GLP-1 action in L6 myotubes is via a receptor different from
the pancreatic GLP-1 receptor
H.
Yang1,
J. M.
Egan1,
Y.
Wang1,
C. D.
Moyes2,
J.
Roth3,
M. H.
Montrose3, and
C.
Montrose-Rafizadeh1
1 Laboratory of Clinical
Physiology, Gerontology Research Center, National Institute on
Aging, National Institutes of Health, Baltimore 21224;
3 Department of Medicine, Johns
Hopkins University, Baltimore, Maryland 21205; and
2 Department of Biology, Queen's
University, Kingston, Ontario, Canada K7L 3N6
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ABSTRACT |
The incretin hormone glucagon-like peptide-1 (GLP-1)-(7
36)
amide is best known for its antidiabetogenic actions mediated via a
GLP-1 receptor present on pancreatic endocrine cells. To investigate
the molecular mechanisms of GLP-1 action in muscle, we used cultured L6
myotubes. In L6 myotubes, GLP-1 enhanced insulin-stimulated glycogen
synthesis by 140% while stimulating
CO2 production and lactate
formation by 150%. In the presence of IBMX, GLP-1 diminished cAMP
levels to 83% of IBMX alone. In L6 myotubes transfected with pancreatic GLP-1 receptor, GLP-1 increased cAMP levels and inhibited glycogen synthesis by 60%. An antagonist of pancreatic GLP-1 receptor, exendin-4-(9
39), inhibited GLP-1-mediated glycogen synthesis in GLP-1
receptor-transfected L6 myotubes. However, in parental L6 myotubes,
exendin-4-(9
39) and GLP-1-(1
36) amide, an inactive peptide on
pancreatic GLP-1 receptor, displaced
125I-labeled GLP-1
binding and stimulated glycogen synthesis by 186 and 130%,
respectively. These results suggest that the insulinomimetic effects of
GLP-1 in L6 cells are likely to be mediated by a receptor that is
different from the GLP-1 receptor found in the pancreas.
glycolysis; glycogen synthesis; muscle; transfection; glucagon-like
peptide
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INTRODUCTION |
GLUCAGON-LIKE peptide-1 (GLP-1) is an incretin hormone
secreted from L cells of the intestine in response to nutrient
ingestion (6). GLP-1 helps to regulate plasma glucose levels by
enhancing insulin secretion from the pancreatic
-cells (14) as well
as diminishing blood levels of both glucagon and somatostatin (11). The
insulinotropic effects of GLP-1 are mediated via the pancreatic GLP-1
receptor, which is linked to activation of adenylyl cyclase (26).
Recent evidence suggests that GLP-1 may also have peripheral effects to
enhance glucose utilization in insulin-sensitive tissues (i.e., fat,
muscle, and liver) (3, 5). Recent studies have shown that GLP-1
stimulates glycogen synthesis and increases glycolysis and glucose
oxidation in isolated rat soleus muscle and liver (24, 25). These
observations were strengthened by evidence for specific GLP-1 binding
sites on membranes of rat myocytes (4). However, it has not been clear
whether the response to GLP-1 in insulin target tissues occurs via the
pancreatic GLP-1 receptor.
In this study, we used L6 myotubes as a model of skeletal muscle (2,
12, 19) to investigate whether GLP-1 has an insulinomimetic effect in
muscle and whether the effect is mediated by the pancreatic GLP-1
receptor isoform. We demonstrate that the insulinomimetic effects of
GLP-1 on parental L6 myotubes are mediated via a receptor that is
functionally different from the pancreatic GLP-1 receptor.
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EXPERIMENTAL PROCEDURES |
Plasmid constructs.
Full-length rat pancreatic GLP-1 receptor clone (Ref. 22; a gift of Dr.
B. Thorens, University of Lausanne, Switzerland) was subcloned in a
plasmid in which GLP-1 receptor and neomycin phosphotransferase (G418
resistance) genes were each driven by individual mouse RNA polymerase
II promoters (pPol2GLPR).
Cell culture and transfection.
Rat L6 myoblasts were grown as described previously (2) in a humidified
95% air-5% CO2 incubator at
37°C, in alpha minimum essential medium (AMEM; Paragon Biotech,
Baltimore, MD) containing 10% fetal bovine serum (FBS; GIBCO BRL,
Gaithersburg, MD) and supplemented with 50 U/ml penicillin, 50 µg/ml
streptomycin, and 2 mM glutamine. The medium was changed every 2 days.
After reaching confluence, the cells were induced to differentiate by
exposure to 2% FBS in AMEM for 2 days.
