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Department of Metabolism, Nutrition and Hormones (M.I.L.-D., M.M., M.L.V.-P., I.V.), Fundación Jiménez Díaz, 28040 Madrid, Spain; and Laboratory of Experimental Medicine (W.J.M.), Brussels Free University, B-1070 Brussels, Belgium
Address all correspondence and requests for reprints to: Dr. Isabel Valverde, Fundación Jiménez Díaz, Departamento Metabolismo, Nutrición y Hormonas, Avda. Reyes Católicos, 2, 28040-Madrid, Spain.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Male Wistar rats, kept on a standard pellet diet (UAR, Panlab,
Barcelona, Spain) and tap water ad libitum, were used. The
non-insulin-dependent diabetes mellitus models [diabetic rats treated
with STZ on the first day of birth (STZ-N0) and on the 5th
of birth (STZ-N5)] were induced in rats as in Portha
et al. (6), by ip injection of STZ, as follows: the
STZ-N0 group received 100 µg/g BW dissolved in 25 µl of
a citrate-NaOH buffer (50 mM, pH 4.5), on the day of birth;
and the STZ-N5 group received 80 µg/g BW 5 days after
birth; at the age of 6–7 weeks, the rats were subjected to an iv
glucose tolerance test (2.8 µmol/g BW) to select those animals with a
glucose use coefficient below 2.5 x 10-2 x
min-1. The insulin-dependent diabetes mellitus model
(STZ-adult) was induced in adult rats by a single dose of STZ (60
µg/g BW) ip administered, the rats being used 1 week later. Normal
rats were also included. All rats were used after fasting overnight,
and for normal rats, they were also studied in postprandial condition.
Isolated hepatocytes were prepared as in Hue et al. (7).
Glycogen-synthase a and -phosphorylase a activities
Isolated hepatocytes (4 x 106), previously
preincubated for 10 min at 37 C in Krebs-Ringer bicarbonate buffer
(KRB) (118 mM NaCl, 4.8 mM/l KCl, 1.2
mM KH2PO4, 1.2 mM
MgSO4, 2.5 mM CaCl2, and 25
mM NaHCO3), pH 7.4, containing 0.1% BSA and
5.6 mM D-glucose, were incubated for 1–10 min
in 100 µl KRB containing 0.1% BSA and D-glucose (0–16.7
mM) in the absence and presence of GLP-1, insulin, or
glucagon. The samples were immediately frozen. For the enzyme
activities assays, the frozen samples were placed at 4 C and
homogenized in 400 µl of medium containing 50 mM
glycylglycine, 100 mM NaF, 35 mM EDTA, and
0.5% glycogen (wt/vol) at pH 7.4.
Glycogen synthase a activity was measured following the
described procedure (7); in brief, 20 µl homogenate (
360 µg
protein), corresponding to 0.8 x 106 cells, were
added to 100 µl of reaction mixture containing 72 mM
glycylglycine, 12% glycogen (wt/vol), 12 mM
Na2SO4, and 0.3 mM
UDP-[U-14C]glucose (0.1 µCi/tube), and were incubated
for 1–15 min at 20 C. For kinetics studies, the concentrations of
UDP-glucose ranged from 0.1–5 mM. The reaction was stopped
at 4 C by the addition of 200 µl 0.5 N KOH; and, after adding 35 µl
10% glycogen (wt/vol) as carrier, the total glycogen was extracted by
2 ml ethanol (66% final) during 15 min. The samples were centrifuged
at 600 x g for 5 min, and the precipitate was washed
once with 5 ml 66% ethanol and centrifuged at 3000 x
g during 30 min. The pellet dissolved in water was examined
for its radioactive content in a scintillation counter.
For total glycogen synthase activity, the reaction mixture included 7.2 mM glucose-6-phosphate, and Na2SO4 was omitted, as described (8). In some experiments, glucose-6-phosphate (0.1–10 mM) was also tested in the absence of Na2SO4.
