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Streptozotocin- and alloxan-diabetes in mice
EUROPEAN JOURNAL OF PttARMACOLOGY 7 (1969) 89-96. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM
STREPTOZOTOCIN-
AND ALLOXAN-DIABETES
in M I C E
Claus RERUP Department o[ Pharmacology, University o f Lund, Sweden and Finn TARDING Research Laboratories, Novo Therapeutisk Laboratorium A IS, Copenhagen, Denmark
Receded 27 January 1969
Accepted 12 March 1969
C.RERUP and F.TARDING, Streptozotocin- and alloxan-diabetes in mice, European J. Pharmacol. 7 (1969) 89-96. In non-fasted albino (NMRI) mice intravenous diabetogenic doses were 200 mg/kg body weight of streptozotocin or 70 mg/kg of aUoxan monohydrate. Differences in diabetes development were as follows. Maximal blood glucose levels during the phase of acute hyperglycaemia were observed 120 min after streptozotocin and 45 min after alloxan injection. Liver glycogen was depleted more slowly after streptozotocin than after alloxan. Tolbutamide given 5 or 60 min following streptozotocin injection clearly affected the blood sugar response. Elevated plasma insulin levels were found during the hypoglycaemic phase following injection of either drug. Plasma insulin and blood glucose levels were poorly correlated following streptozotocin treatment in contrast to findings in alloxan treated animals. Streptozotocin hypoglycaemia was more severe than alloxan hypoglycaemia. During the chronic hyperglycaemia the blood glucose levels were relatively stable within individuals following diabetes induction by means of either drug but there were large individual variations. However, alloxan-diabetic mice resumed and surpassed their original body weight with time, streptozotocin-diabetic animals did not. Both types of diabetes were normally sensitive to exogenous insulin. Streptozotocin-diabetes appeared to be irreversible. It is concluded that the development and chronic state of diabetes in mice following streptozotocin and alloxan, respectively, are not identical. Streptozotocin Alloxan
Plasma insulin Blood glucose
1. INTRODUCTION Streptozotocin, an antibiotic obtained from streptomyces achromogenes, is composed o f 1-methyl-1nitrosourea and glucose (Herr et al., 1967). The compound has a wide range o f pharmacological properties. Thus, it possesses antibiotic, diabetogenic and both antitumoural (Evans et al., 1965) and cancer inducing properties (Arison and Feudale, 1967). Streptozotocin induces diabetes in the rat, dog (Rakieten et al., 1963), hamster, m o n k e y and the Swiss mouse (Schein et al., 1967). There is general agreement that streptozotocin-diabetes - like alloxandiabetes - is brought about by a b e t a - c y t o t o x i c action
Tolbutamide Liver glycogen
in the islets of Langerhans although several findings suggest that the mechanisms o f diabetogenesis are different. Ketosis or elevation of plasma-free-fattyacid levels does not occur in streptozotocin-diabetic rats (Mansford and Opie, 1968). Streptozotocin has a higher specificity as a /3-cytotoxic agent in the rat (Arison et al., 1967; Junod et al., 1967). Streptozotocin but not alloxan, depleted mouse liver nicotinamide adenine dinucleotide (NAD) levels (Schein and Loftus, 1968). This last effect could be prevented by pretreatment with nicotinamide. These findings prompted a study o f streptozotocin- and alloxan-diabetogenesis using one strain of mice under similar laboratory conditions.
90
C.RERUP and F.TARDING
Table 1 Effect of increasing intravenous doses of streptozotocin to non-fasting mice on diabetogenesis, mean body weight change and mortality. No. of mice
Streptozotocin dose mg/kg
Diabetes frequency after 7 days
Mean body weight change after 2 weeks, g
Mortality after 2 weeks
5 5 5 5
70 140 200 300
0/5 1/5 4/4* 3/3**
+ 3.2 + 3.5 - 1.0 - 2.0
0/5 0/5 1/5 4/5
* One mouse died in hypoglycaemia. ** Two mice died in hypoglycaemia. 2. MATERIAL AND METHOD
3. RESULTS
Female albino NMRI mice* weighing 2 0 - 2 5 g were used throughout. During all experimental conditions the animals had free access to a standard pellet diet** and tap water. Streptozotocin*** was dissolved in redistilled water and given within four minutes from the time of dissolving. Alloxan monohydrate was dissolved at pH 5 (HC1) and given at a dose of 70 mg/kg body weight within 10 min from dissolving. Tolbutamide was dissolved at pH 8.3 and given at a dose of 80 mg/kg body weight. All drugs were injected intravenously via a tail vein. Blood sampling (25 /al and 250 /al for determinations of blood glucose and plasma insulin levels, respectively) was performed by orbital puncture using commercial constriction pipettes (Rerup and Lundquist, 1966). Blood glucose levels were determined enzymatically (Marks, 1959), plasma insulin levels immunologicallyt (Heding, 1966) and liver glycogen enzymatically (Rerup and Lundquist, 1967). Diabetes was considered to be manifest in mice with blood glucose levels of 200 mg/100 ml or above. Animals lacking this criterion were discarded.