For stable transfection, subconfluent (50% confluent) L6 myoblasts
were transfected with 2 µg of Sst II
linearized pPol2GLPR using Lipofectamine reagent and Opti-MEM I medium
(GIBCO BRL). After overnight incubation, medium was
changed to AMEM with 10% FBS for an additional 24 h. Geneticin (G418;
GIBCO BRL) at an effective concentration of 600 µg/ml was then added
to the medium to select the G418-resistant cells. After 5 days, single
colonies (clonal mutant lines) or a mixed population (containing ~300
colonies) was passaged to maintain a subconfluent cell density. After
10 days, G418 was removed from the medium, cells were expanded for experiments, and cell aliquots were frozen. The presence of transfected pancreatic GLP-1 receptor mRNA was confirmed by Northern blot in all
clones and in the mixed population (data not shown).
Intracellular cAMP levels.
Cells grown in 12-well plates were washed 3 times and incubated with 1 ml of Krebs-Ringer phosphate buffer (KRP) containing 128 mM NaCl, 5 mM
KCl, 1.3 mM MgCl2, 1.2 mM
CaCl2, 25 mM HEPES (pH 7.4), 1 mM
NaPO4 (pH 7.4), 2.5 mM glucose,
and 0.1% BSA for 4 h at 37°C. Cells were then exposed to KRP
containing various concentrations of GLP-1-(7
36) amide (Bachem,
Torrance, CA) for 30 min at 37°C, with or without 1 mM IBMX. After
treatment, cells were washed three times with ice-cold Dulbecco's
phosphate buffer saline (DPBS) and lysed with ice-cold 0.6 M perchloric
acid for 5 min. Aliquots of cell lysate were transferred to microfuge
tubes, and the pH was adjusted to 7.0 with 5 M
K2CO3.
After 5 min of centrifugation (2,000 g), the supernatant was vacuum dried
and redissolved in 200-500 µl of RIA buffer (500 mM Tris, pH
7.5, and 4 mM EDTA, pH 8.0). After addition of 0.15 mM sodium
bicarbonate (20-50 µl) and 0.15 mM zinc sulfate (20-50
µl), samples were incubated for 15 min on ice and then centrifuged
for 5 min at 2,000 g. Aliquots of the
supernatant were assayed using the
[3H]cAMP assay kit
(Amersham). Data are presented in picomoles or nanomoles of cAMP per
milligram of protein. Each well contained comparable amounts of
cellular protein (data not shown).
Glycogen synthesis.
A modification of the method of Myers (17) was used. In brief, L6
myotubes in 12-well dishes were washed twice with KRP and incubated in
KRP containing 2.5 mM glucose for 3 h at 37°C. The buffer was then
replaced with fresh KRP containing
D-[U-14C]glucose
(0.29 mCi/mmol; Amersham), with or without GLP-1 or other peptide
analogs and/or porcine insulin (Calbiochem, San Diego, CA), for
30 min at 37°C. The cells were then washed three times with
ice-cold DPBS and lysed in 400 µl of 20% KOH. The lysate was
transferred to microfuge tubes containing 100 µl of carrier glycogen
(type VII from muscle, Sigma, St. Louis, MO) at a final concentration
of 1 mg/ml. The lysate was boiled and centrifuged, and the glycogen was
precipitated with 2.5 volumes of 100% ethanol with overnight
incubation at
20°C. After one repetition of the precipitation step, the glycogen pellet was dissolved in 200 µl of
water and counted in 10 ml of Ecoscint A (National Diagnostics, Atlanta, GA).
Glucose oxidation and production of lactate and other glycolytic
intermediates.
L6 myotubes grown in Nunc 25 cm2
flasks were washed three times with KRP and incubated in KRP for 1 h at
37°C. The buffer was then replaced with fresh KRP containing
D-[U-14C]glucose
(1.8 mCi/ml final concentration) with and without GLP-1 and/or
insulin. Each flask was capped with fitted plastic caps containing GF/C
filters (Whatman, Maidstone, UK) to trap
CO2 (16). After 1 h of incubation
at 37°C, 200 µl of 1 M hyamine hydroxide was injected onto the
filter and 200 µl of concentrated perchloric acid was added to the
cells. After 90 min of shaking, the filter was added to 10 ml of
Ecoscint A containing 10 µl of glacial acetic acid (to reduce
chemiluminescence) and counted. For measurements of lactate and other
glycolytic intermediates, cells were scraped from the flask and
transferred to 15-ml tubes. After the lysate had been centrifuged
(2,500 g), the perchloric
acid-containing supernatant was transferred and neutralized (10 µl
0.5% phenol red indicator and ~300 µl 10 M KOH) and then incubated
overnight at
20°C. The lysate was clarified by
centrifugation, and the supernatant was loaded onto an anion-exchange
column (AG-X8, Bio-Rad, Richmond, CA) and washed three times with
distilled water. The resin was counted in 10 ml of Ecoscint A with 30 µl of glacial acetic acid to reduce color quench. The anion exchange
column binds all negatively charged metabolites, including lactate,
pyruvate, and other glycolytic intermediates. We consider that the
majority of the glycolytic products will be lactate. However,
copurification of other glycolytic intermediates was not excluded.
