Biotechnol. Appl. Biochem. (2003) 38, 183–191 (Printed in Great Britain)
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Temporal response pattern of biochemical analytes in experimental diabetes Daniela M. Mori*, Amanda M. Baviera*, Lizeti Toledo de Oliveira Ramalho†, Regina C. Vendramini*, Iguatemy L. Brunetti* and Maria T. Pepato*1 ˆ ˆ *Departamento de An´alises Cl´ınicas, Faculdade de Ciencias Farmaceuticas de Araraquara, Universidade Estadual Paulista Julio de Mesquita Filho – UNESP, Brasil, and †Departamento de Morfologia, Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista Julio de Mesquita Filho – UNESP, Brasil
The activities of the enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), creatine kinase (CK), amylase (AMS) and angiotensin converting enzyme (ACE) have been used to assess the toxic effects of xenobiotics that have hypoglycaemic action in hepatic, pancreatic, renal and muscle tissue. Using a validated experimental model of diabetes mellitus in rats, we ascertained whether this syndrome itself affected the serum activities of these enzymes over a 53-day period. Levels of hepatic enzymes AST, ALT and ALP were higher in the streptozotocin (STZ)diabetic rats (group D), but were controlled by insulin therapy (group DI). AMS was reduced in group D and unchanged in group DI rats. Proteinuria was detected 1 day after STZ administation and partially controlled by insulin (group DI); its early presence in group D rats, and the lack of any change in serum ACE in this group, indicates that proteinuria is the better marker for microangiopathy. Microscopic examination of liver, kidney, heart and skeletal muscles (soleus and extensor digitorum longus) revealed various alterations in group D rat tissues, which were less pronounced in group DI. The liver, pancreas and kidney tissue-damage was consistent with the altered serum levels of AST, ALT, ALP and AMS and proteinuria. We conclude that: (i) rigorous control is required when these serum-enzyme levels are used as indicators of tissue toxicity in experimental diabetes, and (ii) LD, CK and bilirubin serum levels, which are unaffected by diabetes, can be used when testing effects of xenobiotics on tissues.
Introduction The diversity of cell types encountered in the various tissues and organs of the body are enabled to perform their specific tasks by the presence of unique structures and metabolic pathways within them, or particular patterns of enzymes.
With the exception of the plasma-specific enzymes, such as those involved in coagulation, all enzyme activity found in plasma or serum originates in tissues. A change in such an activity may reflect an increase of its production or, more frequently, cell damage and/or blocking of its normal route of clearance, with or without exposure to drugs and/or toxins [1]. Thus, activity in the serum of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), creatine kinase (CK), amylase (AMS) and angiotensin converting enzyme (ACE) have been used to evaluate the toxic effects of xenobiotic products (natural or synthetic) known for their hypoglycaemic action, in tissues such as liver, muscle, pancreas and vascular, both in this laboratory [2] and elsewhere [3–6]. However, it is possible that diabetes mellitus, in itself, affects the serum levels of these enzymes, given that insulin deficiency promotes a number of changes in the tissues [7]. Employing a validated model of experimental diabetes in rats, we investigated the temporal response pattern of various biomolecules, including the enzymes referred to above, and evaluated their potential as toxicity markers in tests of natural and synthetic hypoglycaemic agents.
