J Appl Physiol 97: 2339 –2346, 2004;
doi:10.1152/japplphysiol.01219.2003.
TRANSLATIONAL PHYSIOLOGY
Levodopa with carbidopa diminishes glycogen concentration, glycogen
synthase activity, and insulin-stimulated glucose transport
in rat skeletal muscle
Jill L. Smith,1 Jeong-Sun Ju,1 Bithika M. Saha,1 Brad A. Racette,2,3 and Jonathan S. Fisher1
1
Department of Biology, Saint Louis University, St. Louis 63103; 2Department of Neurology and Neurological Surgery
(Neurology), Washington University School of Medicine, St. Louis 63110; 3American Parkinson Disease Association
Advanced Center for Parkinson Research, St. Louis, Missouri 63110
Submitted 13 November 2003; accepted in final form 12 July 2004
(PD) is a chronic progressive neurological
disorder that affects up to one million North Americans with
50,000 more individuals diagnosed each year (4). Because the
disease causes degeneration of dopamine-producing substantia
nigra neurons, levodopa, a dopamine precursor that is able to
cross the blood-brain barrier to enter the central nervous
system, is used to replenish depleted dopamine (2, 4, 6).
Levodopa is the most commonly prescribed and most effective
medication for controlling the symptoms of PD (4).
Several investigators have reported high rates of glucose
intolerance among patients with PD (2, 3, 27). For example, in
two separate studies of 30 and 57 patients with PD, respectively, ⬃50% of the patients displayed abnormal oral glucose
tolerance (2, 27). Similarly, abnormal intravenous glucose
tolerance was found in four of eight patients with PD (3).
Notably, hyperglycemic effects of levodopa and dopamine
have been documented in humans and laboratory animals (3,
17, 18, 33). The decarboxylase inhibitor carbidopa is given
with levodopa to prevent the conversion of levodopa to dopamine in peripheral tissues, because dopamine does not cross
the blood-brain barrier (4). However, carbidopa does not prevent accumulation of dopamine in skeletal muscle in animals
treated with levodopa (9, 30).
Glucose transport is stimulated by insulin in skeletal muscle
through activation of the insulin receptor tyrosine kinase and
phosphorylation of members of the insulin receptor substrate
(IRS) family (reviewed in Ref. 35). Tyrosine-phosphorylated
IRS proteins provide scaffolding sites for activation of signaling cascades, leading to increased glucose transport. One
signaling pathway essential to insulin-stimulated glucose transport is dependent on docking of the p85 regulatory subunit of
phosphatidylinositol 3-kinase (PI3K) on tyrosine-phosphorylated IRS, which activates the PI3K p110 catalytic subunit.
Downstream effectors of PI3K include Akt, which is thought to
participate in the stimulation of glucose transport by insulin (1)
and promotes glycogen synthesis through inactivation of glycogen synthase (GS) kinase-3␤ (8).
Skeletal muscle, the predominant insulin-responsive tissue,
responds to dopamine by elevating endogenous cAMP, an
effect that is blocked by propranolol, a ␤-adrenergic antagonist
(31). ␤-Adrenergic action and cAMP have been implicated as
negative regulators of insulin action (20, 23, 26, 38, 43). GS,
the rate-limiting enzyme for the storage of intracellular glucose
as glycogen in skeletal muscle (25), is activated in the presence
of insulin and inhibited by agents that increase intracellular
cAMP concentrations ([cAMP]) (7, 25, 38). GS is unresponsive to insulin under various conditions of insulin resistance,
Address for reprint requests and other correspondence: J. L. Smith, Dept. of
Biology, St. Louis Univ., 3507 Laclede Ave., St. Louis, MO 63103 (E-mail:
[email protected]).
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.
