PACAP stimulates insulin secretion but inhibits insulin
sensitivity in mice
Karin
Filipsson1,
Giovanni
Pacini2,
Anton J. W.
Scheurink3, and
Bo
Ahrén1
1 Department of Medicine, Lund
University, SE-205 02 Malmö, Sweden;
2 Institute of Systems Science and
Biomedical Engineering (LADSEB-Consiglio Nazionale delle Ricerche),
I-35127 Padua, Italy; and
3 Department of Animal Physiology,
University of Groningen, 9750 Haren, The Netherlands
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ABSTRACT |
Although pituitary
adenylate cyclase-activating polypeptide (PACAP) stimulates insulin
secretion, its net influence on glucose homeostasis in vivo has not
been established. We therefore examined the action of PACAP-27 and
PACAP-38 on insulin secretion, insulin sensitivity, and glucose
disposal as derived from the minimal model of glucose disappearance
during an intravenous glucose tolerance test in anesthetized mice.
PACAP-27 and PACAP-38 markedly and equipotently potentiated
glucose-stimulated insulin secretion, with a half-maximal effect at 33 pmol/kg. After PACAP-27 or PACAP-38 (1.3 nmol/kg), the acute (1-5
min) insulin response was 3.8 ± 0.4 nmol/l (PACAP-27) and 3.3 ± 0.3 nmol/l (PACAP-38), respectively, vs. 1.4 ± 0.1 nmol/l after
glucose alone (P < 0.001), and the total area under the curve for insulin
(AUCinsulin) was potentiated by
60% (P < 0.001). In contrast,
PACAP-27 and PACAP-38 reduced the insulin sensitivity index
(SI) [0.23 ± 0.04 10
4
min
1/(pmol/l) for PACAP-27
and 0.29 ± 0.06 10
4
min
1/(pmol/l) for PACAP-38
vs. 0.46 ± 0.02 10
4
min
1/(pmol/l) for controls
(P < 0.01)].
Furthermore, PACAP-27 or PACAP-38 did not affect glucose elimination
determined as glucose half-time or the glucose elimination rate after
glucose injection or the area under the curve for glucose. Moreover,
glucose effectiveness and the global disposition index
(AUCinsulin times
SI) were not affected by
PACAP-27 or PACAP-38. Finally, when given together with glucose,
PACAP-27 did not alter plasma glucagon or norepinephrine levels but
significantly increased plasma epinephrine levels. We conclude that
PACAP, besides its marked stimulation of insulin secretion, also
inhibits insulin sensitivity in mice, the latter possibly explained by
increased epinephrine. This complex action explains why the peptide
does not enhance glucose disposal.
pituitary adenylate cyclase-activating polypeptide; glucose
effectiveness; minimal model
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INTRODUCTION |
THE NEUROPEPTIDE pituitary adenylate cyclase-activating
polypeptide (PACAP) was originally isolated from the ovine hypothalamus (26) and has later been shown to be expressed in several tissues throughout the body (6). The peptide exists in two forms: PACAP-38 and
PACAP-27, of which the latter is equivalent to
PACAP-38-(1
27) (27). In the pancreas, PACAP has been
localized to intrapancreatic nerve ganglia and to single nerves in the
exocrine parenchyma, around blood vessels, and in conjunction with
islets (2, 15), and one study has also indicated that PACAP occurs in
islet endocrine cells (34). Furthermore, PACAP receptors have been
demonstrated in the pancreas and in insulin-producing cells (2, 6, 17, 33). Moreover, PACAP has been demonstrated to stimulate insulin secretion in vivo in humans and dogs (13, 21), in vitro in the perfused
rat pancreas (10, 35), and in insulin-producing cells (2, 3, 17, 22,
34), mainly through activating adenylate cyclase (3, 22). Hence, PACAP
is a pancreatic neuropeptide with a stimulatory action on insulin
secretion. However, the potential involvement of PACAP in glucose
homeostasis is not established, since the peptide has also been
demonstrated to stimulate the secretion of glucagon (10, 13, 15, 29,
35) and catecholamines (6, 21) as well as to induce glycogenolysis (29,
36), the net effect being hyperglycemia. Also, both the stimulation of
glucagon and catecholamines and the hyperglycemia induced through stimulation of glycogenolysis might indirectly influence insulin secretion and the glucose disposal rate. Therefore, the integrated influence of PACAP on the glucose homeostasis might be complex.
The aim of the present study was to examine in more detail the in vivo
influence of PACAP on insulin secretion, insulin sensitivity, and
glucose disposal after an intravenous glucose challenge in anesthetized
mice. To that end, we exploited the minimal model of glucose
disappearance for glucose and insulin data analysis after an
intravenous glucose tolerance test (8, 14) with seven samples over 50 min in mice. This method allowed estimation of acute insulin secretion,
total insulin secretion, insulin sensitivity (the ability of insulin to
enhance glucose disappearance and to inhibit glucose production),
glucose effectiveness (glucose disappearance per se at basal insulin
without any change in insulin), and the glucose elimination rate. We
also examined the influence of PACAP on glucagon secretion and on
plasma levels of norepinephrine and epinephrine, and, finally, we
compared the influences of PACAP-27 with those of PACAP-38.
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METHODS |
Animals. Nonfasted NMRI mice
(Bomholdtgaard Breeding and Research Center, Ry, Denmark), weighing
20-25 g, were used throughout the study. The animals were fed a
standard pellet diet and tap water ad libitum.
Intravenous glucose tolerance test.
