-0273). Adult male
Wistar rats (260 –300 g) were obtained from the University of British
Columbia Animal Care Unit. The synthetic glucocorticoid hormone Dex
(1 mg/kg) or an equivalent volume of ethanol was administered by
intraperitoneal injection to nonfasted rats, and animals were euthanized at
4 h. In the human body, the basal daily secretion of cortisol is ⬃6 – 8
mg/m2 (in a 70-kg adult male, this translates to ⬃0.2 mg/kg). In response
to stress, cortisol release is increased up to 10-fold of the basal value (2
mg/kg) (13). For exogenous administration, dosing with corticosteroids
depends on the disease condition and varies from 75 to 300 mg/day
(⬃1– 4 mg/kg). Previous studies using an euglycemic-hyperinsulinemic
clamp have determined that this dose of Dex induces whole body insulin
resistance within 4 h (20, 43).
Euglycemic-hyperinsulinemic clamp. Whole animal insulin resistance was assessed using an euglycemic-hyperinsulinemic clamp, as
previously described (43). This procedure involves the simultaneous
intravenous infusion of insulin [HumulinR (3 mU 䡠 min⫺1 䡠 kg⫺1), to
inhibit endogenous hepatic production] and D-glucose (50%) for 3 h;
the quantity of exogenous glucose required to maintain euglycemia is
a reflection of the net sensitivity of target tissues (mainly skeletal
muscle) to insulin. At regular intervals, a small amount of blood taken
from the tail vein was analyzed for glucose (using a glucometer,
AccuSoft Advantage). The glucose infusion rate was adjusted accordingly to maintain euglycemia.
Tissue-specific responses to insulin. To assess tissue-specific insulin resistance, the skeletal muscle (gastrocnemius and soleus muscles
from the hind leg) and heart from control animals and 4-h Dex-treated
animals were evaluated for total and phospho-insulin receptor substrate-1 (IRS-1) and Akt before and after 15 min of injection of
rapid-acting insulin into the tail vein (8 units HumulinR) using
Western blot analysis (18, 23). At this early time point, there was no
significant reduction in blood glucose with insulin.
Myocardial glycogen content. Frozen cardiac tissue was powdered,
weighed, incubated at 85°C (10 min) with 1 N NaOH, and followed
by neutralization with 1 N HCl. The neutralized sample was subjected
to further acid hydrolysis with 6 N HCl (85°C, 2 h) (32). After
neutralization with 5 N NaOH, samples were subjected to glucose
analysis using a glucokinase assay kit. The presence of cardiac
glycogen was confirmed by visualization after periodic acid-Schiff
staining (21). Where indicated, animals were administered mifepristone [RU-486 (20 mg/kg)] 1 h before Dex. After 4 h of Dex, hearts
were isolated and frozen, and powdered tissue was used to measure
cardiac glycogen. We have previously reported that phosphorylation
of AMPK was inhibited by acute intralipid infusion (19). To determine the effect of AMPK inhibition on cardiac glycogen accumulation, animals were anesthetized with pentobarbital sodium, and the
left jugular vein was cannulated. Intralipid (5%, 1.2 ml 䡠 kg⫺1 䡠 h⫺1) or
vehicle (saline) was then infused over a period of 6 h, after which
hearts were removed for the determination of glycogen. In some
animals, Dex was administered 2 h after the intralipid infusion was
initiated.
Subcellular compartmentalization of glucose transporter 4. Membrane fractions were isolated by a previously described method using
a sucrose density gradient (14). With Western blot analysis, identification of glucose transporter 4 (GLUT4) protein was done using
rabbit polyclonal GLUT4 as the primary antibody and mouse antirabbit horseradish peroxidase as the secondary antibody. Na⫹-K⫹ATPase was used as a plasma membrane marker.