GLP-1 binding.
L6 myotubes grown in 12-well dishes were washed and incubated with
serum-free AMEM for 4 h at 37°C. The cells were then washed twice
with binding buffer (10 mM Tris, pH 7.4, 120 mM NaCl, 1.2 mM
MgSO4, 5 mM KCl, and 15 mM sodium
acetate) and incubated at 4°C overnight with 0.5 ml of binding
buffer containing 2% BSA, 500 U/ml aprotinin, 25,000 counts/min (cpm)
125I-labeled GLP-1 (2,000 Ci/mmol;
Amersham), and a range of concentrations of unlabeled GLP-1. We only
used freshly prepared 125I-GLP-1,
within 3 wk of the reference date. The buffer was then removed, and
cells were washed three times with ice-cold DPBS and lysed with 0.5 ml
of 0.5 N NaOH-0.1% SDS. The radioactivity in the lysate was counted in
a gamma counter (10/600 Plus, ICN, Costa Mesa, CA). Specific binding
was determined by subtraction of the radioactivity associated with
cells incubated with a large excess of unlabeled GLP-1 (0.5 µM).
Ca2+
measurements.
Cells attached to coverslips were loaded with fura 2 by incubation for
1 h at 37°C with 6 µM fura 2-AM. The coverslips were mounted in a
microscope chamber and perfused at 35-37°C with KRP containing
various additives. The temperatures of the chambers and microscope
objective were kept constant by jacketed water circulation. Cells were
studied on a Zeiss Axiovert microscope with a water immersion ×50
Leitz objective, numerical aperture 1.0. Excitation light was provided
by a 75-W xenon lamp attenuated to 20% power by neutral density
filters. Under these conditions, photobleaching of fura 2 was not
detectable. Fura 2 fluorescent emission was measured at 420-570 nm
in response to alternating excitation wavelengths of 350 ± 10 nm
and 380 ± 10 nm by a computer-controlled filter wheel. Pairs of
images were collected every 6 s. Fluorescence was detected by a
Hamamatsu intensifier charge-coupled device camera operating at
constant intensifier and camera gain. Data were collected as four-frame
averages (128 ms/image), which were eight-bit digitized and analyzed by
a Perceptics image processor (Knoxville, TN). In each experiment, up to
20 cells in the camera field could be selected for real-time analysis.
Data from each of the selected cells in the field were collected,
stored separately, and treated as independent observations.
Statistical analysis.
Data are means ± SE. We used the Student's two-tailed
t-test, and differences were
considered significant when P < 0.05.
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RESULTS |
GLP-1 regulates glycogen synthesis in parental L6 myotubes.
To investigate the effects of GLP-1 on glycogen synthesis in muscle, L6
cells were differentiated to myotubes and then treated with or without
insulin in the presence or absence of GLP-1 for 30 min. Insulin alone
(1.0 or 100 nM) significantly increased glycogen levels (1.41 ± 0.08-fold and 2.40 ± 0.28-fold, respectively; n = 13, P < 0.001). As shown in Fig.
1, in the absence of insulin or at 0.1 nM insulin, GLP-1 stimulation of glycogen synthesis was
slight (
1.2-fold, P < 0.05 and
P = 0.06, respectively). GLP-1 (10 nM)
enhanced insulin-mediated glycogen synthesis at 1 or 100 nM insulin
(P < 0.01 and
P < 0.05, respectively). The results suggest that GLP-1 has insulinomimetic effects in L6 myotubes.

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Fig. 1.
Effects of glucagon-like peptide-1 (GLP-1) on basal and
insulin-mediated glycogen synthesis in L6 cells. Cells were incubated
in Krebs-Ringer phosphate buffer (KRP) containing
D-[14C]glucose
in presence (solid bars) or absence (open bars) of GLP-1 (10 nM) and
with a range of concentrations of insulin (0-100 nM) for 30 min,
and [14C]glycogen
synthesis was measured as described in EXPERIMENTAL
PROCEDURES. Data are normalized as percent of response
to 100 nM insulin in absence of GLP-1 (0.7 ± 0.1 nmol glycogen/mg
protein) and are means ± SE from triplicate measurements in 13 separate experiments. Significant differences between presence and
absence of GLP-1: * P < 0.05, ** P < 0.01. Basal glycogen
synthesis was significantly different from glycogen synthesis in
presence of 1 or 100 nM insulin (P < 0.001). Glycogen synthesis in presence of 0.1 nM insulin was not
significantly different from basal.