The mice were anesthetized with an intraperitoneal injection of
midazolam (Dormicum, 0.4 mg/mouse; Hoffmann-La Roche, Basel,
Switzerland) and a combination of fluanison (0.9 mg/mouse) and fentanyl
(0.02 mg/mouse; Hypnorm; Janssen, Beerse, Belgium). Thereafter, a blood
sample was taken from the retrobulbar, intraorbital, capillary plexus
in heparinized tubes, whereafter D-glucose (1 g/kg; British Drug Houses, Poole, UK) was injected rapidly
intravenously either alone or together with synthetic ovine PACAP-27 or
synthetic ovine PACAP-38 at various dose levels (both peptides from
Peninsula Europe Laboratories, Merseyside, UK). In two other
experimental series, human insulin (Actrapid; Novo Nordisk, Bagsvaerd,
Denmark) was injected intravenously (0.25 U/kg) together with glucose,
or PACAP-27 (1.3 nmol/kg) was injected intravenously under baseline
conditions (i.e., without any concomitant glucose injection; controls
were given saline). The volume load was 10 µl/g body wt. New blood
samples were taken after 1, 5, 10, 20, 30, and 50 min. In the
experiments on circulating glucagon and catecholamines, synthetic ovine
PACAP-27 was injected intravenously at a dose level of 1.3 nmol/kg
alone or together with glucose (1 g/kg), and blood was sampled
immediately before and at 1, 5, 20, and 50 min after the injection. The
samples for glucose and insulin were taken in heparinized tubes, the
samples for glucagon were taken in chilled tubes containing aprotinin, and the samples for catecholamines were taken in chilled tubes containing heparin and EDTA. After immediate centrifugation at 4°C,
plasma was separated and stored at
20°C or
80°C until
analysis.
Analysis. Plasma insulin was
determined radioimmunochemically with the use of a guinea pig anti-rat
insulin antibody, 125I-labeled
porcine insulin as tracer, and rat insulin as standard (Linco Research,
St. Charles, MO). Free and bound radioactivity was separated by use of
an anti-immunoglobulin G (IgG) (goat anti-guinea pig) antibody (Linco).
The sensitivity of the assay is 12 pmol/l, and the coefficiency of
variation is <3% at both low and high levels. Plasma glucose was
determined with the glucose oxidase method. Plasma glucagon was
determined radioimmunochemically with the use of guinea pig
antiglucagon antibodies specific for pancreatic glucagon,
125I-labeled glucagon as tracer,
and glucagon standard (Linco). Free and bound radioactivity was
separated by use of an anti-IgG (goat anti-guinea pig) antibody
(Linco). The sensitivity of the assay is 7.5 pg/ml, and the
coefficiency of variation is <9% at both low and high levels. Plasma
catecholamines were determined by liquid chromatography in combination
with electrochemical detection, as previously described (28).
Data analysis. Insulin and glucose
data from the seven-sample intravenous glucose tolerance test were
analyzed with the minimal model technique (9). The model assumes a
first-order nonlinear insulin controlled kinetic and accounts for the
effect of insulin and glucose itself on glucose disappearance after
exogenous glucose injection. It provides the parameter
SI (insulin sensitivity index), which is defined as the ability of insulin to enhance glucose disappearance and inhibit glucose production (8), and the parameter SG, which is the glucose
effectiveness, representing glucose disappearance per se from plasma
without any change in dynamic insulin (1). The parameter
SG is composed of a glucose
disposal action of basal insulin, the parameter BIE (basal insulin
effect), and the parameter GEZI (glucose effectiveness at zero
insulin), which is the glucose disposal without any influence of
insulin (19). Acute insulin secretion (AIR) was calculated as the mean
of suprabasal 1- and 5-min insulin levels, and the areas under the
curve for insulin (AUCinsulin)
and glucose (AUCglucose) were
assessed using the trapezoidal rule of suprabasal values. Finally, we
also calculated a unitless index called GDI (global disposition index)
by multiplying SI times
AUCinsulin. The glucose
elimination rate after the glucose injection
(KG) was calculated using the
half-time
(t1/2) for minutes 1-20 after glucose
injection after logarithmic transformation of the individual plasma
glucose values. In the experiment on basal levels,
AUCinsulin, the AUC for glucagon
(AUCglucagon), and
AUCglucose were calculated using
the trapezoid rule of data with basal levels subtracted.
Statistics. Means ± SE are shown.
Statistical analyses were performed with the SPSS for Windows system.
Analysis of the normal distribution was performed with the
Kolmogorov-Smirnov goodness-of-fit test. Statistical comparisons
between groups were performed with the nonparametric Mann-Whitney
U-test, since several of the
parameters did not display normal distribution. Pearson's product
moment correlation was used to estimate linear relationships between normally distributed variables. The coefficient of variation of estimated parameters was given by the percent ratio between the SD and
the absolute values. The SD was obtained by the square root of the main
diagonal of the covariance matrix calculated during the least square
estimation procedure (30).
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RESULTS |
Circulating insulin and glucose after intravenous
glucose or saline. In the control mice not given PACAP,
the rapid intravenous injection of glucose (1 g/kg) raised plasma
insulin levels from 438 ± 32 to 2,088 ± 191 pmol/l after 1 min
(n = 54;
P < 0.001; Fig.