Materials and methods Experimental protocol A group of 50 male 6-week-old Wistar rats, supplied freely with food and water and weighing 147.0 + − 11.0 g, were placed in metabolic cages and allowed to adapt for 3 days. Key words: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, streptozotocin (STZ)-diabetic rat, seric enzyme, streptozotocin. Abbreviations used: AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; LD, lactate dehydrogenase; CK, creatine kinase; AMS, amylase; ACE, angiotensin converting enzyme; STZ, streptozotocin; w, weight-change relative to day of STZ or saline administration; N, normal; D, diabetic; DI, insulin diabetic; EDL, extensor digitorum longus. 1 To whom correspondence should be addressed (e-mail
[email protected]). C
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At the start of the experiment (t−3 ), the following initial physiological and biochemical measurements were made: body weight, food and fluid intake and urine excretion, the urine being divided into aliquots for the evaluation of urinary urea and proteinuria, and blood from the tip of the tail was collected in Eppendorf tubes, with or without 15 µl of liquaemin sodium (Roche, Indianapolis, IN, U.S.A.), for the determination of plasma glucose or serum enzymes AST, ALT, ALP, LD, CK, AMS and ACE, respectively. After 3 days of adaptation (t0 ) 40 animals (body weight 148.9 + − 10.9 g) were deprived of food for 14–16 h, anaesthetized with diethyl ether and injected with streptozotocin (STZ), at 50 mg/kg body weight, in 0.01 M citrate buffer (pH 4.5), via the jugular vein. The control group of 10 rats, chosen at random from the original 50, were fasted in an identical manner and had a volume of 0.9 % saline solution, equal to that of the STZ, injected by the same route. All 50 rats were then returned to their metabolic cages and given free access to food and water. One day after injection of STZ or saline, the following variables were measured: change in body weight (w, weight-change relative to day of STZ or saline administration), urinary volume and proteinuria. At 3 days after the injections (at t3 ), the enzyme activities, plasma glucose, urinary glucose and urea, proteinuria, water and food intake, urinary volume and w were measured. From the 40 STZ-diabetic rats, 10 pairs with symptoms similar in severity were selected by matching body weight, serum glucose level, urinary glucose excretion and urinary urea as closely as possible. One rat from each pair was randomly assigned to the STZ-diabetic group (D, n = 10), while the other was assigned to the STZ-diabetic control group which was to receive insulin (DI, n = 10). The remaining 20 rats, which could not be matched closely, were not used. The rats of group DI were treated twice a day (9:00 and 18:00 h) for 47 days, by subcutaneous injection of 3 units of NPH (neutral protamine Harguerdon) insulin (Biohulin NU-100; 100 units/ml; Biobr´as, Montes Claros, MG, Brazil), this treatment being initiated on day 6 after STZ administration. In paired tests, we measured the activities of serum enzymes, plasma glucose, urinary glucose and urea, intake of food and water, every 15 days, while proteinuria, urinary volume and w were measured approx. every 7 days until 53 days after STZ or saline injection (47 days of the treatment with insulin). The rats were killed in the morning (08:00–09:30 h) by decapitation, and samples of the free-running blood collected for the determination of plasma levels of glucose, serum bilirubin and all serum enzymes. Approx. 2 h before being killed, blood samples were collected from the tail of each rat to determine serum CK alone. The blood samples used to determine plasma glucose were collected in Eppendorf tubes with liquaemin sodium, stood on ice for 30 min, and then were centrifuged at 1000 g for 10 min to C
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obtain the plasma. The blood samples used to quantify serum enzymes were collected in tubes containing gel separator (Becton Dickinson, San Jos´e, CA, U.S.A.), and the serum separated from the whole blood by centrifuging at 1000 g for 10 min. The measurements of plasma glucose, serum bilirubin and the seric activities of AST, ALT, ALP and ACE, as well as urinary glucose and urea, were carried out up to 15 days after collection of the samples, which were stored at − 20 ◦ C. LD and CK were measured within 4 and 7 days of collection, respectively, LD having been stored at 25 ◦ C and CK at − 20 ◦ C. All measurements of an individual variable were made on the same day, in groups of 10 samples. Histological analysis Immediately following decapitation, the liver, heart, pancreas, kidney and the soleus and extensor digitorum longus (EDL) muscles were removed from each animal, immediately immersed in 10 % (v/v) formaldehyde and kept in this fixative for 72 h. Next, the tissues were dehydrated in a series of ethanol solutions [70, 80, 90 and 100 % (v/v)], cleared in xylene and embedded in paraffin wax. Thin sections (6 µm) were stained with haematoxylin/eosin and examined under an optical microscope (Zeiss, model Jenaval) (n = 3 per organ per group, samples chosen at random). The slides were examined without the histologist knowing the animal or group from which the tissue was taken. Feeding and housing conditions and ethical approval All rats were fed a normal laboratory commercial stock diet containing (w/w) 16 % protein, 56 % carbohydrate and 8 % fat and were housed under a 12 h:12 h light/dark cycle at 22– 25 ◦ C. The experimental protocols and the treatment and care of the rats followed the rules approved by the Ethics Committees. Chemical analysis Urinary glucose was measured by the o-toluidine method of Dubowski [8], urea by the urease method [9,10] and proteinuria by the modified Bradford method [11]. Plasma glucose and the serum enzymes AST, ALT, ALP were determined in a Bayer Technicon RA-XT autoanalyser. Serum activities of LD [12] and AMS [13] and serum concentration of bilirubin [14,15] were assayed by colorimetric methods; activities of CK [16] and ACE [17] by kinetic spectrophotometric methods. In all these assays, storage times and temperatures were controlled rigorously, in order to minimize deterioration of samples. These enzymic activities and other measurements were performed on a Hitachi U 1100 spectrophotometer. Unless otherwise indicated, all reagents were purchased from Merck or Sigma and were of at least analytical grade.
