Download CENTRAL ADMINISTRATION OF ALLOXAN IMPAIRS GLUCOSE

CENTRAL ADMINISTRATION OF ALLOXAN IMPAIRS GLUCOSE TOLERANCE
IN RATS
Melita Salkovic-Petrisic 1, Zdravko Lackovic 1, Siegfried Hoyer 2, Peter Riederer 3
1
Department of Pharmacology, School of Medicine, University of Zagreb, Salata 11, HR-10
000 Zagreb, Croatia.
2
Department of Pathology, University of Heidelberg, Germany
3
Department of Clinical Neurochemistry, Clinic of Psychiatry and Psychotherapy, University of
Würzburg, Germany
Running title: Central alloxan administration and glucose tolerance
Corresponding author:
* Melita Salkovic-Petrisic
Department of Pharmacology, School of Medicine, University of Zagreb, Salata 11, HR-10 000
Zagreb, Croatia.
telephone:
+385-1-45 90 219
telefax:
+385-1-45 66 843
e-mail:
[email protected]
SUMMARY
By means of oral glucose tolerance test (OGTT), we investigated glucose tolerance in rats pretreated with intracerebroventricular and subcutaneous non-diabetogenic dose of betacytotoxic
drug alloxan 7 days before OGTT. Being normoglycemic and normoinsulinemic pre-OGTT, at
30 minutes post-OGTT, alloxan intracerebroventricularly-treated rats had a lower glucose and a
higher insulin plasma levels in comparison with controls or alloxan subcutaneously treated
animals. Centrally administered alloxan seems to have within the brain effect on the regulation
of peripheral glucose tolerance and insulin secretion.
Keywords:
alloxan, intracerebroventricular, glucose, insulin
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INTRODUCTION
Alloxan induces experimental diabetes by selectively destroying pancreatic  cells when
peripherally administered in high doses (Szkudelski, 2001). Intracerebroventricular (i.c.v.)
administration of low alloxan doses neither produces diabetes nor alters steady-state blood
glucose levels in animals, but does induce alterations in the brain neurochemistry (Lackovic and
Salkovic 1990) and behaviour (Arjune and Bodnar 1990; Lubin and Bodnar, 1988). We report
on decreased blood glucose and increased plasma insulin levels 30 minutes after oral glucose
overload in rats previously treated with an i.c.v. non-diabetogenic dose of alloxan.
MATERIALS AND METHODS
Animals
Alloxan administration. Adult male Wistar rats (Department of Pharmacology, Zagreb
University School of Medicine), weighing 150-200 g, given general anaesthesia (chloralhydrate
300 mg/kg intraperitoneally), were administered a single dose of alloxan monohydrate (500
g/kg) dissolved in saline, injected i.c.v. (2 l/200 g body weight) into the right lateral ventricle,
according to the previously described procedure (Noble et al. 1967). Another group of animals
received the same drug dose (500 g/kg) subcutaneously /s.c./ (200 l/200 g body weight), and
the control animals received saline i.c.v. Animals (5-6 per group) were kept on food and water
ad libitum.
Oral glucose tolerance test (OGTT) was performed seven days following the administration of
alloxan (9.00 – 10.00 a.m.). The 50% D-glucose solution (2.5 g/kg body weight) was given
orally with a plastic orogastric catheter to conscious rats. Blood samples were collected by
puncturing tail vein of each conscious rat sequentially before and 30 min after OGTT. In
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additional experiment, control and alloxan i.c.v.-treated animals (500 g/kg) were rendered to a
24-hour fasting regime prior the OGTT and blood samples were collected sequentially before,
30 and 60 min after OGTT. Animals were sacrificed following the last blood withdrawn.
Biochemical analyses
Plasma glucose concentrations were measured using a commercial Kit (Glucose-PAP Test,
Herbos Diagnostics) by the glucose oxidase method. Plasma insulin concentrations were
measured by the radioimmunoassay (RIA), using the commercial sensitive rat insulin RIA Kit
SRI-13K (DRG International, Inc.).
Statistical analysis
Data are expressed as median and minimum-maximum value range. The blood glucose area
under the curve (AUC 0 - 60 min) was calculated using the trapezoidal rule; the values obtained
by distracting the baseline (pre-OGTT value of each animal) value from values measured at 30
and 60 minutes post-OGTT were used. The significance of difference within each group
between pre- and post-OGTT values was evaluated by Wilcoxon matched pair test, and among
the groups pre- or post-OGTT by Kruskal-Wallis ANOVA median test, followed by MannWhitney U-test. A p<0.05 was considered statistically significant for all tests.
Ethics
The experiments were carried out under the guidelines of The Principles of Laboratory Animal
Care (NIH Publication No. 86-23, revised 1985), according to the Croatian Act on Animal
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Welfare (NN 19/1999), and were approved by the Croatian Ministry of Science, Education and
Sports of the Republic of Croatia (Project No. 0108253).
RESULTS
Pre-OGTT plasma glucose values did not differ between the control, alloxan i.c.v.- and s.c.treated animals (Fig. 1A). In comparison with the baseline value, each group of animals had an
increased plasma glucose value 30 minutes post-OGTT (p<0.05). Post-OGTT plasma glucose
values differed between the groups (p<0.05), showing lowered plasma glucose values in alloxan
i.c.v.-treated rats in comparison with control and alloxan s.c.-treated animals (p<0.05) (Fig. 1A).