3.1. Diabetogenic dose of streptozotocin
* Laboratory Animal Breeding, Laven, Denmark. ** Ferrosan Ltd., Maim6, Sweden. *** Provided through the courtesy of W.E.Dulin, Ph.D., The Upjohn Company, Kalamazoo, Michigan, USA. t Anti-insulin serum from guinea pigs and 12sI-insulin were kindly provided by Mrs. L.Heding, M.Sc., Novo Research Institute, Copenhagen, Denmark.
Due to limitations of the amount of streptozotocin available groups of 5 mice were injected intravenously with increasing doses as shown in table 1. It appears from table 1 that 70 mg/kg body weight was without diabetogenic effect, 140 mg/kg was diabetogenic in one out of five mice. In these two groups there was a normal body weight gain observed during 20 days from streptozotocin treatment. When the dose was increased to 200 mg/kg all mice became diabetic and one died 3 days after the treatment. A further increase of the dose to 300 mg/kg killed 4 out of 5 mice, two during hypoglycaemia and two on the 3rd and 5th day following treatment leaving one diabetic mouse alive. In the last two dose groups the mean change in body weight after drug treatment was negative. When the dose of 200 mg/kg body weight of streptozotocin was repeatedly injected into groups of mice it was invariably followed by a diabetic state. The mortality within one week from injection was 5 - 1 0 per cent and was usually due to the severe hypoglycaemia occurring 8 - 2 4 hr after drug administration. Subsequently streptozotocin at a dose of 200 mg/kg body weight was used and compared with 70 mg/kg of alloxan monohydrate, which has been shown to be a suitable diabetogenic dose at this laboratory (Rerup and Lundquist, 1967).
STREPTOZOTOCIN- AND ALLOXAN-DIABETES IN MICE
91
BLood gtuo0se,mg / 100 rnL Alloxan 300"
Alloxan
200-
100-
o 3'~ ~ ~ ~
~
~
Io
~'3
io
~'8
hours
Fig. 1. The effect of intravenous injection of alloxan monohydrate (70 mg/kg) and streptozotocin (200 mg/kg) on blood glucose levels in non-fasted female NMRI mice. The curves in the left part of the figure differ significantly (p < 0 . 0 0 1 ) e x c e p t at zero and 8 hr. Alloxan and streptozotocin was given at random to groups of 8 mice each, 11 blood samples (25 t~l) per mouse.
A
Blood glucose, mg / 100 ml Strept. 300-
B Strept.
/
aline
Saline
200-
100
Tolbutamide
{~ 0~5 "I
2
rolbutomi de
3 hours
()
i
2
3
hours
Fig. 2. Effect of tulbutamide (80 mg/kg intravenously) on streptozotocin induced blood sugar fluctuations. Mean responses o f l 0 mice (a total of 40 mice). Tolbutamide given 5 min (A) and 60 min (B) following streptozotocin treatment (200 mg/kg). Significance levels P for the difference between saline and tolbutamide treated mice shown between mean responses,
92
C.RERUP and F.TARDING
Table 2 Liver glycogen and blood glucose levels 30 and 90 min following intravenous injection of aUoxan monohydrate (70 mg/kg), streptozotocin (200 mg/kg) and saline, respectively, into non-fasted mice. 10 mice per group. P = probability of random difference versus controls, n.s. = non-significant.
Group
Treatment
1
Saline
2
Streptozotocin
Observation time after injection
Mean blood glucose mg/100 ml 106 + 5*
30min
3.76 +0.41
n.s.
~0.001
2.23 -+0.35
~0.01
243 + 21
4.
Saline
115+ 4
5.
Streptozotocin Alloxan
4.66 + 0.66 n.s.
Alloxan
90 min
P
111 + 5
3.
6.
P
Mean liver glycogen % of wet liver
4.99+0.28
288 + 11
<0.001
2.45 + 0.43
~0.001
255 + 27
<0.001
5.00 + 0.61
n.s.
* Standard error of mean.
3.2. Blood glucose fluctuations The change in blood glucose level in two groups of 8 mice receiving 200 mg/kg o f streptozotocin and 70 mg/kg o f alloxan, respectively, are given in fig. 1. Alloxan evoked a sudden sharp rise in blood glucose level with a peak at 45 min. In contrast the blood sugar response to streptozotocin was delayed, with minimal changes during 45 min. A marked hyperglycaemia with a peak at 2 hr after treatment occurred while in alloxan treated mice the blood glucose level was decreasing towards hypoglycaemia. The lowest blood sugar levels following alloxan treatment were recorded after 6 hr and after at least 10 hr in the streptozotocin treated group. The hypoglycaemia following streptozotocin was more severe than that following alloxan (p < 0.02). In the streptozotocin treated group the lowest individual blood glucose level was 21 mg/100 ml obtained at 10 hr from a mouse which died shortly afterwards. One day (23 hr) after the treatment the mean blood glucose level was slightly hyperglycaemic in both groups, after two days it was clearly diabetic. In this experiment the mean blood glucose levels were not significantly different between groups on day 1 and day 2. 3.3. Liver glycogen levels Liver glycogen level was not significantly decreased 30 rain after streptozotocin injection; blood glucose level was normal. However, 30 min after
alloxan liver glycogen was markedly reduced and blood glucose was elevated, confirming previous work (Lundquist and Rerup, 1967). When liver glycogen was determined 90 min after treatment (about 30 min before the maximum of streptozotocin hyperglycaemia, but about 45 min past the peak o f alloxan hyperglycaemia) streptozotocin injected mice showed a marked liver glycogen depletion, whereas in alloxan treated mice liver glycogen levels had already returned to normal (table 2). Thus it appears that the initial hyperglycaemia following either drug is due, at least in part, to mobilization of glycogen from the liver.