∗ a 216.6 + − 14.0 † ∗ a 67.0 + 14.4 † − ∗ 18.8 + 0.9 † − ∗ a 74.6 + − 4.1 † ∗ a 52.3 + − 3.4 † ∗ a 7.16 + − 0.32 † ∗ a 3.90 + − 0.20 † ∗ a 835.2 + 44.9 † −
364.6 + − 13.8 ∗a 212.2 + − 14.7 a 7.3 + 0.3 − ∗ 19.2 + − 2.6 ∗a 14.6 + − 2.6 1.59 + − 0.30 ∗ 1.08 + − 0.20 ∗a 332.2 + 25.3 − 415.5 + − 11.1 a 276.3 + − 9.5 a 4.9 + 0.3 − a 9.2 + − 0.3 3.6 + − 0.5 1.75 + − 0.10 0 209.4 + − 14.6
D
∗a
DI
300.8 + − 8.7 ∗a 148.4 + − 8.6 ∗ 12.2 + 0.5 − ∗a 40.7 + − 4.3 ∗a 26.6 + − 3.8 1.03 + − 0.13 ∗ 1.19 + − 0.19 ∗a 514.3 + 44.5 − 336.1 + − 6.7 a 198.9 + − 5.6 a 8.5 + 0.4 − 14.2 + − 0.5 4.1 + − 0.4 1.69 + − 0.04 0 168.8 + − 4.29
226.4 + − 11.8 † ∗ a 76.8 + − 12.0 † ∗ 19.3 + 1.2 † − ∗ a 75.9 + − 4.0 † ∗ a 53.9 + − 3.8 † ∗ a 6.94 + − 0.24 † ∗ a 2.63 + − 0.28 † ∗ a 766.5 + 60.7 † −
a
N
177.2 + − 3.4 17.7 + − 2.7 16.2 + − 0.4 ∗a 60.6 + − 2.8 ∗a 35.0 + − 2.3 ∗a 5.19 + − 0.13 ∗a 2.35 + − 0.23 ∗a 625.0 + 43.6 − 178.9 + − 3.1 39.8 + − 2.4 16.1 + − 0.6 23.5 + − 0.6 3.4 + − 0.4 1.69 + − 0.03 0 201.3 + − 22.9 164.3 + − 2.8 − 15.6 + − 1.1 23.5 + − 0.7 3.7 + − 0.4 1.35 + − 0.04 0 237.7 + − 22.3 Body weight (g) w (g) Food intake (g/24 h per100 g of bw) Liquid intake (ml/24 h per100 g of bw) Urinary vol. (ml/24 h per100 g of bw) Plasma glucose (mg/ml) Urinary glucose (g/24h per100 g of bw) Urinary urea (mg/24h per100 g of bw)
153.8 + − 1.2 − 17.9 + − 1.0 25.4 + − 0.7 2.9 + − 0.6 1.42 + − 0.04 0 229.7 + − 26.9
162.5 + − 2.7 − 18.6 + − 1.1 24.9 + − 1.2 3.0 + − 0.6 1.30 + − 0.05 0 237.3 + − 26.2
D DI N DI Group. . .
