U . Arnelo et al. Brain Research 887 2000 391 –398
393
measured using a biochemical analyzer YSI Model 2700 2.8. Statistical analyses
select, Yellow Springs Instruments, Yellow Springs, OH. After the rats were decapitated, the brains were rapidly
The effects of IAPP infusion on change in body weight removed and dissected on ice. The hypothalamus, hip-
were evaluated by one-way analysis of variance ANOVA. pocampus, striatum, left cortex, and right cortex were snap
Planned comparisons of mean body weight change at the 5 frozen in liquid nitrogen. Both plasma and brain tissues
and 25 pmol kg–min doses with the mean levels for the were stored frozen at 2808C until subsequent extraction
controls were evaluated by direct contrasts of means using and radioimmunoassay or detection of biogenic amines by
the statistical program SYSTAT. Effects of IAPP on daily high-pressure liquid chromatography HPLC.
food intakes were determined by separate repeated mea- sures ANOVA with IAPP dose as the between-group factor
and day of infusion as the within-group factor. Again, 2.5. Extraction and radioimmunoassay of IAPP and
planned comparisons were evaluated by direct contrasts of insulin
means using the statistical program SYSTAT. Effects of IAPP on other parameters were analyzed similarly using a
The extraction and radioimmunoassay procedure previ- 2 factor ANOVA, with the duration of infusion and dose of
ously described in detail [30] was modified to include IAPP as the two factors. Planned contrasts were used to
acidification of plasma samples with acetic acid final determine the effect of low and high doses of IAPP
acetic acid concentration 0.25 M and centrifugation to compared with the 0 dose at for the 2 or 5 day infusion,
remove precipitated proteins before extraction of IAPP and using the statistical program SYSTAT.
insulin from the supernatant on Sep-Pak, C-18 reverse- For each analysis, the difference was considered signifi-
phase cartridges Waters, Milford, MA.. The eluates were cant if P,0.05. Values are expressed as means6S.E.M.
lyophilized and resuspended with 800 ml assay buffer 10 mM KH PO , 60 mM Na HPO
10 mM EDTA, 7.6 mM
2 4
2 4,
sodium azide and 0.3 bovine serum albumin for
3. Results
radioimmunoassay [30] Insulin concentrations were mea- sured using a rat insulin radioimmunoassay kit from Linco
3.1. Effects of IAPP on food intake and body weight Research, Inc. St. Charles, MO. Recovery after extrac-
tion was measured for both peptides, and the results were The lower panels in Fig. 1 show the effects of IAPP on
corrected accordingly. daily food intake during the 2-day Fig. 1A, and 5-day
Fig. 1B treatment periods. On the day before insertion of 2.6. Extraction of brain tissue samples and measurement
the osmotic mini-pumps, daily food intake was similar in of biogenic amines by HPLC
all groups. IAPP significantly decreased food intake dose- dependently during the 2-day and the 5-day treatment
The frozen tissue samples were minced, boiled for 10 periods. The 24 h food intake during the second day in the
min in ten volumes of 1 M acetic acid. N-methyl-5- 2-day treated animals was reduced by 27 in the 5
hydroxytryptamine NM-5HT and 3,4-dihydroxybenzyl- pmol kg–min group, from a control value of 25.262.3 g
amine DHBA, the internal standards for serotonin and to 18.462.6 g P,0.001 and by 44 to 14.064.7 g in the
catecholamines, respectively, were added. The samples 25 pmol kg–min group P,0.001. Likewise, food intake
were then homogenized, centrifuged and the supernatants during the fifth day in the rats receiving IAPP for 5 days
lyophilized and stored at 2208C until subsequent analysis. was reduced by 14 in the 5 pmol kg–min group, from a
The samples were reconstituted in 1 ml phosphate buffer control value of 26.662.7 g to 23.062.6 g P,0.01, and
0.05 M, pH 7.4. 0.6 ml was mixed with 4.4 ml 10 mM by 24 to 20.362.4 g P,0.001 in the 25 pmol kg–min
HCl and further purified on a cation-exchange column group.
before being analyzed for norepinephrine NE and dopa- At the time of osmotic mini-pump insertion, there were
mine DA on a HPLC system, using a reverse-phase no significant differences in body weight among the groups
column under isocratic conditions and an electrochemical receiving treatment for 2 days 35863, 36162, and
detector, as previously described [37]. 35863 g for the groups receiving 0, 5, and 25 pmol kg–
min, respectively or for the groups treated for 5 days 2.7. Measurement of neuropeptides using
35966, 35864, and 35862 g in the groups receiving 0, radioimmunoassays
5, and 25 pmol kg–min, respectively. The upper panels in Fig. 1 show that IAPP infusion dose-dependently caused a
Neuropeptide Y
NPY, neurotensin
NT, and
marked loss of body weight during the 2-day experiment cholecystokinin CCK were measured using previously
P,0.001, as well as during the 5-day experiment P, described radioimmunoassays [18,38,39]. Within and be-
0.001. In the 2-day treated animals, the dose of 5 pmol tween assay variation was less than 8 and 13, respective-
kg–min resulted in a greater weight loss than that found in ly for all three assays.
the control group 12.060.9 g vs. 2.561.6 g, respectively,
394 U
Fig. 1. Effects of 2-day A, and 5-day B infusions of islet amyloid polypeptide 0, 5, and 25 pmol kg–min on daily food intake bottom and body weight change top in ad libitum fed rats. Day
0 denotes the day before insertion of the osmotic mini-pump. Values are means6S.E.M. 5P,0.01, 5P,0.001 vs. 0 pmol kg–min in the 2-day group; †5P,0.05, ††5P,0.01, †††5P,0.001 vs. 0 in the 5-day group.