3.4.Effect of sulphonylurea on the initial hyperglycaemia Tolbutamide, given 5 min after streptozotocin, evoked a marked hypoglycaemia at 30 and 60 min which substantially reduced the initial hyperglycaemic phase occurring at 120 min. When the tolbutamide injection was given closer to the time of peak hyperglycaemia following streptozotocin, i.e. 60 min following injection of the diabetogenic drug, the hyperglycaemia was again reduced and hypoglycaemia was observed 3 hr after streptozotocin injection. These findings contrast with the inability to tolbutamide to reduce the initial hyperglycaemia when the sulphonylurea is given after alloxan (Lundquist and Rerup, 1967).
STREPTOZOTOCIN- AND ALLOXAN-D1ABETES IN MICE
93
Table 3 Blood glucose and plasma immunoreactive insulin levels 9 hr following intravenous injection of streptozotocin (200 mg/kg) and saline, respectively, into non-fasted mice. P = probability of random difference between groups, r = correlation coefficient, n.s. = non-significant.
Group
No. of mice
1
8
2
16
Treatment Saline Streptozotocin
Mean blood glucose mg/100 ml
P
125 + 4* 30 -+ 8
Plasma insulin tt-unit/ml
P
r
<0.001
-0.49, n.s.
9.4 + 4.8
<0.001
79.3 + 8.7
* Standard error of mean
3.5. Hypoglycaemic phase Plasma insulin levels were elevated shortly before and during the streptozotocin induced hypoglycaemia. When recorded at 6 hr during falling blood sugar levels the elevation of plasma insulin levels was moderate but significant over controls. The negative correlation between blood glucose and plasma insulin levels was not significant in contrast to findings 4 hr after alloxan (Lundquist and Rerup, 1967). Nine hours after streptozotocin plasma insulin levels were markly elevated as shown in table 3. 3.6. Chronic diabetic state Most animals were diabetic 48 hr after injection of
g
either drug. However, in several mice chronic hyperglycaemia did not develop until one week after streptozotocin. Blood glucose levels during streptozotocin-diabetes were subject to individual variations, as in alloxandiabetes. The blood glucose level varied between mice from about 250 mg/100 ml to 550 rag/100 ml. The loss in body weight following alloxan injection was only transient and an increase over the initial weight was observed about three weeks after treatment. Following streptozotocin treatment, however, the mice did not regain their original body weight during an observational period of at least two months (fig. 3).
Body weight
30-
Diabetes
25-
induction AUoxan
20¸
Fig. 3. Body weight change of mice treated with streptozotocin (200 mg/kg) and allo×an monohydrate (70 mg/kg) during 56 days. Significance levels (P) for the difference between groups shown between mean weights (10 mice per group).
94
C.RERUP and F.TARDING Foil in brood gtucose I rag/100 mt
150. Strept
n
100.
F
i Anal.ysisof variance:
/
insutin togdose effect
// J/ 50"
P
(diabetes type (divergence
I.curvature
[/
n.s. n.s. n.So
A=n2s 018 -0.1
2 0.3
S 0.7
mU I 20 g mouse togdose
Fig. 4. Sensitivity to exogenous insulin of alloxan- and streptozotocin-diabetic mice. Mean responses of 6 mice (a total of 30 mice). Highly significant logdose response dependence, no divergence of slopes or curvature, no difference in sensitivity to insulin between diabetes types. ~= s/b = index of precision, n.s. = non-significant. Response: fall in blood sugar level 15 rain following intravenous insulin injection.
3.7. Sensitivity to insulin There was no measurable difference between alloxan- and streptozotocin-diabetic mice in their response to intravenously injected insulin. Given at three dose levels to groups of mice with both types of diabetes (6 groups totally) insulin was "assayed against itself" as shown in fig. 4. The data contributing to fig. 4 were analyzed in a similar manner to a six-point assay by means o f a factorial design and the analysis of variance. This showed no measurable difference in sensitivity to insulin between diabetes types, a highly significant regression o f the response on logdose; no significant divergence of slopes was present, which means that there was no difference in tolerance distribution between alloxan- and streptozotocin-diabetic mice. No significant departure from linearity of the regression lines, neither combined nor opposed, was present and the index of precision ~ = s/b was 0.25.