1). Thereafter, plasma insulin levels
rapidly returned toward baseline values. The plasma insulin level
decline exhibited two phases, with a first phase during the first 10 min (t1/2 4.9 ± 0.5 min) and a second phase after the first 10 min
(t1/2 24.7 ± 2.7 min). Also, plasma glucose levels peaked at 1 min after the
intravenous glucose injection (28.5 ± 0.7 vs. 9.6 ± 0.3 mmol/l at baseline, P < 0.001),
whereafter plasma glucose levels declined with an elimination half-life
of suprabasal glucose of 9.7 ± 0.8 min for the first 10 min after
glucose injection (Fig. 1). The half-life of circulating glucose
correlated significantly and inversely to AIR
(r =
0.30,
P = 0.024) but not to the
AUCinsulin [r =
0.06, not
significant (NS)]. However,
AUCinsulin correlated significantly to the plasma glucose level at 50 min after glucose injection (r =
0.41,
P < 0.001). The glucose elimination
rate determined as the KG value
was 4.2 ± 0.1%/min. The KG
value correlated to AIR (r = 0.48, P < 0.001) but not to
AUCinsulin
(r = 0.001, NS). Figure 1 also shows
that the single injection of saline did not significantly increase
circulating insulin or glucose during the 50-min study period.

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Fig. 1.
Plasma insulin and glucose immediately before and at 1, 5, 10, 20, 30, and 50 min after iv injection of glucose (1 g/kg,
n = 54) or saline
(n = 14) in anesthetized mice. Means ± SE are shown.
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Effects of PACAP-27 and PACAP-38 on glucose-stimulated
insulin secretion. When PACAP-27 or PACAP-38 was
administered intravenously at various dose levels together with glucose
at 1 g/kg, it was found that the two peptides equipotently and
equiefficiently potentiated glucose-stimulated insulin secretion (Fig.
2). The half-maximal response for the two
peptides was obtained at a dose level of ~33 pmol/kg. At the
maximally effective dose for both peptides, 130 pmol/kg, insulin
secretion, as determined by the
AUCinsulin, was approximately
twofold potentiated (P < 0.001).

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Fig. 2.
Area under the curve for suprabasal plasma insulin levels during 50 min
[on y-axis, shown as area under
the curve for insulin
(AUCinsulin)] after iv
administration of pituitary adenylate cyclase-activating polypeptide
(PACAP)-27 or PACAP-38 at various dose levels together with glucose (1 g/kg) expressed as percent mean of the respective control animals run
in the same experimental series. Groups without PACAP consisted of 24 animals each, group with PACAP-27 at 1.3 nmol/kg consisted of 16 animals, and group with PACAP-38 at 1.3 nmol/kg consisted of 24 animals, whereas the other groups consisted of 6-12 animals each.
Means ± SE are shown.
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Effects of PACAP-27 and PACAP-38 on glucose
disappearance. Both PACAP-27 and PACAP-38 at 1.3 nmol/kg potentiated the glucose-induced increase in plasma insulin
levels (Fig. 3). Thus plasma insulin levels
in the 1-min sample were increased more than twofold by either of the
peptides (P < 0.001). However, in
spite of the marked increase in plasma insulin after administration of
PACAP together with glucose, glucose elimination rate was not altered,
as is evident from Fig. 3, bottom,
showing an almost identical reduction in circulating glucose after the
1-min peak. This is also evident from the calculation of the
KG value, which was 4.2 ± 0.1%/min in controls vs. 3.9 ± 0.2%/min after glucose plus
PACAP-27 and 4.3 ± 0.2%/min after glucose plus PACAP-38 (NS).

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Fig. 3.
Plasma insulin and glucose immediately before and at 1, 5, 10, 20, 30, and 50 min after iv injection of glucose (1 g/kg) alone or together
with either PACAP-27 or PACAP-38 at 1.3 nmol/kg in anesthetized mice.
Means ± SE are shown. There were 16 mice in each of the two
experimental groups with glucose ± PACAP-27 and 24 mice in each of
the two experimental groups with glucose ± PACAP-38.
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The data for circulating insulin and glucose were used for the
estimation of the parameters of the minimal model. Table
1 shows the mean and SE for all control mice injected
with glucose alone (n = 54). In the
Kolmogorov-Smirnov goodness-of-fit test, baseline insulin,
SI, BIE, and GDI did not show a
normal distribution (P < 0.05),
whereas the other parameters did (P > 0.05). The accuracy of parameter estimation was evaluated with the
coefficients of variation of the estimates, the values of which for the
single groups of experiments are reported in Table 2.
Table 3 shows the parameters estimated from the
minimal model injected with PACAP-27 and PACAP-38. It is seen that
AUCinsulin and AIR were both
significantly increased by PACAP-27 and PACAP-38, whereas AUCglucose was not altered by
either of the two peptides. Furthermore, SI was reduced both by PACAP-27
and PACAP-38, whereas SG was not altered significantly. However, the two components of
SG were both significantly
altered, since both PACAP-27 and PACAP-38 increased GEZI but inhibited
BIE. The unitless GDI, finally, was not significantly affected by
PACAP-27 or PACAP-38, since the reduction in
SI and the increase in
AUCinsulin compensated each
other.
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Table 1.
Baseline, nonfasted levels of glucose and insulin and parameters
calculated from the seven-sample minimal model in control mice
intravenously injected with glucose
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Table 3.
