ࡱ> BDAfo@P\bjbj p p %poo3ppppppplll8D\{2P:1111111$13R51p1pp;52>)>)>) pp1>)1>)D>)):&/,ppf/D P5l'6R/ f0K20{2\/ o6(do6f/ppppo6pf/V5>))11d)" CENTRAL ADMINISTRATION OF ALLOXAN IMPAIRS GLUCOSE TOLERANCE IN RATS Melita Salkovic-Petrisic 1, Zdravko Lackovic 1, Siegfried Hoyer 2, Peter Riederer 3 1Department of Pharmacology, School of Medicine, University of Zagreb, Salata 11, HR-10 000 Zagreb, Croatia. 2Department of Pathology, University of Heidelberg, Germany 3Department of Clinical Neurochemistry, Clinic of Psychiatry and Psychotherapy, University of Wrzburg, 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:  HYPERLINK mailto:melitas@mef.hr melitas@mef.hr SUMMARY By means of oral glucose tolerance test (OGTT), we investigated glucose tolerance in rats pre-treated 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 INTRODUCTION Alloxan induces experimental diabetes by selectively destroying pancreatic b 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 mg/kg) dissolved in saline, injected i.c.v. (2 ml/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 mg/kg) subcutaneously /s.c./ (200 ml/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 additional experiment, control and alloxan i.c.v.-treated animals (500 mg/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 Mann-Whitney 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 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 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 / 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. 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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Life Sci 71: 1681-1694 Szkudelski T (2001) The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 50: 537-546 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 GIabceuvw G H ! < 嵫§zpjc\\ hM{6CJ hM{>*CJ hXZ[qy  hjbrD7n|%w| ( 2 ԾԾԷԷԱԪԤԱԚԾhM{h< h(CJ hW}CJ hM{CJH* h<CJ hM{6CJ hOCJhM{CJOJQJ hCJ hM{CJ hM{>*CJ hM{5CJh//mH sH h'4mH sH hM{mH sH ;'(!!""""&& - -..../// $da$gdM{2 A!J!X!Z![!"0"""""""S####$$($?$D$E$\$g$$A%r%{%~%%&&2&&&&&&&& ''"'m'n'w'''''( ( (S((v++;,`,k,~,,˿ѹ h6b1CJ hhCJ hWCJ h6b1CJ h@M`/a; xڽZL~69gC5H(%B FĔ!lES)"&ID6I'ICS'QiMZPRiP#*ʶؒԻw;s/ ɟ|w}{g !!! 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