3.8. Remission o f the diabetic state Plasma insulin could not be detected for 14 days following treatment up to 3 months in both alloxanand streptozotocin-diabetic mice. After that interval positive plasma insulin values were occasionally recorded in alloxan-diabetic mice and a clear remission of the diabetic state was observed consisting in increased body weight, decreased mean blood sugar levels, and a measurable rise in plasma insulin levels upon intravenously injected corticotropin (,Rerup, to be published). Streptozotocin treated animals did not recover; their body weight did not increase nor did mean blood glucose levels become lower during 100 days, after which time more than half of the mice had succumbed. The diabetic state in streptozotocin treated mice appears thus to be irreversible. 4. DISCUSSION It should be emphasized that in this paper the term "diabetogenesis" is used in a broad sense and
STREPTOZOTOC1N-AND ALLOXAN-DIABETESIN MICE does not refer to beta-cytotoxic action alone but includes all effects which may lead to a chronic diabetic condition due to insulin deficiency. The above findings concerning the effects of streptozotocin and alloxan show both similarities and differences. The triphasic pattern of the blood sugar response, the depletion of liver glycogen prior to the peak of the initial hyperglycaemia, elevated plasma insulin levels during hyperglyceamic and individually adjusted blood glucose levels during the chronic diabetic state are seen after administration of either drug. On the other hand, the marked difference in the time of the initial hyperglucaemia peak, the inability of tolbutamide to influence alloxan induced hyperglycaemia (Lundquist and Rerup, 1967) as opposed to the sulphonylureas effect when given after streptozotocin, a more severe hypoglycaemia following streptozotocin, and the apparent irreversibility of streptozotocin diabetes show that the two drugs have markedly different effects regarding induction and chronic state of diabetes. The latter is supported by the findings of Schein and Loftus (1968) that the injection of streptozotocin, but not alloxan, was followed by a marked depletion of nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH) in mouse liver. These authors also showed that it was the 1-methyl-l-nitrosourea moiety of the molecule being responsible for liver NAD and NADH depletion. Both streptozotocin and 1-methyl-l-nitrosourea show antitumoural effects, but the latter is not diabetogenic. This means that the addition of glucose to l-methyl-l-nitrosourea to form streptozotocin is essential for a diabetogenic effect, for which reason Schein and Loftus (1968) ascribe a "carrier function" to glucose. More difficult to explain is the initial delay of the change in blood glucose level following streptozotocin treatment. Whether the drug is transformed into another beta-cytotoxic agent or is subject to some kind of storage before exerting its toxic effect remains to be investigated. Possibly streptozotocin is bound to the beta-cell membrane for a period before triggering the diabetogenic response. The hypoglycaemia following streptozotocin treatment may be explained in the same way as alloxan hypoglycaemia: neither drug interacts directly with insulin but both destroy the mechanism of insulin release in the beta-cell leading to large amounts of
95
stored insulin entering the blood. Whether insulin synthesis is deranged solely by the drugs or partly by exhaustion secondary to the destruction of the insulin releasing mechanism is not clear. Preliminary results (Rerup, unpublished) obtained in alloxan poisoned mice have shown that insulin does not disappear from the body in a single exponential clearance. Lack of insulin is the main cause of the chronic diabetic state in both alloxan and streptozotocin treated animals. A possible peripheral insensitivity to insulin is unlikely in view of the finding that 0.8 milliunits of insulin per 20 g mouse evoked a clear blood sugar lowering response. This is a dose which in normal mice lowers the blood glucose level somewhat less. In normal mice, however, insulin treatment produces hypoglycaemia and activation of counteracting mechanisms suggesting that the sensitivity to exogenous insulin was not different in normal, streptozotocin- and alloxan-diabetic mice. The two drugs appear to have basically different actions in the chronic diabetic state. Streptozotocindiabetic mice were in a poor general condition, their body weight never resumed the initial level, and their blood glucose levels stayed practically unchanged. Alloxan-diabetic animals, on the other hand, do resume and surpass their preinjection body weight and reach the weight of normals after 3 weeks. Their hyperglycaemia is reduced and they are finally able to produce - and upon challenge with injected corticotropin to increase - insulin secretion again (Rerup, to be published). There are species differences in the response to alloxan and streptozotocin. In rats, Arison et al. (1967) found a lower mortality (8 per cent) following streptozotocin than after alloxan (37 per cent), which is in contrast with the present findings in mice. The above authors consider the possibility of alpha-cell damage by streptozotocin to explain the low toxicity and suggest a possible application of streptozotocin for the investigation of regeneration of beta-cells. Here again the findings in rats are in contrast to the above results in mice. Finally, the data of Mansford and Opie (1968) give support to the view that streptozotocin and alloxan do produce different types of diabetes. These authors found in rats ketosis and abnormally high glycolytic intermediates during alloxan-, but not streptozotocin-diabetes.