P,0.001. The dose of 25 pmol kg–min caused an even tween the study groups in the 2-day experiment. However,
greater weight loss of 16.062.0 g P,0.001. In the 5-day in the 5-day study, the IAPP-treated rats had lower plasma
experiment, the weight loss of 1.362.1 g in the 5 pmol insulin levels than the controls.
kg–min group and 11.762.1 g in the 25 pmol kg–min group were significantly different from the 4.461.2 g
3.3. Effects of IAPP on neurotransmitters and weight gain of the control group P,0.05 and P,0.001,
neuropeptides respectively.
As shown in Table 2, IAPP increased NE in the 3.2. Effects of IAPP on blood glucose, plasma IAPP,
hypothalamus in the 2-day, 5 pmol kg–min group and and insulin levels
5-HIAA in the hypothalamus in the 5-day, 5 pmol kg–min group compared to the control groups at the respective
Table 1 shows that plasma IAPP concentrations were time points both P,0.05.
increased dose-dependently at the end of the 2-day and Table 3 summarizes the concentrations of NPY, NT, and
5-day infusion periods 2-day, P,0.001; 5-day, P,0.001. CCK measured in the various brain regions. Compared to
Blood glucose levels were similar in all treatment groups. the 5-day control animals, the 5-day, 25 pmol kg–min
There were no differences in insulin concentrations be- group had significant increases in CCK in the hypo-
U . Arnelo et al. Brain Research 887 2000 391 –398
395 Table 1
Concentrations of blood glucose, plasma IAPP, and plasma insulin, following 2 and 5 days of IAPP administration at 0, 5 and 25 pmol kg-min 0, 5, and
a
25 Treatment groups
2 days 5 days
0 n510 5 n59
25 n59 0 n59
5 n59 25 n59
Blood glucose mmol l 10.960.2
10.260.4 9.960.5
10.160.3 9.760.4
9.860.5 Plasma IAPP pmol l
7.262.0 64.465.0
198.0634.5 12.863.1
48.263.5 160.5617.5†††
Plasma insulin pmol l 209.2633.4
183.3626.5 175.4626.9
291.3635.9 163.0615.5††
147.0621.3†††
a
Values are means6S.E.M. 5P,0.05, 5P,0.001vs. 0 in the 2-day group; ††5P,0.01, †††5P,0.001vs. 0 in the 5-day group.
Table 2 Concentrations of serotonin 5-HT, 5-hydroxyindoleacetic acid 5-HIAA, dopamine DA, 3,4-dihydroxyphenylacetic acid DOPAC, homovanillic acid
HVA, norepinephrine NE, and 3-methoxy-4-hydroxyphenylglycol MHPG in the hypothalamus hypo, hippocampus hippo, striatum stri, left cortex
a
l. cx and right cortex r. cx of rat brain, following 2 and 5 days of IAPP administration at 0, 5 and 25 pmol kg–min 0, 5 and 25 Treatment groups
2 days 5 days
0 n510 5 n59
25 n59 0 n59
5 n59 25 n59
5-HT nmol g hypo
8.160.5 8.760.4
7.460.6 6.160.7
7.760.7 7.460.2
hippo 3.460.1
3.560.2 3.060.3
3.360.3 3.460.1
3.460.2 stri
4.060.2 4.060.1
3.760.4 3.460.3
3.560.1 3.860.2
l. cx 2.660.1
2.560.1 2.360.1
2.760.2 2.660.1
2.760.1 r. cx
2.460.1 2.660.1
2.260.1 2.560.2
2.760.1 2.760.1
5-HIAA nmol g hypo
3.360.2 4.060.3
3.660.3 2.560.4
3.260.3† 3.160.3
hippo 2.760.2
3.260.2 3.260.3
2.260.3 2.460.3
2.160.2 stri
4.260.2 4.760.2
4.260.4 3.060.4
3.460.4 3.860.6
l. cx 1.060.1
1.360.1 1.160.0
1.060.1 1.060.1
1.060.1 r. cx
1.260.1 1.260.1
1.060.0 1.060.1
1.060.1 1.160.1
DA nmol g hypo
1.260.4 1.760.6
1.660.4 1.260.8
0.860.4 0.560.2
hippo nd
nd nd
nd nd
nd stri
26.766.8 32.0610.1
29.867.9 3.861.1
4.060.9 8.760.6
l. cx nd
nd nd
nd nd
nd r. cx
nd nd
nd nd
nd nd
DOPAC nmol g hypo