Immunoprecipitation and Western blot analysis. After Dex treatment, heart homogenates (500 ␮g protein) were immunoprecipitated
using a rabbit monoclonal total Akt substrate of 160 kDA (AS160)
antibody (3 h, 4°C). The immunocomplex was pulled down with
protein A/G-Sepharose for 3 h, separated, boiled for 5 min at 95°C in
Laemmli buffer, and subjected to SDS-PAGE. Western blot analysis
using antibodies against AS160, phospho-(Ser/Thr) Akt substrate
(PAS), phospho-Akt (Ser473, Thr308), total and phospho-AMPK-␣
(Thr172), and phospho-acetyl-CoA carboxylase (ACC; Ser79) was then
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Glucose-6-phosphate content. Glucose-6-phosphate was determined in perchloric acid extracts of frozen ventricular tissue using
standard spectrophotometric techniques (6, 26).
Glucose uptake in cardiomyocytes. Ventricular calcium-tolerant
myocytes were prepared by a previously described procedure (41).
Cells were plated on laminin-coated culture plates (60 mm, to a
density of 300,000 cells/well). Glucose uptake was evaluated using
radiolabeled 2-deoxyglucose (9). Briefly, myocytes were incubated in
a glucose-free DMEM containing 0.2% fatty acid-free BSA and
pyruvate (1.0 mM) as the energy source. Dex (100 nM) was then
added to the incubation media for 20 min. A radioactive mixture
(containing 5 ␮Ci of [2-deoxy-3H]glucose) was added to the plates,
and incubation was continued for another 10 min. After removal of the
buffer, cardiomyocytes were washed with cold PBS (2⫻) and lysed
using NaOH, and lysates were used to determine the radioactivity.
2-Deoxyglucose uptake was expressed as nanomoles per milligram
per minute.
Materials. The ECL detection kit was purchased from Amersham
Canada. Akt, phospho-Akt (Ser273/Thr308), AMPK, phospho-AMPK-␣,
GS, phospho-GS, GSK-3-␤, phospho-GSK-3-␤, AS160, PAS, Na⫹-K⫹ATPase, and GAPDH antibodies were obtained from Cell Signaling
(Danvers, MA). GLUT4 antibody was purchased from Abcam (Cambridge, MA). All other chemicals were obtained from Sigma Chemical.
Statistical analysis. Values are means ⫾ SE. Wherever appropriate,
one-way ANOVA followed by Tukey or Bonferroni tests or the unpaired
Student’s t-test was used to determine differences between group mean
values. The level of statistical significance was set at P ⬍ 0.05.
RESULTS
Cardiac tissue has limited influence on whole body insulin
resistance induced by Dex. We have previously reported that
an injection of Dex for 4 h was not associated with either
hyperinsulinemia or hyperglycemia (43). Nevertheless, using
the euglycemic-hyperinsulinemic clamp, a direct measure of
insulin sensitivity, Dex lowered the glucose infusion rate
necessary to maintain euglycemia (Fig. 1A). We assessed the
effects of Dex on the responses of skeletal muscle and cardiac
tissue to insulin. In skeletal muscle, both basal and insulinstimulated phosphorylation of IRS-1 (Fig. 1C) and Akt (Fig.
1B) were reduced after 4 h of Dex. These effects were not
observed in cardiac tissue, which demonstrated a normal response to insulin when IRS-1 (Fig. 1E) and Akt (Fig. 1D)
phosphorylation were measured. After 4 h of Dex, total IRS-1
and Akt did not change in skeletal and cardiac muscle compared with control (Fig. 1, B–D). Thus, after Dex, cardiac
tissue has a limited contribution toward the development of
whole body insulin resistance.