For glycogen phosphorylase a activity (7), homogenate (100 µl) was mixed with 100 µl reaction mixture containing 200 mM NaF, 100 mM glucose-1-phosphate, 2% glycogen (wt/vol), and 1 mM caffeine, and was incubated 30 min at 30 C. The reaction was stopped at 4 C with 500 µl 10% trichloroacetic acid. After adding 3 ml H2O, the supernatant was separated by centrifugation at 2000 x g during 5 min, and its content in Pi was determined by colorimetry (9).
Presentation of results
Results are expressed as mean ± SEM, together
with the number of observations. The statistical significance of the
increments was assessed by Student’s t test. ANOVA was also
performed when appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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provides information on the
metabolic status and basal activity of glycogen synthase a
in the four groups of rats concerned in this study.
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and mentioned below refer to the
measurements made at the 15th min of the enzymatic assay, at which time
the reaction velocity had virtually reached its steady-state value
(Fig. 1).
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and 3, GLP-1 increased glycogen synthase
a activity in hepatocytes from either fed (P
< 0.001, F = 7.99, df = 188, by ANOVA) or fasted normal rats
(P < 0.001, F = 7.96, df = 166), and from
fasted STZ-N0 (P < 0.001, F = 20.75,
df = 130), STZ-N5 (P < 0.01, F =
4.04, df = 127), and STZ-adult (P < 0.001, F
= 10.97, df = 143) rats. In fed normal rats, insulin increased
glycogen synthase a activity (P < 0.001,
F = 5.95, df = 94) in a dose-related manner comparable with
that seen with GLP-1. Thus, the relative extent of glycogen synthase
a activation was at 10-9 M and at
10-7 M, respectively, similar in the case of
GLP-1 (22 ± 5%, n = 20; and 21 ± 3%, n = 21)
and insulin (20 ± 4%, n = 16; and 22 ± 3%, n =
15). The results illustrated in Figs. 2 and 3 refer to data obtained
after 5 and 10 min of exposure of the hepatocytes to the hormone,
because no significant difference was observed between the values
obtained at these two times of incubation. At 10-9
M concentration of GLP-1 or insulin, the activation of
synthase a was already observed after 1 min exposure of the
hepatocytes to the hormones, representing 106 ± 9% (n = 12)
of the paired value recorded after 5 min of stimulation by either GLP-1
or insulin.
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, P <
0.001, F = 22.89, df = 289). Indeed, at all hormone
concentrations, the mean relative extent of enzyme activation was lower
in fed rats than in overnight-fasted animals, the former value
averaging 63.3 ± 2.5% of the latter one (n = 6). This
difference could be related, in part at least, to the lower
(P < 0.05) basal value for glycogen synthase
a activity in fasted rats (261 ± 32 mU/g protein) than
in fed animals (334 ± 7 mU/g protein). Actually, there was no
significant difference between the absolute values for the
GLP-1-induced pair increment in reaction velocity found in fed and
fasted rats, except at 10-10 M concentration
of the hormone, in which case such an increment was higher
(P < 0.01) in fed rats (71.0 ± 0.2 mU/g, n
= 20) than in fasted animals (45.4 ± 0.1 mU/g, n = 17). The
concentration-dependency of the enzymatic response to GLP-1 was not
different, however, in fed and fasted rats. Thus, the increment in
reaction velocity above basal value at 10-12 M
GLP-1 represented about half of that recorded at 10-9
M GLP-1, the latter concentration causing a
close-to-maximal activation of glycogen synthase a. The
10-12 M/10-9 M ratio
for the hormone-induced increase in enzymatic activity indeed averaged
45.0 and 48.0%, in fasted and fed rats, respectively.