N
D
171.7 + − 2.7 22.3 + − 2.7 ∗ 18.8 + − 1.5 ∗a 62.4 + − 4.1 ∗a 36.0 + − 3.0 ∗a 5.14 + − 0.18 ∗a 2.45 + − 0.26 ∗a 592.6 + 34.6 −
a
a
DI N
D
∗ a
53 [47] −3
25 [19] After STZ administration (insulin treatment)
3
Before STZ.administration
In order to evaluate how diabetes may influence the activities of serum enzymes, a completely reliable experimental model of diabetes must be employed. The induction of diabetes by injection of STZ, in rats maintained throughout in metabolic cages, was proved to fulfil these requirements in our validation tests (Table 1). It can be seen that, on the initial day (t−3 ), the three groups of rats (N, D and DI) had similar mean body weights and metabolic variables, while on day 3 after STZ administation (t+3 ), most of the variables measured in the caged rats had increased in both the D and DI groups, relative to the normal group N (P < 0.05). Moreover, groups D and DI exhibited similar mean severity of diabetes. On days 25 and 53 after induction of diabetes by STZ, group D showed significantly increased values (P < 0.05) of all these variables except body weight, both compared with group N and compared with initial values (at t−3 ). As expected in growing rats, all groups exhibited a increase in body weight, but the growth seen in group D was significantly less than that in group N. Group DI, evaluated on days 19 and 47 of insulin treatment, had lower values than group D for the physiological and metabolic variables, plasma glucose actually being similar to that in group N, while the increase in body weight was higher than in group D (P < 0.05). Activities of the liver transaminases AST and ALT increased markedly in the serum of group D rats, from day 25 after onset of diabetes, compared with group N (inter-group) or with pre-diabetic values on day t−3 (intragroup); see Figure 1. This effect was mostly eliminated by insulin treatment. The same general trend was observed for serum ALP (another liver enzyme), although in this case the activity was aggravated earlier, from day 3 of diabetes onset, causing an initial rise in group DI that was corrected by the subsequent insulin treatment. The amount of bilirubin, whether conjugated with glucuronic acid in the liver (direct) or not conjugated (indirect), as well as the total, was found to be similar in each of the groups N, D, DI, taken in pairs (Figure 1). Liver tissue was subjected to histological analysis (Figure 2), in order to check whether the alterations to liver enzyme levels in the serum were accompanied by compatible changes in the tissue. In normal rats, polyhedral hepatocytes were observed to have a central nucleus and visible nucleolus; the cytoplasm was well-stained with basophil
Table 1 Metabolic variables in non-diabetic rats and STZ-diabetic rats, treated and untreated with insulin
Results
Number of days. . .
Statistical analysis Data are expressed as means + − S.E.M. They were analysed by two-way ANOVA, and the Newman–Keuls test for subsequent multiple comparisons. The unpaired Student’s t test was used to analyse bilirubin data. The significance level was P < 0.05.
All values are means + − S.E.M. (n = 10). All parameters but body weight (bw), w and plasma glucose are given per 100 g of body weight. Two-way ANOVA and Newman–Keuls test used for comparisons (P < 0.05) between groups for identical time periods (∗ different from N; †different from D) and over time within groups (a ) different from the first day (t−3 ) in same group.
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(A)
(C)
(B)
(D)
Figure 1 Effect of chronic STZ-diabetes on levels of liver enzymes (A) AST (B) ALT (C) ALP and (D) bilirubin in rat serum
䊏 (A, B and C) and N (D), normal group; 䉱 (A, B and C) and DI (D), STZ-diabetic group treated with 6 units/day insulin from day 6 after STZ administration; 䊉 (A, B and C) and D (D), STZ-diabetic group. All values are means + − S.E.M. (n = 10). Two-way ANOVA and Newman–Keuls test was used for comparisons (P < 0.05) between groups for identical periods (*different from N; †different from D); and over time within groups (a ) and different from first day (t−3 ) in the same group. Bilirubin data were analysed by unpaired Student’s t test. Abbreviation: U, units.