96
C.RERUP and F.TARD1NG
REFERENCES Arison, R.N., E.I.Ciaccio, M.S.Glitzer, J.A.Cassaro and M.P. Pruss, 1967, Light and electron microscopy of lesions in rats rendered diabetic with streptozotocin, Diabetes 16, 51. Arison, R.N. and E.L.Feudale, 1967, Induction of renal tumour by streptozotocin in rats, Nature 214, 1254. Evans, J.S., G.C.Gerritsen, K.M.Mann and S.P.Owen, 1965, Antitumour and hyperglycemic activity of streptozotocin (NSC 37917) and its cofactor U-15774, Cancer Chemotherapy Rept. 48, 1-6. Heding, L., 1966, A simplified insulin radioimmunoassay method, in "Labelled proteins in tracer studies", L.Donato et al. (eds.), Euratom, Brussels, 345. Herr, R.R., H.K.Jahnke and A.D.Argoudelis, 1967, The structure of streptozotocin, J. Amer. Chem. Soc. 89, 4808. Junod, A., A.E.Lambert, L.Orci, R.Pictet, A.E.Gonet and A.E.Renold, 1967, Studies of the diabetogenic action of streptozotocin, Proc. Soc. Exptl. Biol. Med. 126, 201. kundquist, 1. and C.Rerup, 1967, On the development of alloxan-diabetes in mice, Europ. J. Pliarmacol. 2, 35.
Mansford, K.R.L. and L.Opie, 1968, Comparison of metabolic abnormalities in diabetes mellitus induced by streptozotocin or by alloxan, Lancet 7554, 670. Marks, V., 1959, An improved glucose oxidase method for determining blood, CSF, and urine glucose levels, Clin. Chim. Acta 4, 395. Rakieten, N., M.L.Rakieten and M.V.Nadkarni, 1963, Studies on the diabetogenic actions of streptozotocin (NSC37917), Cancer Chemotherapy Rept. 29. 91. Rerup, C. and l.Lundquist, 1900, Blood glucose level in mice, 1. Evaluation of a new technique of multiple serial sampling, Acta Endocr. 52, 357. Rerup, C. and l.Lundquist, 1967, Non-specific reaction of current glucose oxidase preparations with glycogen and its application for glycogen determination in tissues, Acta Pharmacol. et Toxicol. 25, 41. Schein, P.S., D.A.Cooney and M.L.Vernon, 1967, The use of nicotinamide to modify the toxicity of streptozotocindiabetes without loss of antitumour activity, Cancer Re~ 27, 2324. Schein, P.S. and S.Loftus, 1968, Streptozotocin: Depression of mouse liver pyridine nucleotides, Cancer Research 28, 1501.
STREPTOZOTOCIN-
AND ALLOXAN-DIABETES
in M I C E
Claus RERUP Department o[ Pharmacology, University o f Lund, Sweden and Finn TARDING Research Laboratories, Novo Therapeutisk Laboratorium A IS, Copenhagen, Denmark
Receded 27 January 1969
Accepted 12 March 1969
C.RERUP and F.TARDING, Streptozotocin- and alloxan-diabetes in mice, European J. Pharmacol. 7 (1969) 89-96. In non-fasted albino (NMRI) mice intravenous diabetogenic doses were 200 mg/kg body weight of streptozotocin or 70 mg/kg of aUoxan monohydrate. Differences in diabetes development were as follows. Maximal blood glucose levels during the phase of acute hyperglycaemia were observed 120 min after streptozotocin and 45 min after alloxan injection. Liver glycogen was depleted more slowly after streptozotocin than after alloxan. Tolbutamide given 5 or 60 min following streptozotocin injection clearly affected the blood sugar response. Elevated plasma insulin levels were found during the hypoglycaemic phase following injection of either drug. Plasma insulin and blood glucose levels were poorly correlated following streptozotocin treatment in contrast to findings in alloxan treated animals. Streptozotocin hypoglycaemia was more severe than alloxan hypoglycaemia. During the chronic hyperglycaemia the blood glucose levels were relatively stable within individuals following diabetes induction by means of either drug but there were large individual variations. However, alloxan-diabetic mice resumed and surpassed their original body weight with time, streptozotocin-diabetic animals did not. Both types of diabetes were normally sensitive to exogenous insulin. Streptozotocin-diabetes appeared to be irreversible. It is concluded that the development and chronic state of diabetes in mice following streptozotocin and alloxan, respectively, are not identical. Streptozotocin Alloxan
Plasma insulin Blood glucose
1. INTRODUCTION Streptozotocin, an antibiotic obtained from streptomyces achromogenes, is composed o f 1-methyl-1nitrosourea and glucose (Herr et al., 1967). The compound has a wide range o f pharmacological properties. Thus, it possesses antibiotic, diabetogenic and both antitumoural (Evans et al., 1965) and cancer inducing properties (Arison and Feudale, 1967). Streptozotocin induces diabetes in the rat, dog (Rakieten et al., 1963), hamster, m o n k e y and the Swiss mouse (Schein et al., 1967). There is general agreement that streptozotocin-diabetes - like alloxandiabetes - is brought about by a b e t a - c y t o t o x i c action
Tolbutamide Liver glycogen
in the islets of Langerhans although several findings suggest that the mechanisms o f diabetogenesis are different. Ketosis or elevation of plasma-free-fattyacid levels does not occur in streptozotocin-diabetic rats (Mansford and Opie, 1968). Streptozotocin has a higher specificity as a /3-cytotoxic agent in the rat (Arison et al., 1967; Junod et al., 1967). Streptozotocin but not alloxan, depleted mouse liver nicotinamide adenine dinucleotide (NAD) levels (Schein and Loftus, 1968). This last effect could be prevented by pretreatment with nicotinamide. These findings prompted a study o f streptozotocin- and alloxan-diabetogenesis using one strain of mice under similar laboratory conditions.