Parkinson’s disease; insulin resistance; diabetes; dopamine
PARKINSON’S DISEASE
http://www. jap.org
8750-7587/04 $5.00 Copyright © 2004 the American Physiological Society
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Smith, Jill L., Jeong-Sun Ju, Bithika M. Saha, Brad A. Racette,
and Jonathan S. Fisher. Levodopa with carbidopa diminishes glycogen concentration, glycogen synthase activity, and insulin-stimulated glucose transport in rat skeletal muscle. J Appl Physiol 97:
2339 –2346, 2004; doi:10.1152/japplphysiol.01219.2003.—We hypothesized that levodopa with carbidopa, a common therapy for
patients with Parkinson’s disease, might contribute to the high prevalence of insulin resistance reported in patients with Parkinson’s
disease. We examined the effects of levodopa-carbidopa on glycogen
concentration, glycogen synthase activity, and insulin-stimulated glucose transport in skeletal muscle, the predominant insulin-responsive
tissue. In isolated muscle, levodopa-carbidopa completely prevented
insulin-stimulated glycogen accumulation and glucose transport. The
levodopa-carbidopa effects were blocked by propranolol, a ␤-adrenergic antagonist. Levodopa-carbidopa also inhibited the insulin-stimulated increase in glycogen synthase activity, whereas propranolol
attenuated this effect. Insulin-stimulated tyrosine phosphorylation of
insulin receptor substrate (IRS)-1 was reduced by levodopa-carbidopa, although Akt phosphorylation was unaffected by levodopacarbidopa. A single in vivo dose of levodopa-carbidopa increased
skeletal muscle cAMP concentrations, diminished glycogen synthase
activity, and reduced tyrosine phosphorylation of IRS-1. A separate
set of rats was treated intragastrically twice daily for 4 wk with
levodopa-carbidopa. After 4 wk of treatment, oral glucose tolerance
was reduced in rats treated with drugs compared with control animals.
Muscles from drug-treated rats contained at least 15% less glycogen
and ⬃50% lower glycogen synthase activity compared with muscles
from control rats. The data demonstrate ␤-adrenergic-dependent inhibition of insulin action by levodopa-carbidopa and suggest that
unrecognized insulin resistance may exist in chronically treated patients with Parkinson’s disease.
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LEVODOPA INHIBITS INSULIN ACTION IN MUSCLE
METHODS AND MATERIALS
Materials. Purified porcine insulin was from Eli Lilly (Indianapolis, IN). Levodopa and carbidopa were from Sigma Chemical (St.
Louis, MO). 3H-labeled 2-deoxyglucose (2-DG) and 14C-labeled
mannitol were from American Radiolabeled Chemicals (St. Louis,
MO). Enzymes and other chemicals were purchased from Roche
Diagnostics (Indianapolis, IN) and Sigma Chemical.
Animals. Male Wistar rats (Charles River Laboratories, Wilmington, MA) weighing ⬃130 g were used for the study. The temperature
of the animal room was maintained at 21°C, and a 12:12-h light/dark
cycle was set. Rats were allowed free access to food and water. The
Saint Louis University Animal Care Committee approved all procedures.
Muscle incubations. The acute effects of 30 ␮M levodopa with 100
ng/ml carbidopa on insulin-stimulated glycogen accumulation were
examined in isolated epitrochlearis muscles. Levodopa and carbidopa
concentrations were chosen to approximate one to two times the
plasma concentrations of the drugs found in humans who have taken
a single dose of levodopa-carbidopa (200 mg-20 mg or 200 mg-50
mg) (5, 37). Rats were anesthetized after an overnight fast via an
intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt),
and epitrochlearis muscles were removed. Each epitrochlearis was
incubated (14, 26) with shaking in 25-mm-diameter vials containing 2
ml of incubation medium with a gas phase of 95% O2-5% CO2 for 3 h
at 35°C in one of five groups. The basal medium contained oxygenated Krebs-Henseleit buffer (KHB), 28 mM mannitol, 0.1% BSA, and
12 mM glucose. This glucose concentration is in the range of blood
glucose levels for rats undergoing oral glucose tolerance tests (15),
and plasma glucose concentrations in fed Wistar rats (9.3 ⫾ 0.5 mM,
mean ⫾ SE, n ⫽ 8; 13) have been reported to approach this value.
