<title>1. INTRODUCTION</title> <p>Islet amyloid polypeptide is a 37-amino-acid
peptide, which is produced in the <italic>β</italic>-cells in the pancreatic islets [##REF##3035556##1##–##REF##10341286##3##]. It is
coreleased with insulin [##REF##7479680##4##], and exogenous administration of IAPP inhibits
insulin secretion [##REF##1977811##2##, ##REF##10341286##3##, ##REF##2055243##5##–##REF##8074211##7##]. Several studies have also shown that exogenous
administration of IAPP at supraphysiological doses inhibits glucagon secretion [##REF##9005972##8##–##REF##16914214##11##].
IAPP of the human form may lead to fibril formation, which causes amyloid
deposition in the islets resulting in <italic>β</italic>-cell dysfunction and diabetes [##REF##1573849##12##–##REF##17192466##14##].
We have previously shown that mice with <italic>β</italic>-cell specific overexpression of the
human form of IAPP (hIAPP) have defective insulin secretion and disturbed islet
topography with centrally located glucagon producing <italic>α</italic>-cells [##REF##9833947##15##, ##REF##17482289##16##]. Whether
these mice in addition have disturbed glucagon secretion is, however, not
known. Therefore, the aim of the present study was to examine the glucagon
response to an oral protein load in these mice compared to wildtype mice. Since
IAPP has been shown to inhibit gastric emptying [##REF##16914214##11##], also the gastric emptying
rate in the transgenic and wildtype mice was evaluated with the previously described
acetaminophen-test [##REF##16627575##17##], to control for any differences in gastric emptying between
the groups.</p>
<title>2. METHODS</title> <title>2.1. Animals</title> <p>Hemizygous transgenic mice
with islet <italic>β</italic>-cell expression of hIAPP on a C57BL/6J/6xDBA/2 background were
generated as previously described [##REF##8622964##18##]. Transgenic status was determined by PCR
using oligonucleotide primers directed against the hIAPP transgene [##REF##11118007##19##]. The
transgenic mice and their wildtype controls were kind gifts of Dr Steven E
Kahn, University of Washington, Seattle, Wash, USA.
Transgenic and wildtype mice were transported from the animal facility of the University of Washington,
Seattle, to the In Vivo Department, Biomedical Center,
Lund University, Lund, Sweden, after embryo transfer performed
at Taconic A/S, Ry, Denmark. The animals were
cross-bred for &gt;16 generations to C57BL/6J mice. The animals were kept in a
12-hour light schedule (lights on at 0600 am) and given a standard pellet diet
(fat 11.4%, carbohydrate 62.8%, protein 25.8% on an energy base, total energy
12.6 kJ/g) and tap water ad libitum. The Ethics Committee in Lund/Malmö
approved the study.</p> <title>2.2. Experiments</title> <p>Following a four-hour period after removal of food from the cage, female
transgenic and wildtype animals
were anesthetized with an intraperitoneal injection of midazolam (Dormicum,
Hoffman-La-Roche, Basel, Switzerland, 0.2 mg/mouse) as well as a combination of
fluanison (0.4 mg/mouse) and fentanyl (0.02 mg/mouse; Hypnorm, Janssen, Beerse,
Belgium). Thirty minutes later, a blood sample was taken from the retrobulbar,
intraorbital, capillary plexus in heparinized tubes. Then, whey protein (100%
Anywhey, 75 mg, Optimum Nutrition, Lindesberg, Sweden) and acetaminophen
(paracetamol; Sigma Chemical Co, St Louis, Mo, 2 mg) dissolved in saline (total
volume 500 <italic>μ</italic>L) were administered through a gastric tube (outer diameter 1.2 mm). After 15, 30, 60, and 120 minutes, blood samples, 75 <italic>μ</italic>L each, were
collected. Blood was kept in heparinized tubes containing 5 <italic>μ</italic>L Trasylol
(aprotinin; 10000 KIE/mL; Bayer HealthCare AG, Leverkusen, Germany),
immediately centrifuged whereupon plasma was separated and stored at −20°C until
analysis for glucose, glucagon, insulin, and acetaminophen.</p> <title>2.3. Analyses</title> <p>Plasma glucagon was determined with radioimmunoassay (Linco Res, St Charles, Mo, USA) with a 
guinea pig antiglucagon antibody, radioiodine labelled glucagon as tracer and glucagon standard. CV of
the assay is 8% and the sensitivity of the assay is 10 pg/mL. The antibodies do
not cross-react with GLP-1. Plasma insulin was determined with radioimmunoassay
(Linco) with a guinea pig antirat insulin antibody, radioiodine labelled human
insulin as tracer and rat insulin as standard. Plasma acetaminophen was