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125I-GLP-1 binding in L6 myotubes.
As shown in Fig. 2,
125I-GLP-1 binding was saturable,
and the data were well fitted with a single binding site having a
dissociation constant
(Kd) of 1.84 ± 0.35 nM (n = 7) and a maximum
receptor number of 5,444 ± 1,472 receptors/cell (Table
1). Specific binding, as determined by
subtraction of the nonspecific binding (662 ± 94 cpm) from the
total binding (812 ± 103 cpm, n = 11), was 150 ± 22 cpm. The specific binding was 0.58 ± 0.08%
(n = 11; the amount of specific
binding was significantly greater than zero,
P < 0.001) of total radioactivity
added and 20 ± 2.8% of total binding. These values are
similar to the values previously obtained from 3T3-L1 adipocytes (15).

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Fig. 2.
Saturation binding isotherm of
125I-GLP-1 in L6 cells.
125I-GLP-1 binding was measured in
intact L6 cells in presence of various concentrations of unlabeled
GLP-1. Specific binding was calculated as total binding minus
nonspecific binding (latter measured in presence of 500 nM unlabeled
GLP-1). The results were converted to fmol bound after calculation of
specific activity of labeled GLP-1, and results in each experiment were
normalized to amount bound at 10 nM GLP-1 (10.4 ± 3.0 fmol, mean ± SE, n = 9). Curve is best fit
for single binding site with a dissociation constant
(Kd) of 1.84 ± 0.35 nM. Data are means ± SE from triplicate measurements in
9 separate experiments.
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Table 1.
GLP-1 binding, Kd, and number of GLP binding sites in
parental and GLP-1 receptor-transfected L6 myotubes
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Signal transduction via GLP-1 receptor in L6 myotubes.
To investigate the signal transduction mechanisms activated by GLP-1 in
L6 myotubes, we first measured intracellular cAMP levels. GLP-1 (10 nM)
alone had no significant effect on cAMP levels (Fig.
3A).
However, in the presence of 1 mM IBMX (a phosphodiesterase inhibitor),
GLP-1 (10 nM) lowered cAMP levels to 83 ± 4% of IBMX control
(n = 3, P < 0.05). This small but
significant decrease in cAMP levels by GLP-1 is opposite to the known
effect of the activated form of the pancreatic GLP-1 receptor (26).

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Fig. 3.
Effects of GLP-1 on intracellular cAMP
(A) and intracellular free
Ca2+ levels
(B) in L6 cells.
A: cells were incubated in KRP
containing 100 nM insulin in presence or absence of 10 nM GLP-1, with
or without IBMX (1 mM), for 30 min. Data are percentage of control (1 mM IBMX in absence of GLP-1, 6.7 ± 1.4 pmol cAMP/mg protein) and
are means ± SE from 7 separate experiments performed in duplicate.
* P < 0.05. B: intracellular free
Ca2+ was measured using a
fluorescence-imaging microscope, and cells attached to coverslips were
loaded with fura 2 dye. Ratio of fluorescence at 350-nm excitation to
that at 380-nm excitation is shown on
y-axis vs. time. Boxed areas indicate
periods when cells were exposed to either 100 nM GLP-1 or 10 µM
carbachol (carb). A representative experiment (averaged responses of 20 cells) is shown. Similar results were obtained in 4 monolayers examined
in 2 separate experiments.
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In COS cells overexpressing pancreatic GLP-1 receptor, it has been
shown that GLP-1 can couple to increases of intracellular Ca2+ concentration (26). We also
measured intracellular Ca2+ levels
in L6 myotubes by imaging of intracellular fura 2 fluorescence. As
shown in Fig. 3B, exposure of L6
myotubes to 100 nM GLP-1 had no effect on free intracellular
Ca2+ concentrations measured in 20 cells. Similar results were obtained with 10 nM GLP-1 (data not shown).
Because effects of GLP-1 might require the presence of insulin, in one
experiment GLP-1 effect was tested in the presence of 100 nM insulin
and was shown to have no effect on intracellular
Ca2+ levels (data not shown).