Muscles in the insulin group were incubated in the basal medium
containing 60 ␮U/ml insulin, a concentration of insulin that produces
half-maximal insulin-stimulated glucose transport (14) and is consistent with plasma insulin concentrations of fed Wistar rats (13). The
insulin-levodopa-carbidopa medium contained 60 ␮U/ml insulin, 30
␮M levodopa, and 100 ng/ml carbidopa. The levodopa-carbidopa
medium contained 30 ␮M levodopa and 100 ng/ml carbidopa. The
insulin-levodopa-carbidopa-propranolol medium contained 60 ␮U/ml
insulin, 30 ␮M levodopa, 100 ng/ml carbidopa, and 10 ␮M propranolol. Vials were wrapped in aluminum foil before and during muscle
J Appl Physiol • VOL
incubations to prevent breakdown of levodopa, a light-sensitive compound. After the incubations, muscles were quickly blotted and
trimmed at 4°C, clamp-frozen with tongs cooled in liquid nitrogen,
and stored at ⫺80°C before analysis of glycogen accumulation and
GS activity as described below.
Muscle glucose transport determination. Rats were anesthetized
after an overnight fast as described above, and the epitrochlearis,
soleus, and flexor digitorum brevis muscles were dissected out. Soleus
muscles were split longitudinally into thin strips to enhance diffusion
of oxygen and nutrients into the tissue. Muscles were incubated as
described above for 1 h at 35°C in a basal medium containing
oxygenated KHB, 32 mM mannitol, 0.1% BSA, and 8 mM glucose.
Muscles were then preincubated for 30 min at 35°C in fresh medium
containing 0.1% BSA, 8 mM glucose, and 32 mM mannitol with no
insulin, 60 ␮U/ml insulin, or 60 ␮U/ml insulin with 30 ␮M levodopa
and 100 ng/ml carbidopa. Muscles were then washed for 10 min at
30°C in glucose-free medium containing 0.1% BSA, 40 mM mannitol, and levodopa-carbidopa and/or insulin if they had been present in
the previous step. Next, muscles were incubated for 20 min in medium
containing 0.1% BSA, 8 mM 2-DG, 2 ␮Ci/ml 3H-labeled 2-DG, 32
mM mannitol, 0.3 ␮Ci/ml [14C]mannitol, and insulin or levodopa if
they had been present in previous incubations. To assess the potential
involvement of ␤-adrenergic signaling in levodopa-carbidopa actions,
10 ␮M propranolol was included in incubation media for some
epitrochlearis muscles that were incubated with levodopa-carbidopa.
Muscles were blotted on filter paper, clamp-frozen as described
above, and stored at ⫺80°C. Frozen muscle samples were weighed
and homogenized in Kontes ground-glass tubes in 1 ml ice-cold 0.3 M
perchloric acid, and the homogenate was centrifuged (14,000 g for 10
min at 4°C). For determination of glucose transport, 200 ␮l of the
supernatant was added to 1 ml of scintillation solution (Packard
Biosciences, Meriden, CT). Samples were counted for 3H and 14C in
an LS-6000 liquid scintillation spectrophotometer (Beckman, Fullerton, CA). 14C-labeled mannitol was used to correct for extracellular
space.
Acute in vivo effects of levodopa-carbidopa. For in vivo levodopacarbidopa treatments, levodopa and carbidopa were dissolved in NaCl
at a 10:1 ratio of levodopa to carbidopa to mimic a therapeutic dose.
The doses of levodopa and carbidopa used in this study are equivalent
to 116 mg in a 70-kg human, a common therapeutic dose of levodopa
in a patient with early PD. Carbidopa was in complex with 2-hydroxypropyl-␤-cyclodextrin (HBC), which renders the carbidopa water soluble. Vehicle solutions for control animals contained HBC at
the same concentration as in the drug solution. After an overnight fast,
animals were treated intragastrically with a single dose of either
levodopa-carbidopa (0.165 mg levodopa and 0.0165 mg carbidopa per
100 g body wt) or vehicle. Rats were anesthetized 15 min later as
described above before they were fed 1 ml/100 g body wt of either
20% glucose in 0.9% NaCl or 0.9% NaCl without glucose. Fifteen
minutes after the glucose or NaCl feeding, the epitrochlearis and
triceps muscles were excised, clamp-frozen with tongs cooled in
liquid nitrogen, and stored at ⫺80°C along with plasma samples from
each animal. Plasma glucose and insulin concentrations, muscle
cAMP levels, muscle GS activity, and IRS-1 activation were later
assayed.