determined with a colorimetric assay (Cambridge Life Science, Ely, Cambridgeshire, UK). Plasma glucose was determined
with the glucose oxidase method.</p> <title>2.4. Calculations and statistics</title> <p>Means ± SEM are shown. Statistical
comparisons were performed with the Student's <italic>t</italic>-test. For estimation of
glucagon secretion, the increase in plasma glucagon levels during the first 15
minutes after protein gavage was estimated by subtracting baseline glucagon
values from the 15-minute glucagon values. The area under the glucagon and
insulin curves (AUCs) were also calculated using the trapezoid rule.</p>
<title>3. RESULTS</title> <title>3.1. Glucagon response to oral protein</title> <p>
##FIG##0##Figure 1## (upper left panel) shows
plasma glucagon levels during the oral protein challenge. Baseline glucagon
levels were higher in the transgenic animals (41 ± 4.0 pg/mL, <italic>n</italic> = 6) than in the
wildtype animals (19 ± 5.1 pg/mL, <italic>n</italic> = 5, <italic>P</italic> = .015). Glucagon levels at 15, 30,
and 60 minutes after protein administration did not differ significantly
between the groups, whereas the levels after 120 minutes were, again,
significantly, higher in the transgenic animals (<italic>P</italic> = .008). Glucagon
secretion was estimated as the change in glucagon levels during the first 15
minutes after protein gavage. This 15-minute glucagon response to protein
administration was impaired in the transgenic animals, being 21 ± 2.7 pg/mL in
transgenic mice versus 38 ± 5.7 pg/mL in wildtype mice (<italic>P</italic> = .027). The suprapasal AUC for
glucagon for the entire 120-minute study period did not differ significantly
between the groups, being 4.3 ± 1.1 <italic>μ</italic>g/mL × 120 minutes in wildtype mice versus
3.9 ± 0.9 <italic>μ</italic>g/mL × 120 minutes in transgenic mice.</p> <title>3.2. Insulin and glucose responses to oral protein</title> <p>Baseline insulin levels were 50 ± 5.1 påmol/L in wildtype animals and 46 ± 4.9 pmol/L in transgenic mice (NS). The
insulin response to protein ingestion was impaired in transgenic mice; the
suprabasal AUC for insulin for the 120-minute study period was 27.2 ± 3.1 nmol/L
× 120 minutes in wildtype animals versus 16.5 ± 3.9 nmol/L × 120 minutes in
transgenic animals (<italic>P</italic> = .018) (##FIG##0##Figure 1##, upper right). Baseline glucose
levels were 7.2 ± 0.3 mmol/L in wildtype animals and 7.8 ± 0.2 mmol/L in transgenic
mice (NS); glucose levels did not change significantly during the test (##FIG##0##Figure
1##, lower left panel).</p> <title>3.3. Acetaminophen response to acetaminophen administration</title> <p>
##FIG##0##Figure 1## (lower right) shows the acetaminophen concentrations during
test. Plasma acetaminophen increased to a maximum level at 15 minutes after
administration, thereafter it gradually fell. There was no significant
difference between the groups in plasma acetaminophen.</p>
<title>4. DISCUSSION</title> <p>This study evaluated the islet
hormone responses to oral protein ingestion in mice with <italic>β</italic>-cell specific
overexpression of human IAPP. It was found that the insulin response to protein
was impaired in transgenic mice. This confirms that these mice have impaired
insulin secretion, as previously was reported also after oral glucose challenge
[##REF##9833947##15##]. The main novel finding in this report is, however, that the transgenic
mice have also changes in the glucagon levels. Thus, the mice were found to
have higher baseline glucagon levels than their wildtype counterparts and yet
they have a reduced glucagon response to protein administration. The mechanism
of the high-baseline glucagon remains to be established. It is, however, consistent
with the disturbance in islet topography in these mice. Thus, we have
previously shown that the islets of these mice have enlarged population of
glucagon producing <italic>α</italic> cells as opposed to the reduced <italic>β</italic>-cell immunostaining in
these animals which is associated with significantly reduced islet insulin
content [##REF##17482289##16##]. This hyperglucagonemia may be the result of the reduced islet insulin,
in view of the inhibitory influence of insulin on glucagon secretion. At the
same time, the glucagon response to the protein administration was impaired,
which may be explained by the transgene, because IAPP is known to inhibit