0.860.1 0.760.1
0.860.1 0.860.1
0.860.2 1.060.1
hippo nd
nd nd
nd nd
nd stri
9.260.8 9.460.9
9.760.8 11.761.8
12.461.0 13.161.0
l. cx 0.260.0
0.260.0 0.360.1
0.460.1 0.460.1
0.460.1 r. cx
0.260.0 0.260.0
0.260.0 0.460.1
0.460.1 0.460.1
HVA nmol g hypo
nd nd
nd nd
nd nd
hippo nd
nd nd
nd nd
nd stri
5.460.7 5.460.7
5.360.5 7.960.9
8.161.1 8.260.9
l. cx 0.460.0
0.360.0 0.360.0
0.360.1 0.360.1
0.360.1 r. cx
0.360.0 0.460.0
0.360.1 0.160.1
0.460.0 0.360.1
NE nmol g hypo
3.460.9 4.460.8
2.960.7 0.960.4
0.860.4 0.560.0
hippo 0.560.1
0.960.3 0.560.1
0.260.1 0.260.0
0.360.1 stri
0.260.1 0.360.1
0.260.1 0.160.0
0.260.0 0.160.0
l. cx 0.160.0
0.160.0 0.160.0
0.160.0 0.160.0
0.160.0 r. cx
0.160.0 0.160.0
0.160.0 0.160.0
0.160.0 0.160.0
MHPG nmol g hypo
nd nd
nd nd
nd nd
hippo nd
nd nd
nd nd
nd stri
nd nd
nd nd
nd nd
l. cx 0.260.0
0.260.0 0.260.0
0.260.1 0.260.1
0.260.1 r. cx
0.260.0 0.260.0
0.360.1 0.260.1
0.260.1 0.260.1
a
Values are means6S.E.M. 5P,0.05 vs. 0 in the 2-day group. †5P,0.05 vs. 0 in the 5-day group. nd5not detectable.
396 U
Table 3 Concentrations of neuropeptide-Y NPY, neurotensin NT, and cholecystokinin CCK in the hypothalamus hypo, hippocampus hippo, striatum stri,
a
left cortex l. cx and right cortex r. cx of rat brain, following 2 and 5 days of IAPP administration at 0, 5 and 25 pmol kg–min 0, 5 and 25 Treatment groups
2 days 5 days
0 n510 5 n59
25 n59 0 n59
5 n59 25 n59
NPY pmol g hypo
91.267.1 104.866.5
101.865.5 74.865.8
90.467.1 105.869.6††
hippo 31.061.4
32.461.9 32.161.8
24.562.6 25.862.7
27.163.9 stri
37.762.7 36.662.5
41.062.2 33.864.0
35.061.4 35.964.9
l. cx 24.761.2
22.961.7 28.561.8
24.161.3 23.262.9
25.063.5 r. cx
27.261.7 26.861.8
28.561.4 21.861.8
23.362.2 20.360.8
NT pmol g hypo
80.264.2 79.066.2
77.762.8 63.166.3
70.067.2 74.664.5
hippo 8.860.5
10.060.4 10.360.5
7.460.9 7.260.6
7.360.7 stri
5.460.4 5.060.3
6.960.5 4.060.3
4.560.5 5.260.6
l. cx 2.060.2
1.960.1 1.860.2
1.860.1 1.960.2
1.860.2 r. cx
1.960.1 1.860.1
1.860.2 1.960.1
1.960.2 1.860.2
CCK pmol g hypo
93.066.0 90.066.2
88.366.2 68.567.6
68.866.1 87.964.2†
hippo 80.769.3
95.065.5 103.664.6
88.9611.2 101.967.4
103.669.6 stri
84.7612.3 106.3610.1
120.8614.2 64.9612.6
105.266.9† 108.2614.1†
l. cx 88.8614.2
74.767.0 87.669.8
80.4613.8 81.068.6
88.1610.8 r. cx
73.0611.4 78.667.6
86.368.4 73.767.5
75.8611.2 96.9616.2
a
Values are means6S.E.M. 5P,0.05 vs. 0 in the 2-day group. †5P,0.05, ††5P,0.001 vs. 0 in the 5-day group.
thalamus and striatum both P,0.05 and NPY in the would stimulate the release of more insulin and IAPP into
hypothalamus P,0.01. The 5-day, 5 pmol kg–min the circulation.
group also had an increase in CCK in the striatum P, Insulin levels were reduced after 5 days of IAPP
0.05. In the 2-day, 25 pmol kg–min group, NT was infusion but not at 2 days. This is likely to be a result of
increased in the hippocampus compared to the 2-day the prolonged reduction in food intake, since loss of body
control group P,0.05. weight is associated with lower insulin levels [15]. Alter-
natively, the reduction in insulin concentrations could reflect a direct inhibition of food-stimulated insulin secre-
tion by IAPP. Although the effect of IAPP on b-cell
4. Discussion function is controversial, a number of studies have re-