Chronic in vivo levodopa-carbidopa administration and OGTT.
Animals were randomly assigned to either a control group or a
levodopa-carbidopa treatment group (n ⫽ 6/group). The two groups
were treated intragastrically twice a day (8:00 AM and 5:00 PM), 7
days/wk, for 4 wk with levodopa-carbidopa (as described above for
the single drug treatment) or vehicle. Subsequent to 4 wk of drug
treatment, rats were fasted overnight before administration of an
OGTT as previously described (15). Blood samples were collected
from small tail vein incisions using heparinized capillary tubes that
were then spun for separation of plasma. Immediately after collection
of the first plasma samples (designated ⫺30 min), rats were given a
single intragastric dose of levodopa-carbidopa or vehicle (as described
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including diabetes (14, 19, 21). Under conditions in which GS
activity is low and/or insulin does not increase GS activity,
nonoxidative glucose disposal and glycogen accumulation in
skeletal muscle are severely limited (14, 21, 38), and it has
been suggested that impaired glycogen metabolism is a primary defect in diabetes (32). Thus GS activity and glycogen
accumulation are key markers of insulin action in skeletal
muscle and are susceptible to ␤-adrenergically mediated negative regulation.
We hypothesized that levodopa-carbidopa would inhibit
insulin effects in skeletal muscle and that some of the levodopa-carbidopa effects would be dependent on ␤-adrenergic
action. We used an in vitro skeletal muscle preparation to
assess the role of levodopa-carbidopa in glycogen metabolism
and glucose transport and examine possible mechanisms for
levodopa-mediated insulin resistance. We also evaluated the
acute effects of a single dose of levodopa-carbidopa on plasma
glucose levels, muscle [cAMP], IRS-1 activation, and GS
activity. To determine chronic in vivo effects of levodopacarbidopa, additional rats were treated with levodopa-carbidopa for 4 wk, after which an oral glucose tolerance test
(OGTT) and assays for muscle glycogen content and GS
activity were performed.
LEVODOPA INHIBITS INSULIN ACTION IN MUSCLE
J Appl Physiol • VOL
muscles were averaged to yield an n ⫽ 1 for each animal, except for
anterior tibialis, for which values for right and left muscles were not
averaged together.
RESULTS
Effects of levodopa-carbidopa on insulin action in isolated
skeletal muscle. A physiological concentration (60 ␮U/ml) of
insulin increased glycogen accumulation by ⬃40% above
baseline in epitrochlearis muscle (P ⬍ 0.05; Fig. 1). Levodopacarbidopa prevented insulin-stimulated glycogen accumulation, and this inhibition was prevented by propranolol, a
␤-adrenergic antagonist. Insulin stimulated an increase in GS
activity above the basal level in the epitrochlearis by ⬃30%
(P ⬍ 0.05; Fig. 2A), and levodopa-carbidopa prevented the
insulin-stimulated increase in GS activity (P ⬍ 0.05). The
decrease in insulin-stimulated GS activity in the presence of
levodopa-carbidopa was partially prevented by propranolol.
Levodopa-carbidopa treatment tended to increase phosphorylase activity (Fig. 2B), although this effect was not statistically
significant for the small sample size. Levodopa-carbidopa
prevented the insulin-stimulated increase in glucose transport
(P ⬍ 0.05; Fig. 3, A-C), and this effect was blocked by
propranolol in the epitrochlearis (P ⬍ 0.05; Fig. 3C). As shown
in Fig. 4, A and B, levodopa-carbidopa had no effect on
insulin-stimulated phosphorylation of Akt on Thr308 or Ser473.