Carbachol (0.1 mM) was used as a positive control to confirm the
ability of these cells to mobilize
Ca2+ in response to receptor
activation and to confirm Ca2+
responsiveness of fura 2 in each cell. The results suggest that intracellular Ca2+ change is not
involved in GLP-1 signal transduction in myotubes during short-term
GLP-1 treatment. To exclude the possibility that a small but undetected
rise in intracellular Ca2+ was
responsible for GLP-1 stimulation of glycogen synthesis, one control
experiment assessed the effect of carbachol on glycogen synthesis.
Carbachol, which raised intracellular
Ca2+, inhibited basal and
insulin-mediated glycogen synthesis by 19 and 53%
(n = 1), respectively.
These results suggest that a lowering of cAMP levels is associated with
GLP-1 action in L6 myotubes and that
Ca2+ mobilization plays little or
no role in GLP-1 signaling in these cells.
GLP-1 regulates glycolysis and glucose oxidation in parental L6
myotubes.
To examine the potential role of GLP-1 in the regulation of
cAMP-independent processes, we examined glycolysis and glucose oxidation in L6 cells by measuring the production of lactate and CO2, respectively. These processes
have been shown to be cAMP-independent in muscle (9, 18). As shown in
Fig. 4, 100 nM insulin for 1 h induced
significant increases in both CO2
and lactate production (2.0-fold over basal). Similarly, GLP-1 (10 nM)
significantly increased basal glycolysis and glucose oxidation by
~1.5-fold. In the presence of 1 nM insulin, glycolysis and glucose
oxidation were increased by 154 ± 13.5 and 155.5 ± 15.7%
(n = 3), respectively. In the presence
of 1 nM insulin, GLP-1 (10 nM) had a small additive effect on
CO2 production (117 ± 3.5%
vs. insulin alone, P < 0.05, n = 3), whereas the same effect on
lactate production did not reach significant levels (112.8 ± 12.3%
vs. insulin alone, P > 0.05, n = 6). These results suggest that
GLP-1 has insulinomimetic effects in L6 myotubes that may also involve
cAMP-independent signaling pathways.

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Fig. 4.
Effects of GLP-1 on glucose oxidation in L6 cells. Cells were incubated
in KRP containing
D-[14C]glucose
in presence of GLP-1 (solid bars) or in absence of GLP-1 (10 nM) with
(hatched bars) or without (open bars) insulin (100 nM) for 1 h at
37°C.
[14C]CO2
(A) or
[14C]lactate
(B) was measured as described in
EXPERIMENTAL PROCEDURES. Data are
presented in counts/min (cpm) per mg protein and are means ± SE
from duplicate measurements of 3 separate experiments.
* P < 0.05;
** P < 0.01.
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Transfection of L6 myotubes with pancreatic GLP-1 receptor.
The physiological responses of L6 cells to GLP-1, (i.e., decreased cAMP
and increased glycogen synthesis, lactate, and
CO2 production) could not be
explained by our current understanding of early postreceptor pathways
of pancreatic GLP-1 receptor signaling. The results suggest that in L6
myotubes a novel receptor is expressed that responds to GLP-1 (with or
without expression of the pancreatic type GLP-1 receptor) or that the
pancreatic GLP-1 receptor isoform uses different signal transduction
mechanisms in L6 cells and in pancreas. The transfection of the cloned
pancreatic GLP-1 receptor into L6 cells allowed the direct comparison
of receptor signaling between the overexpressed pancreatic GLP-1
receptor and the endogenous GLP-1 receptor. As shown in Fig.
5, both GLP-1 receptor-transfected L6 cell
lines and the parental L6 line differentiated to myotubes. This
suggests that all transfectants (L6/pancGLPR) retained the intact
differentiation properties of parental cells. These results are
important for further analysis of pancreatic GLP-1 receptor function in
transfected cells, since it allows comparison of GLP-1 function within
a cellular milieu similar to parental L6 cells.

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Fig. 5.
Visualization of various differentiated L6 cell lines. Cells were
viewed by differential interference contrast imaging in a Zeiss
Axiovert microscope with a ×40 objective. Parental
(A) and GLP-1 receptor-transfected
L6 cells [mixed population
(B),
clone
5 (C), and
clone
2 (D)]. All GLP-1
receptor-transfected cells
(B-D)
differentiated to myotubes normally (similar to
A, parental L6 cells), which suggests
that transfected cells conserved physiological features of parental L6
cells. Scale in D applies to
A-D.
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Binding characteristics of various L6 cell lines.