Baseline, nonfasted levels of glucose and insulin and parameters
calculated from the seven-sample minimal model in mice intravenously
injected with glucose (1 g/kg) either alone or
together with PACAP-27 (1.3 nmol/kg) or PACAP-38 (1.3 nmol/kg)
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Effects of intravenous insulin on circulating insulin
and glucose. To examine whether the increase in plasma
insulin after injection of PACAP-27 or PACAP-38, albeit marked, was
insufficient to increase glucose elimination in mice, a separate
experiment was undertaken in which insulin was injected intravenously
(0.25 U/kg) together with glucose (1 g/kg). Figure 4
shows that this insulin injection increased plasma insulin to 4,783 ± 416 pmol/l, i.e., by the same degree as injection of PACAP-27 or
PACAP-38 at 1.3 nmol/kg (cf. Fig. 3). However, in contrast to the
failure of PACAP-27 or PACAP-38 to augment glucose elimination, insulin clearly increased glucose elimination. Thus the 5-min plasma glucose values were 20.4 ± 0.4 mmol/l in the absence of insulin injection vs. 17.3 ± 0.6 mmol/l in the presence of insulin injection
(P = 0.002), and the corresponding
20-min values were 12.3 ± 0.4 vs. 7.1 ± 0.6 mmol/l
(P < 0.001). Furthermore, insulin
increased the KG value from 3.9 ± 0.2%/min in controls to 6.1 ± 0.2%/min (P = 0.030). The calculated half-life
of circulating insulin for the first 5 min after the insulin injection
was 2.5 ± 0.1 min, which is lower than that previously reported in
humans and pigs (25, 32).

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Fig. 4.
Plasma insulin and glucose immediately before and at 1, 5, 20, and 50 min after iv injection of glucose (1 g/kg) alone or together with human
insulin (0.25 U/kg) in anesthetized mice. Means ± SE are shown.
There were 6 mice in each group.
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Effects of PACAP-27 on glucagon
levels. When PACAP-27 was injected intravenously under
baseline conditions (1.3 nmol/kg), plasma glucagon levels were
increased concomitantly with an increase in insulin and glucose levels
(Fig. 5). Thus the
AUCglucagon during the 50 min was
981 ± 86 pg/ml in 50 min after injection of PACAP-27 vs. 77 ± 8 pg/ml in 50 min after administration of saline
(P < 0.001). Similarly, the
AUCinsulin was 4.9 ± 0.5 nmol/l in 50 min after PACAP-27 vs.
1.0 ± 0.2 nmol/l in 50 min in controls (P < 0.001), and the AUCglucose was
13 ± 2 mmol/l in 50 min after PACAP-27 vs.
79 ± 7 mmol/l in 50 min in controls (P < 0.001). In contrast, when PACAP was injected together with
glucose, no significant change in glucagon levels was evident when
compared with animals injected with glucose alone (Fig.
6).

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Fig. 5.
Plasma insulin, glucagon, and glucose immediately before and at 1, 5, 20, and 50 min after iv injection of saline or PACAP-27 (1.3 nmol/kg)
in anesthetized mice. Means ± SE are shown. There were 8 mice in
each group.
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Fig. 6.
Plasma insulin, glucagon, and glucose immediately before and at 1, 5, 20, and 50 min after iv injection of glucose (1 g/kg) without or with
addition of PACAP-27 (1.3 nmol/kg) in anesthetized mice. Means ± SE
are shown. There were 6 mice in each group.
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Effects of PACAP-27 on catecholamine
levels. When PACAP-27 was injected intravenously (1.3 nmol/kg) together with glucose, plasma epinephrine levels were
significantly elevated at both 1 min
(P = 0.004) and 5 min
(P = 0.013) after injection when
compared with animals injected with glucose alone (Fig.
7, top). The change in plasma epinephrine at 1 min after injection was 0.87 ± 0.34 ng/ml in animals injected with PACAP-27 plus glucose vs.
0.39 ± 0.22 ng/ml in animals injected with glucose
alone (P = 0.039). In
contrast, plasma norepinephrine was not significantly altered in any of
the two groups of animals after the injection (Fig. 7,
bottom).

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Fig. 7.
Plasma epinephrine and norepinephrine immediately before and at 1, 5, 20, and 50 min after iv injection of glucose (1 g/kg) with addition of
PACAP-27 (1.3 nmol/kg) in anesthetized mice. Means ± SE are shown.
There were 5 mice in each group.
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DISCUSSION |
We have examined the net influences of PACAP-27 and PACAP-38 on insulin
secretion, insulin sensitivity, and glucose disposal in normal mice to
study the integrated action of the pancreatic insulinotropic
neuropeptide PACAP on the glucose homeostasis. The net action of PACAP
is of interest not only physiologically but also since it may be
suggested that agents which increase the formation of cAMP may be of
potential interest in the treatment of diabetes. For example,
glucagon-like peptide 1, which raises cAMP, has been considered as a
new regimen in the treatment of type 2 diabetes (16). Our present
results confirm previous studies in dogs and humans that PACAP
stimulates insulin secretion, which also confirms several previous
studies in vitro verifying that PACAP exerts its action directly on the
insulin-producing cells (2, 3, 10, 13, 17, 21, 22, 34, 35). We also show that the insulinotropic action of PACAP-27 and PACAP-38 is equipotent and equiefficient, which confirms previous results in other
models (2, 21, 34, 35).