In alloxan i.c.v.-treated animals blood glucose AUC 0 - 60 min values were significantly lower in
comparison with the control ones (p<0.05), regardless of the fasting or non-fasting regime prior
the OGTT (Table 1).
Pre-OGTT values of plasma insulin did not differ between the groups (Fig. 1B). Post-OGTT
plasma insulin values were different between the groups (p<0.05), showing increased plasma
insulin values in alloxan i.c.v.-treated rats in comparison with control and alloxan s.c.-treated
rats (p<0.05) (Fig. 1B).
DISCUSSION
Central administration of low non-diabetogenic alloxan dose is associated with alterations of
glucose tolerance found in OGTT. Standardization of conditions did not influence this effect,
which was found to be similar in animals kept on free access to food and in those kept on
fasting regime prior the OGTT (Table 1). Observed suppression of plasma glucose increment
could be related to the changes of plasma insulin level, as 30 minutes following OGTT, the
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plasma insulin level of alloxan i.c.v.-treated rats reached a significantly higher concentration
than that in the control animals at the same post-OGTT time point, which was not seen in
alloxan s.c. treated rats (Fig. 1B). Regarding that all animals were kept under the same
experimental conditions and subjected to the same procedures, at the same time of the day, and
exposed to the same stress level, possible unspecific effects present only in alloxan i.c.v.-treated
and not in other rats, could be ruled out.
Alloxan is selectively taken up into the beta cell by a glucose transporter GLUT2(Gorus et al.,
1982; Munday et al., 1993), and GLUT-2 has been recognized as a target molecule for alloxan
(Schulte et al., 2002). GLUT-2 is also expressed in the hypothalamus and several other brain
regions (Leloup et al. 1994; Brant et al. 1993), which may suggest that alloxan administered
i.c.v. might enter those brain cells that express GLUT-2. Furthermore, specific inhibition of
GLUT2 in arcuate nucleus has been found to modulate nervous control of insulin secretion
(Leloup et al., 1998). To check if alloxan i.c.v.-induced metabolic effects in our experiments
could be related to alterations of the brain insulin receptor, we measured its protein expression
in hypothalamus by means of Western blot (mouse anti-insulin receptor /β subunit/ monoclonal
antibody, Chemicon International), but found no differences between the alloxan-treated and
control groups (results not shown). In line with the similarity of metabolic signaling proposed to
influence glucose sensing in hypothalamic neurones and in pancreatic beta cells (Garcia et al.,
2003; Miki et al. 2001; Schuit, 2001), rat hypothalamic glucokinase, involved in glucose
sensing, was found affected 6 days after alloxan i.c.v. administration (Sanders, 2004).
Our results showed that centrally applied betacytotoxic-induced impairment of brain
neurotransmitters (Lackovic and Salkovic 1990) may have systemic metabolic implications. It
could be speculated that in conditions with a normal peripheral metabolic control, alterations of
brain glucose sensing, or its metabolism, could, in circumstances yet to be discovered, lead to
alteration of the peripheral glucose tolerance and insulin secretion.
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ACKNOWLEDGEMENT
Supported by the Ministry of Science and Technology of the Republic of Croatia (0108253) and
Deutscher Academischer Austauch Dienst (DAAD). Dr. V. Trkulja is thanked for discussion on
the manuscript.
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Fig. 1. Rat plasma glucose (A) and insulin (B) levels measured pre- and 30 min post-oral
glucose tolerance test performed seven days following intracerebroventricular and
subcutaneous administration of a non-diabetogenic dose of alloxan (500 g/kg). Results are
presented as median value of data pooled from 2 (insulin) or 3 (glucose) experiments, N= 5-6
animals per group. Open square = median value, black rectangles = 1st and 3rd quartile, bars =
minimum-maximum. ALL, alloxan; icv, intracerebroventricular; sc, subcutaneous; OGTT, oral
glucose tolerance test. *p<0.05 versus control and alloxan s.c.-treated rats postOGTT values, by
Kruskal-Wallis ANOVA median test and Mann-Withney U test. #p<0.05 versus corresponding
pre-OGTT values, by Wilcoxon matched pair test.
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A)
PLASMA GLUCOSE (mmol/l)
10,5
postOGTT
preOGTT
9,5
8,5
#
#
7,5
#
6,5
5,5
*
4,5
control
ALL-icv
B)
ALL-sc
control
ALL-icv
ALL-sc
PLASMA INSULIN (ng/ml)
3,0
postOGTT
preOGTT
2,5
*
2,0
1,5
1,0
0,5
0,0
control
ALL-icv
ALL-sc
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control
ALL-icv
ALL-sc
Table 1. Statistic parameters of plasma glucose AUC
0-60
values in non-fasting and fasting
control and alloxan /ALL/ istracerebroventricularly (i.c.v.)-treated animals. The values were
obtained by distracting the baseline (pre-oral glucose tolerance test /OGTT/) value of each
animal from the corresponding values measured at 30 and 60 minutes post-OGTT.
AUC 0-60
(mmol/l x min -1)
NON-FASTING
control
FASTING
ALL-i.c.v.
control
211.09
58.60 *
(111.35-290.16)
(17.46-92.80)
81.41
23.40
134.59
lower quartile
108.42
74.99
259.05
upper quartile
*
P <0.05 versus corresponding control group, by Mann-Whitney U-test.
median
(min-max)
92.99
(73.62– 128.55)
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ALL-i.c.v.
137.63 *
(21.39-211.56)
103.41
198.73