GLP-1 binding was analyzed in four independent L6/pancGLPR cell lines
and compared with parental L6 cells (Table 1). Scatchard analysis
revealed that the number of GLP-1 receptor binding sites varied among
the different transfectant cells, ranging from 3 times
(clone
2) to 10 times (mixed population)
higher than the number of endogenous GLP-1 receptor sites. The binding
affinity for GLP-1 was similar among parental cells and L6 transfectant clones.
Signal transduction in GLP-1 receptor-transfected L6 cells.
We measured cAMP levels in transfected L6 myotubes expressing the
pancreatic GLP-1 receptor (Fig.
6A). In
contrast to parental L6 cells, 1 nM GLP-1 increased cAMP 7- to 100-fold
in transfectant populations. These results are unlikely to be due to
cell cloning artifacts because similar results were observed in both
the mixed population and the cloned transfectants. The concentration
that elicited half-maximal response was 10 nM GLP-1 for the mixed
population, with a cAMP response of 311.9 ± 62.4 pmol/mg protein
(n = 3). The results show that the
pancreatic GLP-1 receptor is linked to increases in cAMP levels in
transfected L6 myotubes, suggesting that sufficient
Gs, the G protein that activates
adenylyl cyclase, was available for receptor coupling. We also
performed measurements of intracellular cAMP in the presence of GLP-1
and the phosphodiesterase inhibitor IBMX, to eliminate any variation in
phosphodiesterase activity in various cell lines. In these conditions,
a positive correlation (r = 0.985, P < 0.05) was observed between
GLP-1-stimulated levels of cAMP and the number of GLP-1 binding sites
(Fig. 6B). Our data suggest that
heterologous expression of the pancreatic GLP-1 receptor in L6 myotubes
did not saturate the signal transduction mechanism.

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Fig. 6.
Effects of GLP-1 on cAMP levels in L6 cells transfected with with
pancreatic GLP-1 receptor. A:
intracellular cAMP levels were measured after 30-min exposure of GLP-1
receptor-transfected L6 cell lines to various GLP-1 concentrations.
Data are in pmol/mg protein and are means ± SE of 3 independent
experiments performed in duplicate. B:
correlation between cAMP levels after 30-min treatment of GLP-1
receptor-transfected L6 cell lines with 0.1 nM and in presence of 1 mM
IBMX (n = 4). GLP-1 receptor numbers
were derived from Table 1. Coefficient of correlation is
r = 0.985 (P < 0.05).
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GLP-1 inhibits glycogen synthesis in L6/pancGLPR myotubes.
As shown in Fig. 7, insulin increased
glycogen synthesis in the mixed population of L6/pancGLPR cells,
indicating that transfectants respond normally to insulin. However, in
contrast to parental L6 cells, 0.1 nM GLP-1 significantly inhibited
basal and insulin-stimulated glycogen synthesis. Similar results were
observed with all clones of L6/pancGLPR cells (data not shown).

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Fig. 7.
Effects of GLP-1 on glycogen synthesis in L6 cells transfected with
pancreatic GLP-1 receptor. Glycogen synthesis was measured in a mixed
population of GLP-1 receptor-transfected L6 cells treated for 30 min
with (solid bars) or without (open bars) GLP-1 (0.1 nM) in presence of
a range of concentrations of insulin (0-100 nM). Data are
normalized as percent of response to 100 nM insulin in absence of GLP-1
(0.82 ± 0.09 nmol glycogen/mg protein) and are means ± SE from
triplicate measurements in 3 separate experiments.
* P < 0.05 represent
significant differences between presence vs. absence of GLP-1. Basal
glycogen synthesis was significantly different from glycogen synthesis
in presence of 1 or 100 nM insulin (P < 0.05 and P < 0.01, respectively). Glycogen synthesis in presence of 0.1 nM insulin was not
significantly different from basal.
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Exendin-4-(9
39) differentially affects parental L6 and L6/pancGLPR
myotubes.
An antagonist of pancreatic GLP-1 receptor was used to confirm that
effects in transfectants were due to pancreatic GLP-1 receptor. As
shown in Fig. 8, exendin-4-(9
39) (0.1 µM; a generous gift of Dr. John Eng, Veterans Affairs Medical Center,
Bronx, NY) reversed the inhibitory effect of GLP-1 (0.03 nM) on
glycogen synthesis in L6/pancGLPR cells. However, in parental L6
myotubes (Fig. 9), 10 nM exendin-4-(9
39)
stimulated basal and insulin-mediated glycogen synthesis. This suggests
that this antagonist of the pancreatic GLP-1 receptor is an agonist in
parental L6 myotubes, since it mimics the effect of GLP-1 in this cell
line.

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Fig. 8.