However, levodopa-carbidopa prevented insulin-stimulated
IRS-1 tyrosine phosphorylation (Fig. 4C). It appears that inhibition of GS activity by levodopa-carbidopa occurs independent of changes in Akt activity, which is consistent with
Fig. 1. Levodopa-carbidopa decreases insulin-stimulated glycogen accumulation in isolated skeletal muscle. Epitrochlearis (Epi) muscles were incubated
for 3 h in the absence (⫺) or presence (⫹) of 60 ␮U/ml insulin, 30 ␮M
levodopa with 100 ng/ml carbidopa, and 10 ␮M propranolol, a ␤-adrenergic
antagonist. Bars represent means ⫾ SE. *Significantly different from control,
P ⬍ 0.05. †Significantly different (P ⬍ 0.05) from control and groups treated
with levodopa-carbidopa. n ⫽ 14 (basal), 15 (insulin), 14 (insulin and levodopa-carbidopa), 6 (levodopa-carbidopa), and 6 (insulin, levodopa-carbidopa,
and propranolol).
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above). Thirty minutes later (designated time 0), after collection of
another set of plasma samples, rats were fed intragastrically with 1
ml/100 g body wt of 20% glucose in 0.9% NaCl. Additional plasma
samples were taken 15, 30, 60, 90, and 120 min after the glucose
feeding. Plasma samples were stored at ⫺80°C until analysis of
glucose and insulin concentrations.
The twice-daily treatments with levodopa-carbidopa resumed the
morning after OGTTs. Three days after the OGTT, rats were again
fasted overnight and then anesthetized as described above before
collection of epitrochlearis, plantaris, extensor digitorum longus, and
anterior tibialis muscles. Muscles were clamp-frozen with tongs
cooled in liquid nitrogen and stored at ⫺80°C until analysis of
glycogen concentration and GS activity.
Metabolite and insulin assays. Muscle samples were homogenized
as described above for glucose transport assays, and glycogen concentration was determined by the fluorometric amyloglucosidase
method described by Passoneau and Lowry (29). An enzymatic
fluorometric assay derived from the protocol described by Sugiyama
et al. (34) was used to measure tissue cAMP concentration. Plasma
glucose concentrations were assayed spectrophotometrically (29). For
determination of plasma insulin concentrations, an ultra-sensitive rat
insulin ELISA kit purchased from Crystal Chemical (Downers Grove,
IL) was used according to the manufacturer’s instructions.
GS and phosphorylase assays. Muscle samples were homogenized
in ice-cold buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl,
10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10
mM sodium pyrophosphate, 100 mM NaF, 2 mM Na3VO4, 10 ␮g/ml
aprotinin, 10 ␮g/ml leupeptin, 0.5 ␮g/ml pepstatin, and 2 mM phenylmethylsulfonyl fluoride as previously described (14). GS activity
assays were performed as described by Passoneau and Lowry (29). GS
activity was expressed as the fraction of total activity (measured in the
presence of glucose 6-phosphate) that was independent of glucose
6-phosphate. Glycogen phosphorylase activity assays were performed
in the absence or presence of AMP according to the protocol described by Young et al. (42) Glycogen phosphorylase activity was
expressed as the fraction of total activity that was independent of
AMP (i.e., phosphorylase a activity).
Western blot analysis. Muscle samples were homogenized as described for the enzyme activity assays, and the protein concentrations
of the homogenates were measured by the bicinchoninic acid assay
(Pierce, Rockford, IL). Equal amounts of protein were mixed with
SDS denaturing buffer, boiled for 5 min, and subjected to gel electrophoresis. The protein bands in the gel were transferred to nitrocellulose and incubated with polyclonal antibodies against phosphoserine473 and phosphothreonine308 residues of Akt and peroxidaseconjugated goat anti-rabbit secondary antibodies (Pierce) in Trisbuffered saline containing 0.1% Tween-20 and 1% nonfat dry milk.
Both serine473 and threonine308 must be phosphorylated for full
activation of Akt that would lead to an increase in GS activity (40).
The antibody-bound proteins were visualized using an enhanced
chemiluminescence system (Amersham, Piscataway, NJ) and quantitated with Total Lab Software (Newcastle, UK). For IRS-1 analyses,
membranes were incubated with polyclonal antibodies against the
phosphotyrosine632 residue of IRS-1 and peroxidase conjugated rabbit
anti-goat secondary antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA). Tyrosine632 phosphorylation of IRS-1 is important for full
activation of insulin-stimulated phosphatidylinositol 3-kinase activity
and translocation of GLUT-4 (12).
