Materials and methods 4. Blood sampling and brain dissection
392 U
has been seen with plasma levels of IAPP that are at or min, n510; 5 pmol kg–min, n59; 25 pmol kg–min,
very close to physiological concentrations, it has been n59 and the other rats were infused for 5 days IAPP
suggested that IAPP may play a hormonal role in the dose: 0 pmol kg–min, n59; 5 pmol kg–min, n59; 25
control of food intake. pmol kg–min, n59. After 2 or 5 days of treatment, the
Although the mechanisms for an endocrine role of the animals were anesthetized again, and a blood sample was
peptide are poorly understood, experimental data suggests taken from the abdominal aorta for measurement of blood
that the central nervous system is a likely target. There is glucose, plasma IAPP, and plasma insulin. The animals
evidence that IAPP can cross the blood brain barrier [7], were then decapitated with a guillotine and the brains
suggesting that the IAPP in the brain enters from the removed and dissected on ice.
circulation. Brain administration appears to be more potent than systemic administration for reducing food intake
2.2. Animals and housing conditions [3,31]. High-affinity binding sites for IAPP have been
demonstrated in scattered areas within the brain, including A total of 55 male Wistar rats BK Universal Ltd.,
parts of the hypothalamus [35]. IAPP-induced anorexia has Sollentuna, Sweden were individually housed in plastic
been observed following injections of pharmacological shoebox cages on aspen-chip bedding Macrolon [3,
doses, administered either intrahypothalamically in rats Scanbur, Køge, Denmark. Water and standard rodent
[6,12] or intracerebroventricularly in rats [9,23,31] or mice pelleted chow R36, Lactamin, Vadstena, Sweden were
[26]. In addition, intrahypothalamic injections of IAPP available ad libitum throughout the experiments. The
have been shown to inhibit feeding induced by neuro- humidity of the air was maintained between 55 and 60,
peptide-Y NPY in rats [6]. and the temperature was kept at 22618C. The room had a
Intrahypothalamic injections of IAPP have been reported 12-h light and dark cycle, with lights out at 1800. The
to cause neurochemical alterations in the rat brain, with animals were weighed each day, and the daily chow intake
increases in dopamine and serotonin neurotransmission was recorded after being corrected for spillage. The
seen in the hypothalamus and corpus striatum [12]. Sys- experiment was approved by the Animal Research Com-
¨ temic administration of IAPP as a single bolus injection
mittee for
Southern Stockholm
Stockholms Sodra
¨ ¨ ¨
increased concentrations of serotonin in the hypothalamus Forsoksdjursetiska Namnd. Rats were handled according
and decreased levels of the dopamine metabolite 3- to guidelines established by the National Board for Labora-
methoxytyramine in the corpus striatum [11]. However, tory Animals CFN in Sweden.
changes in neuropeptides known to be involved in the regulation of feeding have not been studied following
2.3. IAPP preparation and osmotic mini-pump infusion IAPP administration.
We have previously reported markedly elevated plasma The effects of IAPP were studied after 2 days of
IAPP concentrations in patients with pancreatic cancer, a infusion, when peak inhibition of food intake was expected
disease often associated with anorexia [30]. We have also and at 5 days, when the anorectic effects were expected to
demonstrated that chronic IAPP infusion in rats, at a dose have diminished [2,3]. The Alzet Alza Corp., Palo Alto,
producing plasma levels similar to those of pancreatic CA model 1003D mini-pump was used for the 2-day
cancer patients, causes a reduction of food intake but has infusions and model 2001 was used for the 5-day infu-
no effect on glucose metabolism [3–5]. However, these sions. Synthetic rat IAPP Multiple Peptide Systems, San
experiments did not investigate the mechanisms respon- Diego, CA was dissolved in 75 DMSO 25 saline
sible for the anorectic effects of IAPP. 0.15 M. The osmotic mini-pumps were implanted 5 cm
The aim of the present study was, therefore, to use doses behind the base of the skull and just to the right of the
of IAPP that were anorectic but not diabetogenic to midline under aseptic surgical conditions, following induc-
investigate the effects of chronically-elevated plasma tion of anesthesia with ketamine, 40 mg kg IP Ketalar 50
levels of IAPP on neurotransmitters and neuropeptides in mg ml, Park-Davis, Barcelona, Spain. As soon as the
rat brain. animals awoke, they were placed in their regular cages and
had free access to food and water.
2. Materials and methods 2.4. Blood sampling and brain dissection
2.1. Study design After 2 or 5 days of treatment, the animals were re-
anesthetized with
pentobarbital Pentobarbitalnatrium,
˚ After a minimum of 7 days acclimation, the rats were
Apoteksbolaget, Umea, Sweden, 50 mg kg IP. The ab- randomly allocated into treatment groups and had osmotic
domen was opened along the midline and 2 ml of aortic mini-pumps with IAPP or carrier vehicle implanted sub-
blood was collected into pre-chilled tubes containing cutaneously during a short period of anesthesia. Half the
EDTA 1.5 mg ml and aprotinin Trasylol 400 KIU ml, animals were infused for 2 days IAPP dose: 0 pmol kg–
Bayer, Leverkusen, Germany. Blood glucose levels were
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