Buildup of cardiac glycogen after Dex is coupled to GLUT4
translocation. Even though Dex did not impede cardiac insulin
signaling, we have previously reported a decline in cardiac
glucose oxidation after Dex (43). In the presence of lower
glucose oxidation, we hypothesized that glucose entering into
the heart would be converted into glycogen. Indeed, Dex
induced an approximately twofold increase in the cardiac
glycogen content measured enzymatically (Fig. 2A) or by
histochemical staining (Fig. 2B). This effect of Dex on glycogen was partially related to receptor activation because although RU-486 reduced the Dex-induced increase in cardiac
glycogen, the levels observed were still higher than control
(control: 37.8 ⫾ 0.50 ␮g/g dry wt, Dex: 69.7 ⫾ 1.5 ␮g/g dry
wt, and Dex ⫹ RU-486: 44.6 ⫾ 2.1 ␮g/g dry wt, P ⬍ 0.05). In
addition to a reduction in glucose oxidation, it was unclear
whether changes in glucose transport could also contribute
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Fig. 3. Subcellular localization of glucose transporter 4 (GLUT4) protein.
A and B: after Dex, heart homogenates were prepared and subjected to plasma
membrane (PM; A) and intracellular membrane (IM; B) separation using a
sucrose gradient. Identification of GLUT4 protein was carried out using rabbit
polyclonal GLUT4 as the primary antibody and mouse anti-rabbit horseradish
peroxidase as the secondary antibody. Na⫹-K⫹-ATPase was used as a PM
marker. Quantitative real-time PCR enabled the determination of GLUT4
mRNA in hearts from control and Dex-treated animals. Results are means ⫾
SE of 3–5 rats/group. *Significantly different from control (P ⬍ 0.05).
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in both total (Fig. 4C) and phospho-AMPK phosphorylation
(Fig. 4A) after 4 h of Dex. This change in AMPK total protein
was accompanied by a modest but insignificant increase in
AMPK-␣2 (Fig. 4E) but a significant increase in AMPK-␣1
(Fig. 4D) gene expression. Once activated, AMPK phosphorylates and inactivates ACC, facilitating fatty acid oxidation.
We measured ACC phosphorylation as a measure of AMPK
activity. ACC phosphorylation was significantly increased in
Dex-treated hearts compared with control hearts (Fig. 4B). To
substantiate the role of AMPK in the Dex-induced accumulation of cardiac glycogen, intralipid was used to inhibit AMPK
phosphorylation. Intralipid lowered the cardiac glycogen accumulation that was observed after Dex (control: 37.4 ⫾ 1.8 ␮g/g
dry wt, Dex: 66.2 ⫾ 2.0 ␮g/g dry wt, and Dex ⫹ intralipid:
45.9 ⫾ 0.7 ␮g/g dry wt, P ⬍ 0.05).
Phosphorylation of AS160 is mainly regulated by AMPK.
AS160 regulates GLUT4 translocation to the plasma membrane by retaining this transporter in intracellular membranes,
a function that is lost upon its phosphorylation (22). The major
upstream regulators of AS160 phosphorylation include Akt and
Fig. 4. Changes in 5⬘-AMP-activated protein kinase (AMPK) protein [total (T-AMPK) and phospho-AMPK (P-AMPK)] and gene expression in hearts isolated
from Dex-treated animals. A–C: after Dex, P-AMPK (A), phospho-acetyl-CoA carboxylase (P-ACC; B), and T-AMPK (C) were measured using rabbit P-AMPK
(Thr172), P-ACC (Ser79), and AMPK-␣ antibodies, respectively. D and E: AMPK-␣1 (D) and AMPK-␣2 (E) gene expression was measured using quantitative
real-time PCR. Results are means ⫾ SE of 3–5 rats/group. *Significantly different from untreated control animals (P ⬍ 0.05).
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toward the accumulation of glycogen. Interestingly, Dextreated hearts exhibited a higher glucose-6-phosphate content
compared with control (control: 2.67 ⫾ 0.16 ␮mol/g dry wt
and Dex: 3.31 ⫾ 0.20 ␮mol/g dry wt, P ⬍ 0.05). More
importantly, after 4 h of Dex, measurement of GLUT4 at the
plasma membrane revealed an excess presence of this transporter protein at this location (Fig. 3A). Usually, this observation is accompanied by a decrease in GLUT4 in the intracellular pool. However, in the present study, the Dex-induced
increase in plasma membrane GLUT4 was also accompanied
by an increase in GLUT4 in the intracellular membrane fraction (Fig. 3B). This latter effect was well correlated with
increased GLUT4 mRNA (Fig. 3B, inset). Dex also increased
glucose uptake directly in isolated cardiomyocytes (control:
4.2 ⫾ 0.85 nmol䡠mg⫺1 䡠min⫺1 and Dex: 9.86 ⫾ 1.74
nmol䡠mg⫺1 䡠min⫺1, P ⬍ 0.05).
