The effect of exogenous glucose on the activity of lysosomal enzymes in some organs of rabbits (short communication)

Zusammenfassung Titel der Arbeit: Der Einfluß von Exoglukose auf die Aktivität der lysosomalen Enzyme in einigen Organen bei Kaninchen (Kurzmitteilung) Die Aktivitäten der lysosomalen Enzyme wurden in der Leber, den Nieren und den Muskeln bei Kaninchen bestimmt. Die Tiere erhielten zweimal pro Tag während 7 Tagen je I ml/kg Körpergewicht 40% Exoglukoselösung. Nach der Glukoseinjektion wurden die Senkung der BGLUund AP-Aktivität in der Leber; BGRD-, BGAL-, NAGL-, APund LL-Aktivität in der Niere; NAGLund LL-Aktivität im Muskel beobachtet.


Introduction
If we introduce to the blood of animal an excess of exogenous glucose, this sugar becomes itself a metabolic Stressor because its level can be the expression of homeostase (LAYCHOCK, 1990;KOLATAJ et al., 1998;RÄNDLE etal., 1988).We did not find the reports connected with the reactivity of lysosomal enzymes during metabolic Stimulation (DESJARDINS, 1995;STORRIE and DESJARDLNS, 1996).In our experiment we studied the changes of that reactivity in some organs of rabbits on glucose model ofthe biochemical stress.

Material and Methods
The experiment was carried out on twenty 6-month-old male rabbits of the New Zealand White breed, Coming from a farm of the Institute of Animals Production in Nitra (Slovakia).The animals weighed 2.0 -2.2 kg and were maintained in identical conditions of nutrition and nursing.They received an industrial feed mixture, 19% of crude protein (Altromin Standard Diets 1320 Totally Pathogene Free TPF; GmbH International; Germany).All animals had a continuous access to water.The rabbits have been divided into control (n = 10) and experimental group (n = 10).The animals of the experimental group received 40% Solution of glucose in the amount of 1 ml/kg of body weight twice daily at 10 00 -ll 00 a.m. and 6 00 -7 00 p.m. during the 7 days to the ear vein.The control rabbits received analogously the 0.9% NaCl Solution.After 7 days the rabbits were killed by interrupting of spinal cord and slices of the liver, kidney and muscle (musculus longissimus dorsi ) at the height of the last rib immediately were taken to analysis.The slices of the liver have been subjected to perfusion by Solution of 0.9% NaCl cooled to + 5° C and similarly to slices of the kidney and muscle were suspended in 0.1 M phosphate buffer pH 7.0 at the temperature + 5° C at ratios 1 g of tissue/6 ml buffer.The whole was homogenized in glass Potter homogenizer at 200 rotations/min.Differential fractioning of the liver homogenates was carried out aecording to the method of MARZELLA and GLAUMANN (1980), kidney and muscle homogenates aecording to the method of BEAUFAY(1972).The activity of ß-glucuronidase (BGRD -EC 3.2.1.31);ß-galactosidase (BGAL -EC 3.2.1.23);ß-glucosidase (BGLU -EC 3.2.1.21)and N-acetyl-ß-glucosaminidase (NAGL -EC 3.2.1.30)was determined on the basis of p-nitrophenyl Substrate by use the micro-spectrophotometric BARRETT 'S method (1972); acid Phosphatase (AP -EC 3.1.3.2) aecording to HOLLANDER (1970); alanyl aminopeptidase (AAP -EC 3.4.11.2) aecording to PFLEIDERER et al., (1964); leucyl aminopeptidase (LAP -EC 3.4.11.1) aecording to PFLEIDERER and CELLIERS (1963); lysosomal esterase (EL -EC 3.1.1.2) and lysosomal lipase (LL -EC 3.1.1.3)aecording to the modified MAINS method (1960) and activity of cathepsin D (Cath.D -EC 3.4.23.5) was estimated aecording to the method of LANGNER et al., (1973) using 2% azocasein in 6M urea as Substrate.All reagents have been made by Serva (Feinbiochemica GmbH & Co., Heidelberg, Germany).The enzyme activity has been expressed in nmol/mg of protem/hour.
In the lysosomal fraction obtained the protein was also determined by a modified Lowry's method (KIRSCHKE and WIEDERANDERS, 1984).The results were statistically analysed aecording to analysis of variance and Student s tests.

Discussion
It is known that glucose is a main energetic source on the pathways of metabolic processes in the human and in the majority of the mammalians.Its level in the blood serum has already been discussed (LEWIS et al., 1996;RUBIO et al., 1997;KOLATAJ et al., 1998).The activity of lysosomal enzymes in the course of the programmed glucose stress in the rabbits is unknown.On the basis of the results obtained it is possible to suggest that the introduce of exogenous glucose had a significant influence on changes in the activity of BGLU and AP in the liver; BGRD, BGAL, NAGL, AP, LL in the kidney; NAGL and LL in the muscle.The ränge of those changes dependend on the kind of enzyme and the tissue.
The activities of all estimated enzymes of all examined organs except AAP, LAP and Cath.D decreased in comparison with control group.The high glucose concentration in the blood serum may be an indicator of diabetes (ANDERSON, 1997;HENRY, 1996) or an indicator ofthe oxidative stress in the tissues of animai.The reactive oxygen forms may disturb the normal carbohydrate metabolism (RÄNDLE et al., 1988).On this way the glucose happens a metabolic Stressor (HALL and BROWN, 1979;ANDERSON, 1997;NONOGAKI and UGUCHI, 1997).The programmed glucose stress in the young bulls has been presented in our earlier paper (KOLATAJ et al., 1998).We did not meet another Communications connected with the reactivity of lysosomal enzymes under the influence of glucose in animal tissues.
We suppose that the revealed changes in the activity of these enzymes are one of the elements of adaptation reaction to biochemical stress caused by the excess of exogenous glucose.
show that the activities of all estimated enzymes of the liver, kidney and muscle except AAP, LAP and Cath.D decreased in comparison with the values of control group.The data of analysis of variance are presented in Table 4. Statistically confirmed differences in the liver were revealed for BGLU [F = 5.01] and AP [F = 7.90]; in the kidney for BGRD [F = 8.27], BGAL [F = 14.42],NAGL [F = 8.56], AP [F = 13.07] and LL [F = 17.40]; in the muscle for NAGL [F = 5.39] and LL [F = 9.94].

Table 1
The activity of lysosomal enzymes ( x ± S d ) in the liver of the rabbits (in nmol/mg of protein/hour) after the 40% Solution of glucose administration; control = 100% (Aktivität der lysosomalen Enzyme in der Leber von Kaninchen, Kontrollgruppe = 100%)

Table 3
The activity of lysosomal enzymes ( x ± S d ) in the muscle of the rabbits (in nmol/mg of protein/hour) after the 40% Solution of glucose administration; control = 100% (Aktivität der lysosomalen Enzyme im Muskel von Kaninchen, Kontrollgruppe -100%)

Table 4
Analysis of variance for the activities of lysosomal enzymes in the liver, kidney and in the muscle of experimental rabbits; A -between groups; B -within groups; D.F. -in all cases for A -1; for B -18 (Varianzanalyse ftlr die Aktivität der lysosomalen Enzyme in der Leber, der Niere und im Muskel von