In our previous study on the influence of PACAP-38 on insulin secretion
in unanesthetized mice, PACAP-38 did not increase plasma insulin,
either under baseline conditions or in conjunction with glucose, and in
combination with the cholinergic agonist carbachol, PACAP-38 actually
inhibited insulin secretion (15). In contrast, in the present study,
PACAP potently increased plasma insulin levels both under basal
conditions and in the presence of glucose. Although a potentially
diminished insulin elimination by PACAP has not been studied, such an
effect is unlikely to explain the rapid and marked increase in plasma
insulin after PACAP administration, since the magnitude of the increase
far exceeds what is possible, even with a complete inhibition of
insulin elimination. Therefore, a stimulated insulin secretion most
likely explains the marked increase in plasma insulin executed by
PACAP. The mechanism of this potent insulinotropic action of PACAP
cannot be established from the results of this in vivo study. It could
theoretically be due to indirect actions of PACAP through the
liberation of other insulinotropic agents by the neuropeptide. It could
also be due to a compensatory response to a primary reduction in
insulin sensitivity by PACAP. However, the most likely mechanism,
considering the potent insulinotropic action of PACAP on isolated
islets and insulin-producing tumor cells (2, 3, 10, 17, 22, 34, 35), is
a direct action on the islets. Furthermore, PACAP-38 also increased
circulating glucagon, which confirms our previous study in
unanesthetized mice (15). This is most likely executed through
stimulation of glucagon secretion from the pancreatic islets. In our
previous study in unanesthetized mice, glucose could not inhibit the
glucagon response to PACAP-38 (15), which is different from the
abolishment by glucose of PACAP-induced glucagon secretion in the
present study in anesthetized mice. It is likely that stress-related
phenomena in unanesthetized mice also account for this difference.
Hence, the present model in anesthetized animals seems to more reliably
show the direct action of PACAP on islet function, when stress-related
actions are circumvented. It should also be emphasized that our present
result of potentiated glucose-stimulated insulin secretion and
increased baseline glucagon secretion in response to PACAP in mice is
identical to the results of our recent report on the action of
PACAP-27 in humans (13).
The main new finding in this study is that, in spite of a marked
insulinotropic action of PACAP, the glucose disposal was not augmented.
This was evident by calculating the area under the curve for plasma
glucose levels during the entire 50-min test as well as the glucose
elimination rate for the 20 min after glucose administration and the
t1/2 of
glucose after glucose administration. We interpret this finding to be
due to the marked (by ~50%) inhibition of insulin sensitivity
induced by PACAP, as evident from the minimal model data of the insulin
sensitivity index. The potentiated insulin secretion and the inhibited
insulin sensitivity thus together compensated each other, the
consequence of which was the unaltered glucose disposal. Consequently,
also the global disposition index, which quantifies the capacity to
compensate a change in insulin secretion or insulin sensitivity with a
comparable inverse change in the other parameter, was not significantly
altered by PACAP.
The reason for the marked reduction in insulin sensitivity by PACAP is
at present not clear. Several hypothetical explanations might be
offered, however. One hypothesis is that the rapid and marked increase
in plasma insulin had compensatorily reduced insulin sensitivity and
thereby prevented its own action to enhance glucose elimination. This
in turn would suggest that the reduction in glucose levels during an
intravenous glucose tolerance test is largely independent of insulin.
Previous reports in rats have indeed indicated this to be the case.
Thus at 3 h after prolonged hyperglycemia in rats, the glucose
elimination after an intravenous glucose challenge has been found to be
unaltered in spite of an exaggerated insulin response (23), and,
furthermore, cholinergic antagonism by atropine has been found to
markedly inhibit glucose-stimulated insulin secretion in rats
preinfused for 48 h with glucose without altering the glucose disposal
rate (7). To examine this intriguing possibility, we administered
insulin intravenously at the dose of 0.25 U/kg together with glucose.
This yielded a circulating insulin level of approximately the same
degree as that seen after PACAP plus glucose. However, we found that
insulin under this condition clearly augmented glucose elimination.
This therefore shows that a rapid reduction in insulin sensitivity
simply due to the rapidly increasing circulating levels of insulin is
not the explanation for the failure of PACAP to augment glucose
disposal. Another hypothesis, which more likely would explain the
reduced insulin sensitivity by PACAP, is that other
insulin-antagonistic actions counteracting the insulin effect have
evolved after PACAP administration. Such insulin-antagonistic actions
could be either PACAP itself, if PACAP directly reduces or counteracts
the action of insulin. It could also, however, be due to other factors,
the secretion of which would be stimulated by PACAP and which in vivo could prevent the action of insulin. An antagonistic action of PACAP
itself on glucose elimination would be supported by data that mice
injected with PACAP had elevated glucose levels when compared with mice
injected with saline (cf. Fig. 5) and by previous reports that PACAP
stimulates the hepatic glucose delivery (29, 36). However, although
this is a possibility under baseline conditions, it is unlikely to be a
mechanism after a glucose challenge, since glucose inhibits liver
glucose delivery (11). The other possible factors that could counteract
the glucose reduction after PACAP administration are glucagon and
catecholamines. Thus previous reports have demonstrated that PACAP
stimulates glucagon secretion from the pancreas (10, 13, 15, 21, 29,
35) and epinephrine secretion from the adrenals (6, 21). To test these
possibilities, we also determined plasma glucagon and catecholamines
after administration of PACAP-27 to mice. We found that, although
PACAP, as expected from results of previous studies (10, 13, 15, 21,
29, 35), increased baseline glucagon levels, the peptide did not increase plasma glucagon in the presence of elevated glucose levels, since plasma glucagon levels after the combined injection of PACAP-27 and glucose were not significantly different from those after injection
of glucose alone. This is interpreted to indicate that glucose
inhibited PACAP-induced glucagon secretion. Therefore, the mechanism of
the impaired insulin sensitivity by PACAP under in vivo conditions in
mice is probably not explained by glucagon. In contrast, plasma
epinephrine levels, but not norepinephrine levels, were elevated after
the combined injection of PACAP-27 and glucose, which might support a
role for epinephrine. Although not directly examined in this study,
such an action of epinephrine could be executed through activation of
the
-adrenoceptors and subsequent formation of cAMP, which is a
well-known mechanism whereby catecholamines induce insulin resistance
(24). However, the mechanism of this action remains to be fully
established.