Dex augments both total and phosphorylated cardiac
AMPK. In addition to Akt signaling, cardiac GLUT4 translocation is also controlled by AMPK (45). In the presence of a
normal Akt signal, we measured AMPK and found an increase
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AMPK. Immunoprecipitation of AS160 followed by Western
blot analysis demonstrated no change in Akt phosphorylation at Ser473 and Thr308 in Dex-treated hearts (Fig. 5, A and
B). However, there was a significant increase in AMPK
phosphorylation at Thr172 (Fig. 5C), which correlated well
with AS160 phosphorylation, as reflected by an increase in
PAS (a measure of phosphorylation at Ser and Thr sites of
AS160; Fig. 5D).
GS undergoes robust dephosphorylation with acute administration of Dex. In addition to the contributions by GLUT4delivered glucose and the reduction of glucose oxidation toward glycogen synthesis after Dex, enzymatic control of this
stored polysaccharide is also an important factor that controls
its accumulation. GS, the rate-limiting enzyme for glycogen
synthesis, is activated upon dephosphorylation (26). In Dextreated hearts, there was a considerable reduction in the phos-
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Fig. 5. AMPK regulation of Akt substrate of 160 kDa (AS160). Animals were treated with Dex, and, at 4 h, hearts from control and Dex-treated animals were
isolated. To examine the association among AS160, Akt, and AMPK, AS160 was first immunoprecipitated using a total AS160 antibody. The immunocomplex
was then immunoblotted with anti-P-Akt [Ser473 (A) and Thr308 (B)], anti-P-AMPK [Thr172 (C)], phospho-(Ser/Thr) Akt substrate [PAS; Ser/Thr (D)], and
anti-AS160 (inset). IP, immunoprecipitation. Results are means ⫾ SE of 3–5 rats/group. *Significantly different from untreated control animals (P ⬍ 0.05).
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DISCUSSION
Fig. 6. Enzyme regulation of cardiac glycogen synthesis subsequent to the
administration of Dex. Animals were killed 4 h subsequent to Dex injection,
and hearts were isolated for the determination of phospho-glycogen synthase
(GS) kinase-3-␤ [P-GSK-3-␤, Ser9 (top)] and total GSK-3-␤ (T- P-GSK-3-␤)
and phospho-GS [Ser641 (bottom)] and total GS (T-GS) using Western blot
analysis. Results are means ⫾ SE of 3–5 rats/group. *Significantly different
from untreated control animals (P ⬍ 0.05).
Chronically, increased levels of endogenous glucocorticoids
are known to cause Cushing’s syndrome, a condition that is
characterized by obesity, insulin resistance, increased lipid
mobilization, and hypertension (28, 58). Exogenous delivery of
glucocorticoids as anti-inflammatory and immunosuppressive
agents is also associated with myocardial failure when administered chronically (35). We attempted to examine the acute
effects of Dex specifically related to cardiac metabolism, given
the injurious effects that excess triglyceride (30) and glycogen
(1) have on the heart. We have previously reported an enlargement in the coronary lipoprotein lipase pool, with a subsequent
increase in fatty acid delivery and augmented cardiac triglyceride accumulation (20, 42, 43). With carbohydrate metabolism, Dex promoted the expression of pyruvate dehydrogenase
kinase 4, which is known to inhibit pyruvate dehydrogenase
and pyruvate flux and, therefore, glucose oxidation (43). Under
these circumstances, we proposed that glucose disposal occurred by its conversion to glycogen. In the present study, our
data suggest that AMPK-mediated glucose entry combined
with the activation of GS and the previously reported reduction
in glucose oxidation (43) act together to promote glycogen
storage.