bodies. By contrast, the hepatocytes of group D rats exhibited nuclear polymorphism, slight polyploidy, visible nuclear chromatin with coarse granules, cell hypertrophy, weak staining of the cytoplasm and fibrosis. These abnormalities were milder in group DI. Clearly, the visible alterations revealed by histology were consistent with the changes in serum activities of liver enzymes. The serum activities of LD and CK, enzymes that originate mostly in muscles, were not significantly different in the three groups (Figure 3). In the case of CK the unusually high activity in group DI, on day 47 of insulin treatment, is presumed to be a chance occurrence. When the cardiac tissue of the STZ-diabetic rats was examined histologically, we observed vascular dilation and congestion, as well as splitting of the endothelium and complete loss of normal capillary morphology. In group DI, these changes were less pronounced. Considering the soleus C
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muscle, we noted rarefaction of the cells and disorganized fibres in the striations in group D, while in group DI there were normal cells, but with some pale fibres around the normal ones. On the other hand, in the EDL muscle spindle, the cells had a normal appearance in both groups D and DI, although in rats of group D, the spindle cells of the EDL were altered. The serum activity of amylase is plotted against time in Figure 4, for the whole STZ-diabetic period of 53 days, and it can be seen that diabetes leads to a considerable fall in AMS activity from day 11 after STZ administation, and that this fall was not reversed by treatment with insulin. On day 3 after STZ administation, a significant fall of AMS activity was observed in group DI, and a non-significant fall in group D. The decrease in activity in group N (relative to day t−3 ) and the divergence between groups D and DI at the end of the experimental period (53 days after STZ administration/
Experimental diabetes and serum enzymes
Figure 3 Lack of effect of chronic STZ-diabetes on levels of muscle enzymes LD (A) and CK (B) in rat serum
Figure 2 Histological preparations of liver tissue from chronic STZ-diabetic rats (A) Normal rat (arrow indicates normal appearance of hepatocyte), (B) STZdiabetic rat treated with 6 units/day insulin for 47 days (arrow indicates polyploid cell) and, (C) STZ-diabetic rat (arrow 1 indicates fibrosis in stroma of tissue, arrow 2 indicates basophil bodies and arrow 3 indicates morphologically altered hepatocytes). Magnification was approx. × 400.
day 47 of insulin treatment) appear to be atypical data points. In histological preparations of the pancreas, organs taken from group D rats exhibited copious secretions in the dilated secretory duct, the latter being enveloped in fibrous stroma and dilated blood vessels. We also discovered morphological changes in the serous acini, with zymogen granules retained in the cytoplasm. The islets of Langerhans exhibited deformed nuclei and little cell differentiation. Similar preparations of pancreas from group DI rats showed some alterations, but they were less obvious.
䊏, Normal group; 䉱, STZ-diabetic group treated with 6 units/day insulin from day 6 after STZ administration; 䊉, STZ-diabetic group. All values are means + − S.E.M. (n = 10). Two-way ANOVA and Newman–Keuls test were used for comparisons (P < 0.05) between groups for identical periods; †different from D. Abbreviation: U, units.
The serum activity of ACE, which is a marker for microangiopathy (including renal), was found to be similar in all three groups throughout the period of the study (Figure 5). Another marker for kidney function change, proteinuria, was above normal from the first day after administration of STZ until the end of the experiment, and this enhanced level was partly corrected by insulin, from day 12 of insulin treatment (day 18 after STZ administration). Microscopic examination of the kidneys revealed, in group D rats, morphological changes in the glomeruli, with randomly placed nuclei and vasodilation of the capillaries. Next to the proximal convoluted tubule were found clusters of cells with clear cytoplasm, forming circular structures like ducts, but without cell membranes. In group DI kidneys, the morphological changes were much less severe. C
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Figure 4 Effect of chronic STZ-diabetes on AMS levels in the rat
䊏, Normal group; 䉱, STZ-diabetic group treated with 6 units/day insulin from day 6 after STZ administration; 䊉, STZ-diabetic group. All values are
means + − S.E.M. (n = 10). Two-way ANOVA and Newman–Keuls test were used for comparisons (P < 0.05) between groups for identical periods [(*) different from N, (†) different from D]; and over time within groups (a) different from first day (t−3 ) in the same group. Down arrow indicates atypical data points for D and DI groups at 53 days after STZ/day 47 of treatment. Abbreviation: U, units.
Discussion A variety of physiological and biochemical variables were measured in rats, by means of metabolic cages, in order to validate our model of experimental diabetes. This model indeed proved adequate, as classic effects of diabetes were observed, partially reversed by insulin treatment (Table 1). Interestingly, animals in the normal control group exhibited a fall in their food and fluid intakes. In the earlier part of the experiment, the rats were visibly agitated in response to daily manipulation, after which they progressively became more used to being handled, so that they moved around less, slept more and were calmer, resulting in lower energy consumption and more efficient metabolism of food. Thus, rates of eating and drinking would be expected to fall, after an initial rise. Toward the end of the experiment, we believe that the continuing fall in both these variables was due not only to the above effect, but also to the normally reduced energy needs as animals reach adulthood. It was found that diabetes raises the levels of activity in the serum of the liver enzymes AST, ALT and ALP (Figure 1), a result that agrees with observations of alloxaninduced diabetic rats [18] and of human diabetes [19]. Such alterations of transaminase activity in the tissues is explicable in terms of energy metabolism, as these enzymes play a role in gluconeogenesis. In diabetes, the stores of glycogen in the liver and muscles are diminished and, in compensation, levels of AST and ALT are raised to produce alternative glucose precursors [20]. In support of this idea, Salimuddin et al. [21] observed that insulin stimulates alanine production in C
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Figure 5 Effect of chronic STZ-diabetes on proteinuria (A) and the absence of effect on serum level of ACE (B)
䊏, Normal group; 䉱, STZ-diabetic group treated with 6 units/day insulin from day 6 after STZ administration; 䊉, STZ-diabetic group. All values
are means + − S.E.M. (n = 10). Two-way ANOVA and Newman–Keuls test were used for comparisons (P < 0.05) between groups for identical periods (*different from N; †different from D); and over time within groups (a ) different from first day (t−3 ) in the same group. Abbreviation: U, units.