Likewise, the 10-12 M/10-9
M ratio between the increments in reaction velocity
attributable to GLP-1 was not significantly different in normal and
diabetics rats (Fig. 3), with an overall mean value of 49.2 ±
6.6%. By ANOVA, the relative increments of glycogen synthase
a activity, as compared with normal rats, were higher in
STZ-N0 (P < 0.01, F = 9.51, df =
246) and lower in STZ-adult rats (P < 0.05, F =
6.70, df = 259); and when compared with STZ-N0 rats,
the relative increments were lower in STZ-N5
(P < 0.001, F = 22.83, df = 247) and in
STZ-adult rats (P < 0.001, F = 49.82, df =
263).
GLP-1 exerted only minor effects upon glycogen phosphorylase
a activity, the mean decrement in reaction velocity not
exceeding about 10% at the highest concentrations of GLP-1
(10-8–10-7 M) tested in these
experiments (Figs. 2 and 3). By ANOVA, only the decrements observed in
STZ-N0 achieved significance (P < 0.001,
F = 6.18, df = 137).
Total activity of glycogen synthase
In hepatocytes from fasted normal rats incubated for 5–10 min
with 16.7 mM D-glucose,
D-glucose-6-phosphate (0.1–10.0 mM) caused a
concentration-related increase in glycogen synthase activity (Fig. 4) up to about 2.3 times the value
recorded at the lowest concentration of the ester.
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. Neither GLP-1 nor insulin (both 10-9 M) affected the total activity of glycogen synthase, as measured in the presence of 10 mM D-glucose-6-phosphate. For instance, in hepatocytes from fasted normal rats incubated for 1–10 min in the presence of 16.7 mM D-glucose, the total activity of glycogen synthase (after exposure to GLP-1 or insulin) averaged 104.5 ± 4.5% (n = 6) of the paired control value found after incubation in the absence of any hormone. A comparable situation prevailed in the diabetic animals.
Influence of D-glucose upon the hormonal response of
glycogen-enzymes
When hepatocytes from fasted normal rats were incubated for 5–10
min at increasing concentrations of D-glucose (0–16.7
mM), the basal activity of glycogen synthase a
progressively increased (Fig. 5
lower panel). The inhibitory effect of glucagon
(10-9 M) was most marked in glucose-deprived
hepatocytes (Fig. 5, upper panel). On the contrary, the
enhancing action of GLP-1 and insulin (both 10-9
M) on glycogen synthase a activity was only
observed in the presence of glucose (P < 0.01 or less,
for both hormones), and it progressively increased as the concentration
of D-glucose was raised to 8.3 and 16.7 mM. At
the highest concentration of D-glucose, 10-9
M GLP-1 exerted a higher increment of the enzyme than
10-9 M insulin (P < 0.01).
The r values, between mean enzymatic activities and hexose
concentration, amounted to 0.9984 (basal activity), 0.5418 (glucagon
response), 0.9969 (insulin response), and 0.9796 (GLP-1 response).
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lower panel).
The stimulatory effect of glucagon (10-9 M)
was most marked in glucose-deprived hepatocytes, and it progressively
decreased as the concentration of D-glucose was raised to
8.3 and 16.7 mM, with an r value of -0.9190 (Fig. 6, upper panel). On the contrary, an inhibitory action of GLP-1
and insulin (both 10-9 M) on glycogen
phosphorylase a activity was only observed at 16.7
mM D-glucose (P < 0.001, in
both cases).
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Kinetics of glycogen synthase a
All reaction velocities so far presented were measured in the
presence of 0.25 mM UDP-glucose. To better characterize the
changes in enzymatic activity caused by either diabetes or the anabolic
hormones, the kinetics of glycogen synthase a were examined
at increasing concentrations of UDP-glucose (0.1–5.0
mM). In hepatocytes from fasted normal rats, incubated for
5–10 min in the sole presence of 16.7 mM
D-glucose, the Michaelis-Menten constant (Km)
for UDP-glucose averaged 0.38 ± 0.12 mM and the
maximal velocity (Vmax), 0.46 ± 0.08 U/g (n = 4
in both cases). As illustrated in Figs. 7 and 8, both GLP-1 and insulin increased
not solely the Vmax but also the Km of the
enzyme, no significant difference being detected between these two
hormones. Pooling the results obtained with the two hormones, the
Vmax averaged 191.4 ± 22.5% (n = 8;
P < 0.001) of the paired basal value, whereas the
Km for UDP-glucose was increased by GLP-1 or insulin to
215.5 ± 35.8% (n = 8; P < 0.005) of the
paired basal measurement.