With insulin resistance, metabolism in multiple organ systems, including the heart, is altered, which is believed to be an
important factor in increased morbidity and mortality (5, 7).
Measurement of insulin sensitivity using the euglycemic-hyperinsulinemic clamp revealed the presence of whole body
insulin resistance with acute Dex treatment, suggesting that
any change in cardiac metabolism that is exhibited by Dex may
be a consequence of the prevalent reduction of insulin sensitivity. Nevertheless, we also determined the responses of skeletal muscle and cardiac tissue to insulin; unlike skeletal muscle, cardiac tissue responded normally to insulin. Under these
conditions, it is possible that the effects of Dex on cardiac
metabolism are also related to direct effects of this glucocorticoid on the heart. Glucocorticoids work through multiple
mechanisms to bring about their desired effects. These include
a specific cytosolic receptor-mediated event and specific and
nonspecific membrane-bound receptor-mediated effects through
which glucocorticoids bring about genomic and nongenomic
outcomes (8, 10, 11). Given our recently reported observation
that Dex activated AMPK (a metabolic switch that plays an
important role in maintaining cellular energy homeostasis)
phosphorylation in isolated cardiomyocytes within 60 min
(20), and the use of RU-486 in our present study to reduce
cardiac glycogen accumulation, our data suggest that the effects of Dex on cardiac metabolism in vivo may be linked to its
direct impact on the heart. At present, the mechanism by which
Dex increases the phosphorylation of AMPK is unknown and
could include changes in the AMP-to-ATP ratio (17) or an
activation of a Ca2⫹/calmodulin-dependent protein kinase kinase (49).
AMPK is known to inhibit anabolic and promote catabolic
processes leading to the conservation of cellular ATP levels. It
does so through multiple mechanisms including increased
delivery and metabolism of both glucose and fatty acids (16,
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cardiac glycogen accumulation after Dex is also dependent on
the phosphorylation states of these two rate-limiting enzymes.
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phorylation of GS (Fig. 6, bottom). The major upstream kinase
that regulates GS phosphorylation is GSK-3-␤, whose activity,
in turn, is reduced by its phosphorylation. As GSK-3-␤ phosphorylation was augmented (Fig. 6, top), our data suggest that
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Increased substrate availability plays an important role in
glycogen synthesis. Thus, the increase in glucose uptake and
glucose-6-phosphate after Dex could be an important contributing feature in glycogen accumulation. Other factors include
alterations in glucose oxidation (43) and changes in the enzymes that control glycogen synthesis or breakdown. In skeletal muscle, glycogen regulation by GSK-3-␤ and GS is well
established. AMPK is known to phosphorylate GS, making it
prone to further phosphorylation by casein kinase-1 and GSK3-␤, leading to its inactivation (40, 50). The relationship
among AMPK, GSK-3-␤, and glycogen has yet to be resolved
in the heart. For example, Mora et al. (33) showed that cardiac
glycogen levels are regulated independently of insulin’s ability
to phosphorylate GSK-3-␤ and stimulate GS. In the present
study, phosphorylation of GS after Dex administration decreased, an effect closely associated with an increase in GSK3-␤ phosphorylation. This effect of Dex on GS in the presence
of increased AMPK phosphorylation was uncommon. As
AMPK has been recently shown to activate GS through a
GSK-3-␤-dependent pathway in HepG2 cells (54), our data
suggest that through multiple mechanisms, AMPK activation
with Dex is associated with glycogen storage.
Limitation of the study. When examining the effects of
AMPK inhibition on the heart, studies have used dominant
negative AMPK overexpression or short interfering RNA to
decrease AMPK activity. One limitation of this study is the use
of intralipid to inhibit AMPK. This is because intralipid, in
addition to its effects on AMPK, is also known to effect insulin
signaling, and therefore glucose metabolism, by decreasing
IRS (Tyr) and Akt (Ser) phosphorylation in the liver and
skeletal muscle (4, 38). Thus, the decrease in cardiac glycogen
after Dex ⫹ intralipid could be a result of both AMPK
inhibition and reduced insulin signaling.