Effect of exendin-4-(9 39) on glycogen synthesis in L6 cells
transfected with pancreatic GLP-1 receptor. Glycogen synthesis was
measured for 30 min in GLP-1 receptor-transfected cells in presence or
absence of 100 nM insulin. GLP-1 (0.03 nM) was added in presence or
absence of 0.1 µM exendin-4-(9 39).
* P < 0.05, *** P < 0.001. Data are
normalized as percent of response to 100 nM insulin alone. Data are
means ± SE from triplicate measurements in 3 separate
experiments.
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Fig. 9.
Effect of exendin-4-(9 39) on glycogen synthesis in parental L6 cells.
Glycogen synthesis was measured for 30 min in L6 parental cells in
presence (solid bars) or absence (open bars) of 10 nM
exendin-4-(9 39), with or without 100 nM insulin.
* P < 0.05, *** P < 0.001 for presence vs.
absence of exendin-4-(9 39). Data are percent of response in presence
of 100 nM insulin alone and are means ± SE from triplicate
measurements in 3 separate experiments.
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Effects of different analogs of GLP-1 in L6 myotubes.
To further characterize the receptor that binds GLP-1 in parental L6
myotubes, a number of peptides related to GLP-1 were used in parental
L6 myotubes to displace 125I-GLP-1
binding at 1 and 500 nM. Table 2 shows that
1 nM exendin-4-(9
39), exendin-4-(1
39), GLP-1-(1
36), and GLP-1
itself displaced 125I-GLP-1
binding by 30-50%. GLP-2 and glucose-dependent insulinotropic peptide were without effect, demonstrating the peptide specificity of
the results. These data suggest that the agonist effect of exendin-4-(9
39) and GLP-1 on glycogen synthesis is likely to be
mediated via a common receptor, albeit with properties different from
those of the pancreatic GLP-1 receptor (compare Figs. 8 and 9). We also
performed glycogen synthesis experiments in the presence of 10 nM
GLP-1-(1
36) and exendin-4-(1
39) (Table
3). Both of these peptides were agonists
and enhanced insulin-mediated glycogen synthesis but had no effects on
basal glycogen synthesis (data not shown). These data suggest a peptide
specificity in L6 cells that is different from what is known for
pancreatic GLP-1 receptor. Table 3 also shows that the response of
cells to GLP-1 was dose dependent; 1 and 10 nM GLP-1 increased
insulin-mediated glycogen synthesis. However, 0.1 nM GLP-1 was without
effect (data not shown). Surprisingly, GLP-1 at 100 nM had no
significant effect on glycogen synthesis. An explanation for this could
be that GLP-1 at this high concentration acts also via the glucagon
receptor, shown by RT-PCR to be present in these cells (data not
shown). Experiments confirmed that basal and insulin-mediated glycogen synthesis were inhibited in the presence of 10 nM glucagon, by 22.6 ± 5.75 and 19.3 ± 7.6%, respectively
(n = 7, P < 0.05), which is in accord with a
classical inhibitory action of glucagon on glycogen synthesis.
 |
DISCUSSION |
An effect of GLP-1 to enhance peripheral glucose utilization in muscle
would be beneficial to patients with either type I or type II diabetes.
However, controversial results exist in the literature as to a direct
effect of GLP-1 on muscle (7, 25). In addition, there is no published
report exploring a potential synergistic effect of GLP-1 on
insulin-mediated glycogen synthesis, glycolysis, or glucose oxidation.
Recent evidence from pancreatic GLP-1 receptor knockout mice showed
that glucose levels after an oral glucose tolerance test were not
modified in the presence or absence of GLP-1 (20) and that whole body
glucose utilization was not modified during a hyperinsulinemic
euglycemic clamp (21). However, other contraregulatory hormones such as
glucagon, somatostatin, and glucocorticoid hormones could have masked a
GLP-1 effect in extrapancreatic tissues of the knockout mice. GLP-1 has
recently been shown to have effects on hormone secretions from the
hypothalamus-pituitary-adrenal axis (1, 13).
To investigate the effect of GLP-1 on a homogenous muscle cell
preparation in which conditions can be precisely controlled, we used L6
cultured cells that differentiate to myotubes. Glucose metabolism
responds to insulin in L6 myotubes. We show that GLP-1 increased
glycolysis and glucose oxidation as well as glycogen synthesis. Our
data support previous observations of glycogenic properties of GLP-1 in
muscle (25) and reveal that these effects of GLP-1 are additive to
those of insulin.