To analyze the data, we have exploited the minimal model approach to
experiments in mice. The frequently sampled intravenous glucose
tolerance test with minimal model analysis was originally developed to
quantitate glucose disposal in dogs and humans after a single
intravenous glucose injection (8, 14). The method provides parameters
characterizing insulin and glucose action on glucose homeostasis (8).
This model has provided a useful and powerful tool for the
understanding of glucose disposal under a variety of conditions, such
as impaired glucose tolerance and type 2 diabetes, and has also been
used in pharmacological studies to examine influences of exogenously
administered substances, such as somatostatin and glucagon-like peptide
1 (5, 8, 9, 12, 14, 19). To the best of our knowledge, the minimal model has been used in small animals (rats) only a few times (14). No
particular constraint should exist that restrains the use of this
technique also in mice, being the only arguments about the length of
the experiment and the limited number of samples (31). The model does
not, however, seem to have problems related to the length of the study
period, since, by the end of the experiments, both insulin and glucose
have returned to preinjection levels, and thus the entire dynamics of
the test are taken into account. In fact, the low dispersion of the
estimates along with the coefficients of variation demonstrated a
precise and accurate assessment of insulin secretion, insulin
sensitivity, and glucose disposal in mice. This seven-sample technique
therefore offers promises to be of importance in future physiological
and pharmacological studies in mice, as well as in phenotypic
characterization of animal models of interest for glucose homeostasis
and diabetes, although this technique needs to be validated against
other methods for determination of insulin secretion and insulin
sensitivity in mice.
We have previously shown in unanesthetized mice that an intravenous
injection of glucose elicits a marked and rapid increase in circulating
insulin with a peak level within the first 2 min and a return to
baseline levels within 6 min (4). The elevation of circulating insulin
was more sustained in the anesthetized animals, which is interpreted as
a lower degree of stress exhibited in these animals, reducing increase
in catecholamines with their inhibiting action on insulin secretion.
The seven-sample technique in anesthetized mice therefore does not seem
to be accompanied by any significant degree of stress, which also is
supported by our finding that there was no increase in basal glucose in
the saline-injected controls.
Glucose elimination is not governed only by insulin-dependent actions.
Thus insulin-independent mechanisms also contribute substantially to
the glucose disposal after glucose administration, mainly by
insulin-independent glucose uptake in the brain and in skeletal muscle
and by suppression of liver glucose output during hyperglycemia (1,
11). The glucose effectiveness calculated by the minimal model
quantifies both of these processes (11), and it has previously been
found that glucose effectiveness accounts for >20% of the variance
in glucose elimination in healthy subjects (20). This is the first time
the minimal model is applied to mice; therefore, there is no previous
history that allows comparisons to deeply evaluate the role of glucose
effectiveness. Although it is generally agreed that the minimal model
yields reliable measurements of insulin sensitivity, it is still the
subject of controversy how to interpret glucose effectiveness in terms
of true physiological implications and limitations (11). Nonetheless, there is a general consensus on its important role in the comprehension of the mechanisms involved in glucose disappearance. In the present study, we found that the glucose effectiveness was not significantly affected by PACAP. Therefore, the global glucose effectiveness is not
compensatorily increased when insulin sensitivity is reduced by PACAP,
which underlines earlier observations that insulin sensitivity and
glucose effectiveness can change independently of each other (18). The
global glucose effectiveness is composed of glucose effectiveness at
basal insulin and at zero insulin. Interestingly, PACAP had significant
effects on both of these parameters. Thus the peptide inhibited glucose
effectiveness at basal insulin, which is a reflection of its inhibitory
influence on insulin sensitivity. In contrast, PACAP increased glucose
effectiveness at zero insulin. This latter effect is similar to
glucagon-like peptide 1 in a previous study in humans (12). However, in
contrast to PACAP, glucagon-like peptide 1 had no influence on glucose
effectiveness at basal insulin and therefore increased global glucose
effectiveness (12). The mechanism of an increase in glucose
effectiveness at zero insulin by PACAP might reside in stimulation of
glucose uptake in tissues like the skeletal muscle or inhibition of
liver glucose production. The molecular mechanism of action by PACAP or
the site of the action cannot be determined by the minimal model,
however, and remains therefore to be established directly. In addition,
it is worth pointing out that, despite being reported in many studies,
the basal insulin effect and glucose effectiveness at zero insulin are
still the subject of debate concerning their true physiological
meaning. For this reason, our conclusions in this respect must be
interpreted as study hypotheses, since it is not possible to date to
draw unquestionable conclusions from the analysis of the components of
glucose effectiveness.
Based on this integrative study on the development of a seven-sample
technique in mice and the influence of PACAP on insulin secretion,
insulin sensitivity, and glucose disposal, we conclude that
1) PACAP stimulates insulin
secretion in a concentration-dependent manner,
2) the insulinotropic actions of
PACAP-27 and PACAP-38 are equipotent,
3) PACAP inhibits insulin
sensitivity without altering the glucose effectiveness,
4) PACAP stimulates glucagon secretion under basal conditions but not after administration of
glucose, 5) PACAP-27 increases
circulating levels of epinephrine but not of norepinephrine,
6) the net effect of PACAP during an intravenous glucose tolerance test is an unaltered glucose disposal in
spite of a marked insulinotropic action, and
7) the minimal model of the
seven-sample technique is a reliable model in anesthetized mice. The
study therefore is consistent with and supports the view that PACAP is
involved in the regulation of insulin secretion but that additional
complex actions are exerted on the net glucose homeostasis. In
contrast, PACAP does not seem to be a good target as a new reagent in
the treatment of diabetes. However, B cell- specific PACAP receptor
agonists, devoid of peripheral influences counteracting the action of
insulin, might offer a good rational for such development, because
PACAP seems to be an extraordinary potent insulinotropic peptide.
 |
ACKNOWLEDGEMENTS |
We are grateful to Lena Kvist for active participation in the
execution of the experiments and to Lilian Bengtsson and Ulrika Gustavsson for expert technical assistance in the determinations of
insulin and glucose.
 |
FOOTNOTES |
The study was supported by the Swedish Medical Research Council (Grant
14X-6834), Ernhold Lundström, Albert Påhlsson, and Novo
Nordisk Foundations, Swedish Diabetes Association, Malmö University Hospital, and the Faculty of Medicine, Lund University.