Statistical analysis. A one-way analysis of variance among experimental groups was performed on all variables. Post hoc comparisons
were performed with Fisher’s protected least-significance difference
tests. A two-tailed Student’s t-test was used to examine body weight
differences between treated and control animals. A level of P ⬍ 0.05
was set for significance for all tests, and all values are expressed as
means ⫾ SE. Statistics were performed with SPSS (Chicago, IL)
software, version 10.0. For analyses of glycogen concentration and
GS activity, and [cAMP] for the in vivo study, results for right and left
2341
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LEVODOPA INHIBITS INSULIN ACTION IN MUSCLE
although control animals gained more weight than animals
treated with levodopa-carbidopa (2 wk: control 260 ⫾ 8 g,
drug 238 ⫾ 14 g; 4 wk: control 352 ⫾ 14 g, drug 324 ⫾ 10 g).
Food intakes were not monitored, so it is possible that drugtreated animals ate less than controls. In an OGTT after the 4
wk of levodopa-carbidopa treatments, blood glucose concentrations were higher (P ⬍ 0.05) in rats treated with levodopacarbidopa compared with controls at 60, 90, and 120 min after
ingestion of glucose (Fig. 6A). There were no significant
differences in plasma insulin concentrations in rats treated with
levodopa-carbidopa compared with the control group except at
120 min, when insulin levels were higher in drug-treated rats
than in controls (Fig 6B). In the animals treated chronically
with levodopa-carbidopa, there was at least 15% less muscle
glycogen content compared with controls (P ⬍ 0.05; Table 1).
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Fig. 2. Levodopa-carbidopa decreases insulin-stimulated glycogen synthase
activity in isolated muscle. Epi muscles were incubated in the absence (⫺) or
presence (⫹) of 60 ␮U/ml insulin, 30 ␮M levodopa with 100 ng/ml carbidopa,
and 10 ␮M propranolol, a ␤-adrenergic antagonist. A: glycogen synthase (GS)
activity (ratio of I form activity to total activity); n ⫽ 7 (basal), 8 (insulin), 7
(insulin and levodopa-carbidopa), and 4 (insulin, levodopa-carbidopa, and
propranolol). B: phosphorylase activity (ratio of phosphorylase a activity to
total activity); n ⫽ 3 (basal), 4 (insulin), 4 (insulin and levodopa-carbidopa),
and 3 (insulin, levodopa-carbidopa, and propranolol). Values are means ⫾ SE.
* Significantly different from control, P ⬍ 0.05; † significantly different from
the mean for insulin treatment, P ⬍ 0.05.
possible protein kinase A-mediated downregulation of GS
activity.
Acute in vivo effects of levodopa-carbidopa. At the early
time point assessed (15 min after glucose feeding), levodopacarbidopa treatment did not cause a significant increase in
plasma glucose concentrations compared with plasma glucose
levels in animals given vehicle alone (Fig. 5A). However, the
single dose of levodopa-carbidopa caused a significant increase
in [cAMP] in triceps muscle compared with muscles from rats
treated with vehicle (P ⬍ 0.05; Fig. 5B). Levodopa-carbidopa
prevented an increase in epitrochlearis GS activity in rats that
were fed glucose (P ⬍ 0.01; Fig. 5C). Figure 5D shows that
levodopa-carbidopa caused a significant decrease in phosphorylation of IRS-1 on Tyr632 (P ⬍ 0.05) in epitrochlearis.
Effects of chronic levodopa-carbidopa treatment. Four
weeks of treatment with levodopa-carbidopa resulted in a
slightly lower body weight (P ⬍ 0.05) in the treated group
compared with the control group. Body weights were similar
before treatment began (control 145 ⫾ 9 g, drug 136 ⫾ 15 g),
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Fig. 3. Levodopa-carbidopa decreases insulin-stimulated glucose transport in
isolated skeletal muscle. Soleus (Sol) muscle strips, flexor digitorum brevis
(FDB) muscles, and Epi muscles were assayed for glucose transport activity in
the absence and presence of insulin and in the absence or presence of
levodopa-carbidopa. For some Epi muscles, propranolol was included with
media containing levodopa-carbidopa. A: Sol, n ⫽ 4/group. B: FDB, n ⫽
4/group. C: Epi, basal (n ⫽ 8), insulin (n ⫽ 6), levodopa-carbidopa⫹insulin
(n ⫽ 7), levodopa-carbidopa⫹insulin⫹propranolol (n ⫽ 4). Bars represent ⫾
SE. *P ⬍ 0.05, significantly lower than the mean for insulin treatment.