90
C.RERUP and F.TARDING
Table 1 Effect of increasing intravenous doses of streptozotocin to non-fasting mice on diabetogenesis, mean body weight change and mortality. No. of mice
Streptozotocin dose mg/kg
Diabetes frequency after 7 days
Mean body weight change after 2 weeks, g
Mortality after 2 weeks
5 5 5 5
70 140 200 300
0/5 1/5 4/4* 3/3**
+ 3.2 + 3.5 - 1.0 - 2.0
0/5 0/5 1/5 4/5
* One mouse died in hypoglycaemia. ** Two mice died in hypoglycaemia. 2. MATERIAL AND METHOD
3. RESULTS
Female albino NMRI mice* weighing 2 0 - 2 5 g were used throughout. During all experimental conditions the animals had free access to a standard pellet diet** and tap water. Streptozotocin*** was dissolved in redistilled water and given within four minutes from the time of dissolving. Alloxan monohydrate was dissolved at pH 5 (HC1) and given at a dose of 70 mg/kg body weight within 10 min from dissolving. Tolbutamide was dissolved at pH 8.3 and given at a dose of 80 mg/kg body weight. All drugs were injected intravenously via a tail vein. Blood sampling (25 /al and 250 /al for determinations of blood glucose and plasma insulin levels, respectively) was performed by orbital puncture using commercial constriction pipettes (Rerup and Lundquist, 1966). Blood glucose levels were determined enzymatically (Marks, 1959), plasma insulin levels immunologicallyt (Heding, 1966) and liver glycogen enzymatically (Rerup and Lundquist, 1967). Diabetes was considered to be manifest in mice with blood glucose levels of 200 mg/100 ml or above. Animals lacking this criterion were discarded.
3.1. Diabetogenic dose of streptozotocin
* Laboratory Animal Breeding, Laven, Denmark. ** Ferrosan Ltd., Maim6, Sweden. *** Provided through the courtesy of W.E.Dulin, Ph.D., The Upjohn Company, Kalamazoo, Michigan, USA. t Anti-insulin serum from guinea pigs and 12sI-insulin were kindly provided by Mrs. L.Heding, M.Sc., Novo Research Institute, Copenhagen, Denmark.
Due to limitations of the amount of streptozotocin available groups of 5 mice were injected intravenously with increasing doses as shown in table 1. It appears from table 1 that 70 mg/kg body weight was without diabetogenic effect, 140 mg/kg was diabetogenic in one out of five mice. In these two groups there was a normal body weight gain observed during 20 days from streptozotocin treatment. When the dose was increased to 200 mg/kg all mice became diabetic and one died 3 days after the treatment. A further increase of the dose to 300 mg/kg killed 4 out of 5 mice, two during hypoglycaemia and two on the 3rd and 5th day following treatment leaving one diabetic mouse alive. In the last two dose groups the mean change in body weight after drug treatment was negative. When the dose of 200 mg/kg body weight of streptozotocin was repeatedly injected into groups of mice it was invariably followed by a diabetic state. The mortality within one week from injection was 5 - 1 0 per cent and was usually due to the severe hypoglycaemia occurring 8 - 2 4 hr after drug administration. Subsequently streptozotocin at a dose of 200 mg/kg body weight was used and compared with 70 mg/kg of alloxan monohydrate, which has been shown to be a suitable diabetogenic dose at this laboratory (Rerup and Lundquist, 1967).
STREPTOZOTOCIN- AND ALLOXAN-DIABETES IN MICE
91
BLood gtuo0se,mg / 100 rnL Alloxan 300"
Alloxan
200-
100-
o 3'~ ~ ~ ~
~
~
Io
~'3
io
~'8
hours
Fig. 1. The effect of intravenous injection of alloxan monohydrate (70 mg/kg) and streptozotocin (200 mg/kg) on blood glucose levels in non-fasted female NMRI mice. The curves in the left part of the figure differ significantly (p < 0 . 0 0 1 ) e x c e p t at zero and 8 hr. Alloxan and streptozotocin was given at random to groups of 8 mice each, 11 blood samples (25 t~l) per mouse.
A
Blood glucose, mg / 100 ml Strept. 300-
B Strept.