Conclusions. In summary, acute Dex administration was
associated with a significant accumulation of myocardial
glycogen. One way by which this process is made possible
is through AMPK-mediated augmentation of glucose uptake, coupled to its regulation of GS, a key enzyme involved
in glycogen synthesis (Fig. 7). Given the contribution of
glycogen storage in eliciting cardiac hypertrophy, ventricular arrythmias, and conduction system disorders, the results
from the present study could help in limiting the deleterious
effects of long-term excesses in endogenous or exogenous
glucocorticoids on the heart.
Fig. 7. Schematic mechanism of how Dex
regulates cardiac glycogen through AMPK.
In the presence of intact insulin signaling
(normal Akt function), Dex, through its effects in enabling AMPK phosphorylation,
regulates GLUT4 translocation by its action
on AS160. The subsequent influx of glucose
augments the cardiac content of glucose-6phosphate (G-6-P). This effect, combined
with the phosphorylation and inactivation of
GSK-3-␤, together with the dephosphorylation and activation of glycogen synthase,
acts in unison to increase cardiac glycogen
content.
AJP-Heart Circ Physiol • VOL
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29, 45). In this context, and related to glycogen, AMPK is
known to inhibit the formation of this storage form of glucose
in skeletal muscle (40, 50, 56). In the heart, both low-flow
ischemia (37) and exercise (34) increase AMPK activity,
which is correlated to a reduction in glycogen content. Unexpectedly, our results suggest that the accumulation of glycogen
with Dex was associated with increased phosphorylation of
AMPK, a phenomenon that is observed in transgenic models of
AMPK activation. In these models, mutation of regulatory
␥-subunits (␥1 R70Q, ␥2 N488I, and ␥2 R531G) increased
AMPK activation and facilitated glycogen accumulation (1, 12,
15, 27). It is possible that the intrinsic property of Dex to block
glucose oxidation counteracts the ability of AMPK to prevent
the storage of this carbohydrate. An additional explanation for
this occurrence could be related to the effect of AMPK to
decrease malonyl CoA, thereby removing its inhibition on
carnitine palmitoyltransferase I and promoting fatty acid oxidation (42). The resultant blockade of glucose oxidation, as
suggested by the Randle hypothesis, could explain the increase
in glycogen storage (44). Whatever the mechanism, our data
suggests that with carbohydrate metabolism, AMPK phosphorylation in the presence of Dex is associated with an anabolic
function.
Under basal conditions, only a small percentage of GLUT4
resides at the plasma membrane, with the remaining fraction
being redistributed in endosomal recycling and GLUT4 storage
compartments (25, 46). Translocation of this transporter protein from the intracellular pool to the plasma membrane is
regulated largely by the phosphatidylinositol 3-kinase-Akt
pathway, in addition to AMPK. These kinases, by phosphorylating and inactivating AS160 (which has multiple phosphorylation motifs at Ser and Thr residues), removes the constraint
that AS160 has on GLUT4, allowing the trafficking of this
transporter to the membrane surface (22, 51, 52). Indeed, the
increase in plasma membrane GLUT4 with Dex was well
correlated to an increase in PAS, a measure of phosphorylation
at Ser and Thr sites of AS160. To determine the contribution of
Akt and AMPK toward this increased AS160 phosphorylation,
we used immunoprecipitation to pull down the trimeric complex containing phospho-Akt, phospho-AMPK, and AS-160
and immunoblotted for the respective phosphoproteins. The
present study suggests that after Dex, in the presence of normal
insulin signaling, AMPK-mediated phosphorylation of AS160
is the predominant factor that controls cardiac GLUT4 movement.
AMPK CONTROL OF CARDIAC GLYCOGEN
GRANTS
This work was supported by an operating grant from the Heart and Stroke
Foundation of British Columbia and the Yukon.
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