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LEVODOPA INHIBITS INSULIN ACTION IN MUSCLE
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Fig. 4. Effects of levodopa-carbidopa treatment on insulin-stimulated phosphorylation of Akt and insulin receptor substrate (IRS)-1. Isolated epitrochlearis muscles were incubated without insulin (control) or with 60 ␮U/ml insulin
in the absence or presence of 30 ␮M levodopa ⫹ 100 ng/ml carbidopa (L/C).
Muscles samples were subjected to SDS-PAGE and immunoblotted using
antibodies specific for phosphorylated Akt and IRS-1. Panels contain representative photographs and quantitation for Akt phosphorylated on Ser 473 (A),
Akt phosphorylated on Thr 308 (B), and IRS-1 phosphorylated on Tyr 632 (C).
Bars represent means ⫾ SE. *Significantly higher than control, P ⬍ 0.05; n ⫽
8 muscles/group.
J Appl Physiol • VOL
Fig. 5. Acute in vivo effects of levodopa-carbidopa. Rats were given a single
intragastric dose of levodopa-carbidopa or vehicle (NaCl). Fifteen minutes
later, rats received glucose in NaCl or NaCl alone as described in METHODS AND
MATERIALS. After an addition 15 min, muscle and plasma samples were taken.
A, plasma glucose concentrations; B, cAMP content of triceps (Tri) muscle; C,
GS activity in Epi muscle; and D, representative photograph and quantitation
for Epi muscle IRS-1 tyrosine phosphorylation. Bars represent means ⫾ SE.
*Significantly higher than control, P ⬍ 0.05; n ⫽ 4 rats/group.
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LEVODOPA INHIBITS INSULIN ACTION IN MUSCLE
Furthermore, GS activity in muscles taken from rats after 4 wk
of treatment with levodopa-carbidopa was ⬃50% lower compared with GS activity in muscles taken from control animals
(P ⬍ 0.05; Table 1).
DISCUSSION
The new information provided by this study is that levodopa-carbidopa decreases insulin-stimulated glucose transport,
glycogen accumulation, and GS activity in skeletal muscle.
These effects of levodopa could be blocked with propranolol,
a ␤-adrenergic antagonist. A single in vivo dose of levodopacarbidopa increased muscle cAMP concentrations, decreased
GS activity, and reduced tyrosine phosphorylation of IRS-1.
Chronic levodopa-carbidopa treatment decreased oral glucose
tolerance and led to lower muscle glycogen concentration and
GS activity. These findings are consistent with previous reports
of hyperglycemic effects of levodopa (3, 17, 18, 33). The
levodopa-related peripheral insulin resistance we found (i.e.,
inhibition of insulin-stimulated glucose transport into and storage by muscle) could contribute to hyperglycemia in animals
treated with levodopa. It appears that levodopa impinges on
J Appl Physiol • VOL
Table 1. Chronic levodopa/carbidopa treatment reduces
muscle glycogen levels and glycogen synthase activity
Glycogen, ␮mol Glucosyl
units/g
Epithrochlearis
Tibialis anterior
Plantaris
EDL
GS Activity, Fraction in I Form
Control
Levodopa/carbidopa
Control
Levodopa/carbidopa
16.8⫾2.7
17.2⫾0.7
19.2⫾0.9
19.1⫾1.0
8.6⫾1.0*
14.8⫾0.5*
16.4⫾0.7*
15.9⫾1.3*
0.32⫾0.03
0.47⫾0.08
0.37⫾0.05
0.57⫾0.19
0.21⫾0.02*
0.22⫾0.04*
0.22⫾0.04*
0.19⫾0.02*
Values are means ⫾ SE. Animals were treated intragastrically twice daily
for 4 wk with levodopa/carbidopa or vehicle. GS, glycogen synthase; EDL,
extensor digitorum longus. *Different from control, P ⬍ 0.05 (n ⫽ 6 rats/
group).