muscle and liver tissue, leading to the restoration of nearnormal levels of these transaminases. Moreover, expression of AST mRNA was augmented in liver tissue from STZdiabetic rats, the effect being reversed by administration of insulin [21]. The results reported here with group DI, in which the serum activities of AST and ALT were reduced by insulin treatment to near-normal values (Figure 1), though deriving from the pools of AST and ALT in the tissues, are at least compatible with the results above. In fact, considering the observations that the serum level of AST reverts to normal and that the corresponding mRNA is modulated by the presence of insulin [21], it is likely that the exogenous insulin is the signal for AST (and possibly ALT), in hepatocytes,
Experimental diabetes and serum enzymes
which in turn leads to a reduced rate of gluconeogenesis and a concomitant fall in glycaemia. The mechanisms by which the serum levels of AST, ALT and ALP are raised in diabetes may involve increased liberation of these enzymes from tissues (mainly liver), owing to oxidative stress and/or the formation of advanced glycosylation end products (AGES) [22,23]. This hypothesis is consistent with the changes observed in the tissues, the weak staining and large volume of the cytoplasm, polymorphism, polyploidy, chromatin granules and fibrosis. Some of these changes, in liver from a group D rat, and the milder effects observed in tissue from group DI, can be seen in Figure 2. Some studies have attributed the rise in serum ALP to the toxic effects of STZ on the liver [24]. However, it is clear from our results that insulin treatment reverses this rise, suggesting that insulin deficiency is the actual cause. The bilirubin in the serum, which is used as an indicator of hepatic and biliary function, suffered no change in the concentration of any of its forms in rats with experimental diabetes (Figure 1). The few published data on bilirubin levels in diabetes are contradictory; there are reports of lower, higher [25,26] and unchanged [27] serum levels in diabetes, in both animals and humans. The serum activity of the enzyme LD was similar in all three groups, N, DI and D (Figure 3). Published data correlating such activity with diabetes are scarce, but our results contradict those of Mansur et al. [26] and Stanely et al. [5], who found the level of this enzyme to be raised in diabetes. Concerning the serum level of CK, it was found, surprisingly, that this activity was affected drastically by the way in which the blood was collected. Thus, when the rats were decapitated on day 53 after STZ administration, the activity measured in groups N, DI and D was 6–10 times higher than on the first day (t−3 ) (results not shown), while no alteration was seen when the blood was collected from the tail (Figure 3). Reports in the literature are contradictory; both higher and lower CK activities have been observed, in serum or tissue, either in experimental or in human diabetes [7,28– 30]. Insulin and the thyroid hormones have been considered to be powerful regulators of the enzyme CK. Low insulin provokes destabilization of its mRNA [31], while insulin treatment has led to recuperation of the loss of CK isoenzyme MB and CK isoenzyme BB activity in diabetic rats [32]. Although we used the same animal model and similar duration of experiment and severity of diabetes in our study of CK activity, we found no significant change in its serum level (Figure 3). Microscopic examination of tissues from the heart and skeletal muscles (soleus and EDL) revealed several alterations in group D, which were corrected, at least
partly, by insulin therapy. Nevertheless, these tissue changes cannot have been enough to affect the serum activities of the enzymes LD and CK. Possibly, the relative activities of their isoenzymes in the tissues are affected. In Figure 4, the rise in seric AMS activity (principally of salivary and pancreatic origin) between day 11 and day 40 after administration of saline, in group N, may be related to fluctuations in the rates of synthesis and secretion of the secretory glands. Such fluctuations have been attributed to aging of the animals [33–36]. The fall in AMS serum activity (Figure 4) corroborates the data of Barneo et al. [3], who observed the same in diabetic animals and proposed a correlation with the severe insulin debt found in diabetes type 1, since this fall was not seen in type 2 cases [3]. The amylase activity was also reduced in the pancreas, showing that diabetes was associated with a lowering of its exocrine function [37]. It is known that insulin