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). In the two types of
diabetic animals, both insulin and GLP-1 again increased the
Vmax, as well as the Km for UDP-glucose
(P < 0.025 or less). The relative magnitude of the
latter hormone-induced changes was less pronounced, however, in
diabetic rats than in normal animals (Fig. 8). Pooling together the
results obtained with either insulin or GLP-1 in each animal model of
diabetes, the hormone-induced increment in Vmax averaged
44.2 ± 8.3% (n = 14), as distinct (P <
0.025) from 91.4 ± 22.5 (n = 8) in normal rats; likewise,
the hormone-induced increase in Km averaged in the diabetic
rats 35.3 ± 11.3% (n = 14), as compared (P
< 0.02) with 115.5 ± 35.8% (n = 8) in normal animals. It
should be noted that even the basal values for Km were
somewhat lower, albeit not significantly so, in either
STZ-N0 rats (0.22 ± 0.04 mM; n = 4)
or STZ-adult rats (0.24 ± 0.07; n = 3) than in normal
animals (0.38 ± 0.12 mM; n = 4). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, the activation of glycogen synthase a reached a steady-state value within 1 min exposure of the hepatocytes to GLP-1, which, at a concentration as low as 10-12 M, caused half-maximal activation of the enzyme.
Second, when comparing fed vs. overnight-starved normal rats, a lower basal activity of glycogen synthase a in fasted animals coincided with a greater relative increment in reaction velocity in response to stimulation of the hepatocytes by GLP-1. These changes are well suited to adapt the rate of glycogen synthesis to the nutritional and, hence, hormonal environment in the fed and starved states.
Third, both the basal activity of glycogen synthase a and the relative extent of its inhibition by glucagon or activation by insulin and GLP-1 were modulated by the extracellular concentration of D-glucose. Once again, such a regulatory process was well suited to ensure an efficient metabolic response of the hepatocytes to changes in glycemia. Indeed, a rise in hexose concentration increased basal glycogen synthase a activity and rendered the enzyme less susceptible to inhibition by glucagon and more sensitive to activation by the anabolic hormones.
Fourth, the activation of glycogen synthase a by either insulin or GLP-1 resulted not solely in an increase in Vmax but also in a decreased affinity of the enzyme for UDP-glucose. The latter change may prevent inappropriately marked activation of glycogen synthase a in situations in which a high circulating level of the hormones would unexpectedly coincide with a low availability of UDP-glucose. Such could be the case, for instance, in insulin-induced hypoglycemia, occurring either in diabetic patients treated with the hormone and/or hypoglycemic sulfonylureas or in subjects harboring an insulinoma.
Last, in diabetic animals, the capacity of insulin or GLP-1 to increase the Vmax of the reaction catalyzed by glycogen synthase a and the hormone-induced change in its affinity for UDP-glucose were both less marked than in normal rats. Although the molecular determinant of the latter alterations remains to be identified, these alterations may represent a further example of diabetes-related modifications in the intrinsic properties of enzymes participating in the regulation of glucose metabolism in hepatocytes. Such alterations are indeed reminiscent of the perturbation of the intrinsic catalytic behavior of glucokinase previously documented in liver homogenates from diabetic animals (10, 11, 12).
In conclusion, this study indicates that the GLP-1-induced activation of glycogen synthase a displays attributes of rapidity, sensitivity, and nutritional dependency that are well suited for both participation in the physiological regulation of enzyme activity and therapeutic purpose.
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Acknowledgments |
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Footnotes |
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2 Research Fellow of the Fundación Conchita Rábago de
Jiménez Díaz.
Received November 19, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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