/
aline
Saline
200-
100
Tolbutamide
{~ 0~5 "I
2
rolbutomi de
3 hours
()
i
2
3
hours
Fig. 2. Effect of tulbutamide (80 mg/kg intravenously) on streptozotocin induced blood sugar fluctuations. Mean responses o f l 0 mice (a total of 40 mice). Tolbutamide given 5 min (A) and 60 min (B) following streptozotocin treatment (200 mg/kg). Significance levels P for the difference between saline and tolbutamide treated mice shown between mean responses,
92
C.RERUP and F.TARDING
Table 2 Liver glycogen and blood glucose levels 30 and 90 min following intravenous injection of aUoxan monohydrate (70 mg/kg), streptozotocin (200 mg/kg) and saline, respectively, into non-fasted mice. 10 mice per group. P = probability of random difference versus controls, n.s. = non-significant.
Group
Treatment
1
Saline
2
Streptozotocin
Observation time after injection
Mean blood glucose mg/100 ml 106 + 5*
30min
3.76 +0.41
n.s.
~0.001
2.23 -+0.35
~0.01
243 + 21
4.
Saline
115+ 4
5.
Streptozotocin Alloxan
4.66 + 0.66 n.s.
Alloxan
90 min
P
111 + 5
3.
6.
P
Mean liver glycogen % of wet liver
4.99+0.28
288 + 11
<0.001
2.45 + 0.43
~0.001
255 + 27
<0.001
5.00 + 0.61
n.s.
* Standard error of mean.
3.2. Blood glucose fluctuations The change in blood glucose level in two groups of 8 mice receiving 200 mg/kg o f streptozotocin and 70 mg/kg o f alloxan, respectively, are given in fig. 1. Alloxan evoked a sudden sharp rise in blood glucose level with a peak at 45 min. In contrast the blood sugar response to streptozotocin was delayed, with minimal changes during 45 min. A marked hyperglycaemia with a peak at 2 hr after treatment occurred while in alloxan treated mice the blood glucose level was decreasing towards hypoglycaemia. The lowest blood sugar levels following alloxan treatment were recorded after 6 hr and after at least 10 hr in the streptozotocin treated group. The hypoglycaemia following streptozotocin was more severe than that following alloxan (p < 0.02). In the streptozotocin treated group the lowest individual blood glucose level was 21 mg/100 ml obtained at 10 hr from a mouse which died shortly afterwards. One day (23 hr) after the treatment the mean blood glucose level was slightly hyperglycaemic in both groups, after two days it was clearly diabetic. In this experiment the mean blood glucose levels were not significantly different between groups on day 1 and day 2. 3.3. Liver glycogen levels Liver glycogen level was not significantly decreased 30 rain after streptozotocin injection; blood glucose level was normal. However, 30 min after
alloxan liver glycogen was markedly reduced and blood glucose was elevated, confirming previous work (Lundquist and Rerup, 1967). When liver glycogen was determined 90 min after treatment (about 30 min before the maximum of streptozotocin hyperglycaemia, but about 45 min past the peak o f alloxan hyperglycaemia) streptozotocin injected mice showed a marked liver glycogen depletion, whereas in alloxan treated mice liver glycogen levels had already returned to normal (table 2). Thus it appears that the initial hyperglycaemia following either drug is due, at least in part, to mobilization of glycogen from the liver.
3.4.Effect of sulphonylurea on the initial hyperglycaemia Tolbutamide, given 5 min after streptozotocin, evoked a marked hypoglycaemia at 30 and 60 min which substantially reduced the initial hyperglycaemic phase occurring at 120 min. When the tolbutamide injection was given closer to the time of peak hyperglycaemia following streptozotocin, i.e. 60 min following injection of the diabetogenic drug, the hyperglycaemia was again reduced and hypoglycaemia was observed 3 hr after streptozotocin injection. These findings contrast with the inability to tolbutamide to reduce the initial hyperglycaemia when the sulphonylurea is given after alloxan (Lundquist and Rerup, 1967).
STREPTOZOTOCIN- AND ALLOXAN-D1ABETES IN MICE
93
Table 3 Blood glucose and plasma immunoreactive insulin levels 9 hr following intravenous injection of streptozotocin (200 mg/kg) and saline, respectively, into non-fasted mice. P = probability of random difference between groups, r = correlation coefficient, n.s. = non-significant.
Group
No. of mice
1
8
2
16
Treatment Saline Streptozotocin
Mean blood glucose mg/100 ml
P
125 + 4* 30 -+ 8
Plasma insulin tt-unit/ml
P
r
<0.001
-0.49, n.s.
9.4 + 4.8
<0.001
79.3 + 8.7
* Standard error of mean
3.5. Hypoglycaemic phase Plasma insulin levels were elevated shortly before and during the streptozotocin induced hypoglycaemia. When recorded at 6 hr during falling blood sugar levels the elevation of plasma insulin levels was moderate but significant over controls. The negative correlation between blood glucose and plasma insulin levels was not significant in contrast to findings 4 hr after alloxan (Lundquist and Rerup, 1967). Nine hours after streptozotocin plasma insulin levels were markly elevated as shown in table 3. 3.6. Chronic diabetic state Most animals were diabetic 48 hr after injection of
g
either drug. However, in several mice chronic hyperglycaemia did not develop until one week after streptozotocin. Blood glucose levels during streptozotocin-diabetes were subject to individual variations, as in alloxandiabetes. The blood glucose level varied between mice from about 250 mg/100 ml to 550 rag/100 ml. The loss in body weight following alloxan injection was only transient and an increase over the initial weight was observed about three weeks after treatment. Following streptozotocin treatment, however, the mice did not regain their original body weight during an observational period of at least two months (fig. 3).