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Fig. 6. Oral glucose tolerance test after chronic levodopa treatment. Rats were
treated twice daily intragastrically with levodopa-carbidopa or vehicle for 4
wk. After an overnight fast, animals in the group treated with drugs received
a single dose of levodopa-carbidopa (at ⫺30 min) followed by an intragastric
glucose load (at 0 min). A, plasma glucose concentrations; B, plasma insulin
concentrations. *P ⬍ 0.05, significant effect of drug treatment. Symbols
represent means ⫾ SE, n ⫽ 6/group.
muscle glucose metabolism despite the presence of carbidopa,
a decarboxylase inhibitor.
Levodopa and its metabolite dopamine have been shown to
cause hyperglycemia in humans in a number of studies (17,
18). In one study, a 1.0-g dose of levodopa given orally to
seven patients with PD caused an increase in fasting plasma
glucose level from 87 to 99 mg/dl within 30 min (3). Four and
five hours after a 100-g glucose load, plasma glucose concentrations were still elevated (133 and 122 mg/dl) in subjects who
had ingested levodopa before consuming glucose compared
with plasma glucose concentrations (83 and 78 mg/dl) in
subjects for whom the oral glucose load was administered
without levodopa (3). In a separate study, 1.0 g of levodopa
caused hyperglycemia in patients with PD who had been
treated for 3 mo with levodopa (33). Furthermore, 12 mo of
chronic levodopa treatment reduced oral glucose tolerance in
these patients, such that mean peak plasma glucose concentrations during oral glucose tolerance tests increased from ⬃165
to ⬃190 mg/dl (33). The year of chronic levodopa treatment
was associated with a threefold increase in mean peak circulating insulin concentration and a twofold increase in insulin
area under the curve during an OGTT (33). Thus in the absence
of a decarboxylase inhibitor, acute and chronic levodopa treatment both appear to cause hyperglycemia (3, 17, 18, 33). The
current study indicates that even in the presence of a decarboxylase inhibitor, levodopa can inhibit insulin action and
impair glycogen metabolism in skeletal muscle. However,
similar studies need to be done in humans.
Dopamine, a metabolite of levodopa, functions through
␤-adrenergic receptors in skeletal muscle (31), although it is
unclear whether dopamine at the concentration (⬃1 ng/g) at
which it is found in skeletal muscle in the absence of exogenous levodopa treatment (11) has a true physiological function.
Muscle cells respond to 0.1 ␮M dopamine with increases in
cAMP concentrations, effects that are blocked by the ␤-adrenergic antagonist propranolol, but not by haloperidol, a dopamine receptor antagonist, or phentolamine, an ␣-adrenergic
receptor antagonist (31). Carbidopa prevents the conversion of
levodopa to its metabolite dopamine (6, 39). However, despite
the action of carbidopa to decrease plasma dopamine concentration in levodopa-treated animals, carbidopa does not prevent
accumulation of dopamine in skeletal muscle (9, 30). Thus it
seems possible that levodopa-carbidopa would cause an increase in [cAMP] in muscle secondary to ␤-adrenergic action
stimulated by a levodopa metabolite.
LEVODOPA INHIBITS INSULIN ACTION IN MUSCLE
GRANTS
This project was supported by the Saint Louis University Office of
Research Services. J. S. Fisher is supported by National Institute of
Diabetes and Digestive and Kidney Diseases Grant K01-DK-066330. B. A.
Racette is supported by National Institutes of Health Grant K23-NS-43351
and the Greater St. Louis Chapter of the American Parkinson Disease
Association.
J Appl Physiol • VOL
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When intracellular [cAMP] increases, protein kinase A activity increases, indirectly or directly increasing phosphorylation of GS and phosphorylase kinase. These actions result in
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We found that levodopa-carbidopa impairs insulin-stimulated glucose transport and glycogen accumulation in muscle.
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2345
2346
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