In L6 myotubes, GLP-1 activates signal transduction pathways different
from those activated by pancreatic GLP-1 receptor. Activation of the
rat pancreatic GLP-1 receptor has been shown to increase intracellular
cAMP; when overexpressed in COS cells (26), this receptor can also
increase intracellular Ca2+. Our
results in L6 myotubes are in accord with previously published data (4)
showing GLP-1 has no effect on basal adenylyl cyclase activity in
muscle. However, in L6 myotubes, we were able to show that an
IBMX-stimulated elevation in cAMP was diminished in the presence of
GLP-1. Because intracellular Ca2+
was not mobilized by GLP-1, the results show clear differences from the
pancreatic GLP-1 receptor.
The results also suggest that GLP-1 has some physiological effects on
L6 myotubes via cAMP-independent mechanisms. GLP-1 stimulated glycolysis (lactate production) and glucose oxidation
(CO2 production), two metabolic
pathways predicted to be cAMP-independent in muscle (9, 18). The
alternative signaling pathway(s) may involve inositolphosphoglycans and
diacylglycerol levels, as shown for BC3H-1 myocytes and liver (8, 23).
Therefore, different rate-limiting steps may control these various
physiological responses.
Our evidence suggests that the effects of GLP-1 in L6 muscle cells are
probably mediated by a receptor distinct from pancreatic GLP-1
receptor. The difference in signal transduction linked to GLP-1 action
could be due either to the pancreatic GLP-1 receptor isoform expressed
in L6 myotubes but coupled to different signal transduction or to a
distinct receptor expressed in muscle that responds to GLP-1. To
distinguish between these two possibilities, we transfected the
pancreatic GLP-1 receptor isoform cDNA into L6 myoblasts. The
transfected myoblasts were able to differentiate into myotubes, so
comparison of signal transduction and function between transfected and
parental L6 cells was made between comparable differentiated cells. In
stable transfectants, activation of pancreatic GLP-1 receptor gave the
predicted response of increasing cAMP levels and inhibiting basal and
insulin-mediated glycogen synthesis. These data suggest that endogenous
receptors that respond to GLP-1 in L6 cells are distinct from the
pancreatic GLP-1 receptor isoform. This effect was not simply due to
overexpression of the GLP-1 receptor, since clones that contain 3 times
(clone
2) to 10 times (mixed population)
more GLP-1 binding sites than the endogenous receptor gave
qualitatively similar results and did not saturate the cellular signal
transduction pathways.
Finally, exendin-4-(9
39), a specific antagonist of pancreatic GLP-1
receptor (10), inhibited GLP-1-mediated glycogen synthesis in L6 cells
transfected with pancreatic type GLP-1 receptors but had opposite
effects in parental L6 cells. Similarly, GLP-1-(1
36), an inactive
peptide on pancreatic GLP-1 receptor, acted as an agonist and increased
glycogen synthesis in parental L6 myotubes. Experiments in which these
peptides were used to compete against binding of
125I-GLP-1 suggest that the
agonistic nature of exendin-4-(9
39), exendin-4-(1
39),
GLP-1-(1
36), and GLP-1-(7
36) is most likely mediated via a common
receptor. Similar results were obtained in 3T3-L1 adipocytes (15) and
rat muscle cells (4).
The results strongly suggest that parental L6 cells express a distinct
receptor that responds to GLP-1, other than the one expressed in
pancreas, since the peptide responsiveness of this receptor is different from that of the pancreatic GLP-1 receptor. Because neither Western nor Northern blots detect a protein with homology to pancreatic GLP-1 receptor and attempts to amplify GLP-1
receptor mRNA using RT-PCR and primers derived from various regions of
pancreatic GLP-1 receptor have failed (data not shown), other
strategies will be necessary to resolve this new receptor. Although the
L6 muscle cell line allows the identification of a receptor with novel
properties, the presence of this novel receptor in normal muscle cells
needs to be investigated. Furthermore, isolation of this new putative
receptor will be needed to further characterize its primary ligand.
In summary, our data show that GLP-1 acts in L6 cells via a receptor
that has signal transduction and ligand specificity different from
those of the pancreatic receptor and that acts in part via cAMP-independent pathways.
 |
ACKNOWLEDGEMENTS |
We thank Lisa G. Adams for expert technical assistance, Michele D. Buckler for secretarial assistance, Dr. Michel Bernier for critical
reading of the manuscript, and Dr. Derek LeRoith for helpful
discussions. We also thank Dr. Amira Klip and Celia Taha for generously
providing the cells and initial input for this study.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and present address of C. Montrose-Rafizadeh: Lilly Research Laboratories, Eli Lilly and Co.,
Lilly Corporate Center, Bldg. 88/416, Drop Code 1543, Indianapolis, IN
46285.
Received 5 March 1998; accepted in final form 19 May 1998.
 |
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