stimulates the production of pancreatic amylase mRNA, being a direct activator of AMS gene expression, which increases the activity by augmenting protein synthesis [38]. Moreover, this hormone increases the flow of pancreatic juice [39], as there are insulin receptors on the cells of the acini of rodent and human pancreas that promote the modulation of enzyme secretion [40]. Further data suggest that insulin plays this important part in the control of pancreatic exocrine function. In histological and morphological analyses it has been demonstrated that acinus cells around the islets of Langerhans are richer in zymogen granules than other acinar cells. The halo of zymogen around the islets has been observed to disappear, and levels of amylase to diminish progressively in experimental diabetes, whereas this activity recovered on treatment with insulin [40]. In our study, loss of AMS activity was not reversed by insulin treatment. The very marked histological changes seen in pancreatic tissue from group D rats, and the milder changes in group DI tissue, are consistent with previous reports [41]. Such alterations suggest cell damage in the pancreatic tissue of all the STZ-diabetic animals (D and DI), the damage in the endocrine region being caused possibly by STZ, given the selective active of STZ on this tissue [42]. However, the experimental method used cannot differentiate between possible reasons for the fall in AMS activity: a direct toxic effect of STZ on the acinus cells, a consequence of the effect of STZ on the β-cells of the islets of Langerhans or both. Proteinuria, a widely-recognized corollary of diabetes [43], was demonstrated in the present study, using metabolic cages. It appeared very early (first day after STZ) in group D rats and was partly eliminated in group DI (Figure 5). Hence, in the context of this experimental model, the measurement of proteinuria may be useful in the testing of xenobiotic compounds to counteract the tubule-toxic effects and/or fibrosis in the tubules that are C
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responsible for the deterioration of kidney function in diabetes [44]. Regarding seric ACE activity, we found no significant difference between the groups of rats, and the apparent divergence between groups D and DI, 3 days after STZ, is presumably due to a random effect (see Figure 5). In the literature, levels of ACE activity in serum and tissue from diabetic animals and humans are not well-defined, as there have been reports of increased, decreased and unchanged activities [4,45–48]. Comparing the methodology described here with that used by the authors who observed changed ACE activity, we can exclude the following as the cause of these differences in results: method of quantifying [49], severity and duration of the diabetic state [4,43,45], type of blood collection and storage [49]. Meanwhile, Bor [49] has reported that liver malfunction in human diabetes can inactivate the ACE in serum. This result corresponds with our findings on transaminases and ALP. Summarizing, we conclude that diabetes in itself leads to raised levels of the enzymes AST, ALT and ALP in the serum, suggesting that rigorous control is needed when these activities are employed as liver toxicity indicators in studies of the effects of xenobiotics used against diabetes. AMS suffers a clear-cut fall in experimental diabetes, whereas LD, CK and bilirubin in serum, which did not show any alterations, may be suitable for evaluation of the toxicity of vegetable and synthetic products in liver and muscle tissue (taking isoenzymes into account). As ACE was not seen to alter in diabetes, proteinuria is proposed for evaluation of renal dysfunction.
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Acknowledgments We thank the Fundac¸a˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP), Fundac¸a˜ o para o Desenvolvimento da Universidade Estadual Paulista (FUNDUNESP) and Programa de Apoio ao Desenvolvimento Cient´ıfico – Faculdade de Ciˆencias Farmacˆeuticas de Araraquara (PADC-FCFAraraquara – UNESP) for financial support. D. M. Mori and Baviera A. M. received fellowships from FAPESP and Programa Institucional de Bolsas de Inicia˜c¸ao Cient´ıfica (PIBIC). We are also grateful to Mrs V. C. O. Alves, Mr F. R. A. Rapussi, Mrs T. N. Nunes and Mrs F. A. Iagame for their technical assistance.
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Received 20 February 2003/19 June 2003; accepted 24 June 2003 Published as Immediate Publication 26 June 2003, DOI 10.1042/BA20030034
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