Address for reprint requests: B. Ahrén, Dept. of Medicine,
Malmö Univ. Hospital, S-205 02 Malmö, Sweden.
Received 14 October 1997; accepted in final form 20 January
1998.
 |
REFERENCES |
1.
Ader, M.,
G. Pacini,
Y. J. Yang,
and
R. N. Bergman.
Importance of glucose per se to intravenous glucose tolerance: comparison of the minimal-model prediction with direct measurements.
Diabetes
34:
1092-1103,
1985[Abstract].
2.
Af Klinteberg, K., A. N. Al-Amin, J. Hannibal, F. Sundler, and B. Ahrén. Effects and localization of the
neuropeptide PACAP and PACAP receptor expression in insulin producing
tissues (Abstract). Diabetologia 39, Suppl. 1: A71, 1996.
3.
Af Klinteberg, K.,
S. Karlsson,
and
B. Ahrén.
Signaling mechanisms underlying the insulinotropic effect of pituitary adenylate cyclase-activating polypeptide in HIT-T15 cells.
Endocrinology
137:
2791-2798,
1996[Abstract].
4.
Ahrén, B.,
and
I. Lundquist.
Effects of selective and nonselective
-adrenergic agents on insulin secretion in vivo.
Eur. J. Pharmacol.
71:
93-104,
1981[Medline].
5.
Ahrén, B.,
and
G. Pacini.
Impaired adaptation of first phase insulin secretion in postmenopausal women with glucose intolerance.
Am. J. Physiol.
273 (Endocrinol. Metab. 36):
E701-E707,
1997[Abstract/Free Full Text].
6.
Arimura, A.,
and
S. Shioda.
Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors: neuroendocrine and endocrine interaction.
Front. Neuroendocrinol.
16:
53-88,
1995[Medline].
7.
Balkan, B.,
and
B. E. Dunning.
Muscarinic stimulation maintains in vivo insulin secretion in response to glucose after prolonged hyperglycemia.
Am. J. Physiol.
268 (Regulatory Integrative Comp. Physiol. 37):
R475-R479,
1995[Abstract/Free Full Text].
8.
Bergman, R. N.
Toward physiological understanding of glucose tolerance. Minimal-model approach.
Diabetes
38:
1512-1527,
1989[Abstract].
9.
Bergman, R. N.,
L. S. Phillips,
and
C. Cobelli.
Physiologic evaluation of factors controlling glucose tolerance in man.
J. Clin. Invest.
68:
1456-1467,
1981[Medline].
10.
Bertrand, G.,
R. Puech,
Y. Maissonnasse,
J. Bockaert,
and
M. M. Loubatières-Mariani.
Comparative effects of PACAP and VIP on pancreatic endocrine secretions and vascular resistance in rats.
Br. J. Pharmacol.
117:
764-770,
1996[Abstract].
11.
Best, J. D.,
S. E. Kahn,
M. Ader,
R. M. Watanabe,
T.-C. Ni,
and
R. N. Bergman.
Role of glucose effectiveness in the determination of glucose tolerance.
Diabetes Care
19:
1018-1030,
1996[Medline].
12.
D,'Alessio, D. A.,
S. E. Kahn,
C. R. Leusner,
and
J. W. Ensinck.
Glucagon-like peptide 1 enhances glucose tolerance by stimulation of insulin release and by increasing insulin-independent glucose disposal.
J. Clin. Invest.
93:
2263-2266,
1994[Medline].
13.
Filipsson, K.,
K. Tornoe,
J. Holst,
and
B. Ahrén.
Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates insulin and glucagon secretion in humans.
J. Clin. Endocrinol. Metab.
82:
3093-3098,
1997[Abstract/Free Full Text].
14.
Finegood, D. T.
Application of the minimal model of glucose kinetics.
In: The Minimal Model Approach and Determinants of Glucose Tolerance, edited by R. N. Bergman,
and J. C. Lovejoy. Baton Rouge, LA: Louisiana State Univ. Press, 1997, p. 51-122.
15.
Fridolf, T.,
F. Sundler,
and
B. Ahrén.
Pituitary adenylate cyclase activating polypeptide (PACAP): occurrence in rodent pancreas and effects on insulin and glucagon secretion in the mouse.
Cell Tissue Res.
269:
275-279,
1992[Medline].
16.
Gutniak, M. K.,
H. Larsson,
S. J. Heiber,
O. T. Juneskans,
J. J. Holst,
and
B. Ahrén.
Potential therapeutic levels of glucagon-like peptide-1 achieved in humans by a buccal tablet.
Diabetes Care
19:
843-848,
1996[Abstract].
17.
Inagaki, N.,
H. Yoshida,
M. Mizuta,
N. Mizuno,
Y. Fujii,
T. Gonoi,
J. Miyazaki,
and
S. Seino.
Cloning and functional characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells.
Proc. Natl. Acad. Sci. USA
91:
2679-2683,
1994[Abstract].