glucagon secretion [##REF##9005972##8##–##REF##16914214##11##]. Hence, high-baseline glucagon and impaired glucagon
response to stimulation are two characteristics of the hIAPP transgene, and may
have different mechanisms.</p> <p>In this study, we also determined
the acetaminophen concentration after acetaminophen administration to determine
whether gastric emptying had been altered in the transgenic mice. Previously,
inhibition by IAPP of gastric emptying has been demonstrated [##REF##16914214##11##] and changes
in gastric emptying would be a mechanism for changes in glucagon secretion
after protein administration. The acetaminophen test has previously been
validated in humans [##REF##10831260##20##] and used in a previous study in mice [##REF##16627575##17##]. It is based
on the poor absorption of acetaminophen from the stomach and the rapid and
almost complete absorption from the small intestine. This implies that plasma
acetaminophen profiles give an estimation of gastric emptying, which also has
been verified as good correlation with isotopic technique measurements of
gastric emptying [##REF##688732##21##, ##REF##11437980##22##]. We found that there was no difference in plasma
acetaminophen profiles between transgenic and wildtype mice. This shows that
the increase in <italic>β</italic>-cell IAPP expression does not affect gastric emptying, and,
therefore, the inhibited glucagon response to oral protein in these mice is not
due to impaired gastric emptying.</p> <p>In conclusion, this study has shown
that <italic>β</italic>-cell specific overexpression of human IAPP increases baseline glucagon
levels and impairs the glucagon response to oral protein in association with
impaired insulin response. This shows that a disturbed <italic>α</italic>-cell function in these
mice is evident in association with the previously described disturbed <italic>β</italic>-cell
function [##REF##9833947##15##, ##REF##17482289##16##].</p>
<p>Recommended by Per Westermark</p> <p>Exogenous administration of islet amyloid polypeptide (IAPP) has been shown to inhibit both insulin and glucagon secretion. This study examined <italic>α</italic>-cell function in mice with <italic>β</italic>-cell specific overexpression of human IAPP (hIAPP) after an oral protein gavage (75 mg whey protein/mouse). Baseline glucagon levels were higher in transgenic mice (41 ± 4.0 pg/mL, <italic>n</italic> = 6) than in wildtype animals (19 ± 5.1 pg/mL, <italic>n</italic> = 5, <italic>P</italic> = .015). In contrast, the glucagon response to protein was impaired in transgenic animals (21 ± 2.7 pg/mL in transgenic mice versus 38 ± 5.7 pg/mL in wildtype mice at 15 minutes; <italic>P</italic> = .027). Baseline insulin levels did not differ between the groups, while the insulin response, as the glucagon response, was impaired after protein challenge (<italic>P</italic> = .018). Glucose levels were not different between the groups and did not change significantly after protein gavage. Acetaminophen was given through gavage to the animals (2 mg/mouse) to estimate gastric emptying. The plasma acetaminophen profile was similar in the two groups of mice. We conclude that disturbances in glucagon secretion exist in mice with <italic>β</italic>-cell specific overexpression of human IAPP, which are not secondary to changes in gastric emptying. The reduced glucagon response to protein challenge may reflect a direct inhibitory influence of hIAPP on glucagon secretion.</p>
<title>ACKNOWLEDGMENTS</title> <p>The authors are grateful to Kristina Andersson,
Lilian Bengtsson, and Lena Kvist for expert technical assistance. This study
was supported by the Swedish Research Council (Grants 6834), The Swedish Diabetes
Foundation, Albert Påhlsson Foundation, The Swedish Diabetes Association,
Region Skåne, and the Faculty of Medicine, Lund University.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Plasma levels of glucagon, insulin, glucose, and acetaminophen following administration 
of whey protein (75 mg) and acetaminophen (2 mg) in female wildtype mice (<italic>n</italic> = 5) and transgenic mice with <italic>β</italic>-cell specific
overexpression of hIAPP (<italic>n</italic> = 6). Means ± SEM are shown. Asterisks indicate
probability level of random difference between the two groups (*<italic>P</italic> &lt; .05).</p></caption></fig>
<graphic xlink:href="EDR2008-304513.001"/>
[]
Exp Diabetes Res. 2008 Jul 7; 2008:304513