18.
Kahn, S. E.,
R. N. Bergman,
M. W. Schwartz,
G. J. Taborsky, Jr.,
and
D. Porte, Jr.
Short-term hyperglycemia and hyperinsulinemia improve insulin action but do not alter glucose action in normal humans.
Am. J. Physiol.
262 (Endocrinol. Metab. 25):
E518-R523,
1992[Abstract/Free Full Text].
19.
Kahn, S. E.,
L. J. Klaff,
M. W. Schwartz,
J. C. Beard,
R. N. Bergman,
G. J. Taborsky, Jr.,
and
D. Porte, Jr.
Treatment with a somatostatin analog decreases pancreatic B-cell and whole body sensitivity to glucose.
J. Clin. Endocrinol. Metab.
71:
994-1002,
1990[Abstract].
20.
Kahn, S. E.,
R. L. Prigeon,
D. K. McCulloch,
E. J. Boyko,
R. N. Bergman,
M. W. Schwartz,
J. L. Neifing,
W. K. Ward,
J. C. Beard,
J. P. Palmer,
and
D. Porte, Jr.
The contribution of insulin independent glucose uptake to intravenous glucose tolerance in healthy human subjects.
Diabetes
43:
587-592,
1993[Abstract].
21.
Kawai, K.,
C. Yokota,
S. Ohashi,
K. Isobe,
S. Suzuki,
T. Nakai,
and
K. Yamashita.
Pituitary adenylate cyclase-activating polypeptide: effects on pancreatic-adrenal hormone secretion and glucose-lipid metabolism in normal conscious dogs.
Metabolism
43:
739-744,
1994[Medline].
22.
Komatsu, M.,
T. Schermerhorn,
S. G. Straub,
and
G. W. Sharp.
Pituitary adenylate cyclase-activating peptide, carbachol, and glucose stimulate insulin release in the absence of an increase in intracellular Ca2+.
Mol. Pharmacol.
50:
1047-1054,
1996[Abstract].
23.
Laury, M. C.,
F. Takao,
D. Bailbe,
L. Penicaud,
B. Portha,
and
A. Ktorza.
Differential effects of prolonged hyperglycemia on in vivo and in vitro insulin secretion in rats.
Endocrinology
128:
2526-2533,
1991[Abstract].
24.
Lönnroth, P.,
J. I. Davies,
I. Lönnroth,
and
U. Smith.
The interaction between the adenylate cyclase system and insulin-stimulated glucose transport. Evidence for the importance of both cyclic AMP-dependent and -independent mechanisms.
Biochem. J.
243:
798-795,
1987.
25.
Meguid, M. M.,
F. Aun,
J. S. Soeldner,
D. A. Albertson,
and
C. M. Boyden.
Insulin half-life in man after trauma.
Surgery
89:
650-653,
1981[Medline].
26.
Miyata, A.,
A. Arimura,
R. R. Dahl,
N. Minamino,
A. Uehara,
L. Jiang,
M. D. Culler,
and
D. H. Coy.
Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells.
Biochem. Biophys. Res. Commun.
164:
567-574,
1989[Medline].
27.
Miyata, A.,
L. Jiang,
R. R. Dahl,
C. Kitada,
K. Kubo,
M. Fujino,
N. Minamino,
and
A. Arimura.
Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38).
Biochem. Biophys. Res. Commun.
170:
643-648,
1990[Medline].
28.
Scheurink, A.,
and
S. Ritter.
Sympathoadrenal responses to glucoprivation and lipoprivation in rats.
Physiol. Behav.
53:
995-1000,
1993[Medline].
29.
Sekiguchi, Y.,
K. Kasai,
K. Hasegawa,
Y. Suzuki,
and
S. Shimoda.
Glycogenolytic activity of pituitary adenylate cyclase activating polypeptide (PACAP) in vivo and in vitro.
Life Sci.
55:
1219-1228,
1994[Medline].
30.
Söderström, T.,
and
P. Stoica.
System Identification. New York: Prentice-Hall, 1989.
31.
Steil, G. M.,
A. Volund,
S. E. Kahn,
and
R. N. Bergman.
Reduced sample number for calculation of insulin sensitivity and glucose effectiveness from the minimal model.
Diabetes
42:
250-256,
1993[Abstract].
32.
Wang, P. R.,
and
Y. W. Chien.
Day-night differences in the kinetics and dynamics of insulin: diabetic versus normal Yucatan minipigs.
Chronobiol. Int.
13:
213-225,
1996[Medline].
33.
Wei, Y.,
and
S. Mojsov.
Tissue specific expression of different human receptor types for pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide: implications for their role in human physiology.
J. Neuroendocrinol.
8:
811-818,
1996[Medline].
34.
Yada, T.,
M. Sakurada,
K. Ihada,
M. Nakata,
F. Murata,
A. Arimura,
and
M. Kikuchi.
Pituitary adenylate cyclase activating polypeptide is an extraordinarily potent intra-pancreatic regulator of insulin secretion from islet
-cells.
J. Biol. Chem.
269:
1290-1293,
1994[Abstract/Free Full Text].
35.
Yokota, C.,
K. Kawai,
S. Ohashi,
Y. Watanabe,
S. Suzuki,
and
K. Yamashita.
Stimulatory effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on insulin and glucagon release from the isolated rat pancreas.
Acta Endocrinol.
129:
473-479,
1993[Medline].
36.
Yokota, C.,
K. Kawai,
S. Ohashi,
Y. Watanabe,
and
K. Yamashita.
PACAP stimulates glucose output from the perfused rat liver.
Peptides
16:
55-60,
1995[Medline].
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