Body weight
30-
Diabetes
25-
induction AUoxan
20¸
Fig. 3. Body weight change of mice treated with streptozotocin (200 mg/kg) and allo×an monohydrate (70 mg/kg) during 56 days. Significance levels (P) for the difference between groups shown between mean weights (10 mice per group).
94
C.RERUP and F.TARDING Foil in brood gtucose I rag/100 mt
150. Strept
n
100.
F
i Anal.ysisof variance:
/
insutin togdose effect
// J/ 50"
P
(diabetes type (divergence
I.curvature
[/
n.s. n.s. n.So
A=n2s 018 -0.1
2 0.3
S 0.7
mU I 20 g mouse togdose
Fig. 4. Sensitivity to exogenous insulin of alloxan- and streptozotocin-diabetic mice. Mean responses of 6 mice (a total of 30 mice). Highly significant logdose response dependence, no divergence of slopes or curvature, no difference in sensitivity to insulin between diabetes types. ~= s/b = index of precision, n.s. = non-significant. Response: fall in blood sugar level 15 rain following intravenous insulin injection.
3.7. Sensitivity to insulin There was no measurable difference between alloxan- and streptozotocin-diabetic mice in their response to intravenously injected insulin. Given at three dose levels to groups of mice with both types of diabetes (6 groups totally) insulin was "assayed against itself" as shown in fig. 4. The data contributing to fig. 4 were analyzed in a similar manner to a six-point assay by means o f a factorial design and the analysis of variance. This showed no measurable difference in sensitivity to insulin between diabetes types, a highly significant regression o f the response on logdose; no significant divergence of slopes was present, which means that there was no difference in tolerance distribution between alloxan- and streptozotocin-diabetic mice. No significant departure from linearity of the regression lines, neither combined nor opposed, was present and the index of precision ~ = s/b was 0.25.
3.8. Remission o f the diabetic state Plasma insulin could not be detected for 14 days following treatment up to 3 months in both alloxanand streptozotocin-diabetic mice. After that interval positive plasma insulin values were occasionally recorded in alloxan-diabetic mice and a clear remission of the diabetic state was observed consisting in increased body weight, decreased mean blood sugar levels, and a measurable rise in plasma insulin levels upon intravenously injected corticotropin (,Rerup, to be published). Streptozotocin treated animals did not recover; their body weight did not increase nor did mean blood glucose levels become lower during 100 days, after which time more than half of the mice had succumbed. The diabetic state in streptozotocin treated mice appears thus to be irreversible. 4. DISCUSSION It should be emphasized that in this paper the term "diabetogenesis" is used in a broad sense and
STREPTOZOTOC1N-AND ALLOXAN-DIABETESIN MICE does not refer to beta-cytotoxic action alone but includes all effects which may lead to a chronic diabetic condition due to insulin deficiency. The above findings concerning the effects of streptozotocin and alloxan show both similarities and differences. The triphasic pattern of the blood sugar response, the depletion of liver glycogen prior to the peak of the initial hyperglycaemia, elevated plasma insulin levels during hyperglyceamic and individually adjusted blood glucose levels during the chronic diabetic state are seen after administration of either drug. On the other hand, the marked difference in the time of the initial hyperglucaemia peak, the inability of tolbutamide to influence alloxan induced hyperglycaemia (Lundquist and Rerup, 1967) as opposed to the sulphonylureas effect when given after streptozotocin, a more severe hypoglycaemia following streptozotocin, and the apparent irreversibility of streptozotocin diabetes show that the two drugs have markedly different effects regarding induction and chronic state of diabetes. The latter is supported by the findings of Schein and Loftus (1968) that the injection of streptozotocin, but not alloxan, was followed by a marked depletion of nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH) in mouse liver. These authors also showed that it was the 1-methyl-l-nitrosourea moiety of the molecule being responsible for liver NAD and NADH depletion. Both streptozotocin and 1-methyl-l-nitrosourea show antitumoural effects, but the latter is not diabetogenic. This means that the addition of glucose to l-methyl-l-nitrosourea to form streptozotocin is essential for a diabetogenic effect, for which reason Schein and Loftus (1968) ascribe a "carrier function" to glucose. More difficult to explain is the initial delay of the change in blood glucose level following streptozotocin treatment. Whether the drug is transformed into another beta-cytotoxic agent or is subject to some kind of storage before exerting its toxic effect remains to be investigated. Possibly streptozotocin is bound to the beta-cell membrane for a period before triggering the diabetogenic response. The hypoglycaemia following streptozotocin treatment may be explained in the same way as alloxan hypoglycaemia: neither drug interacts directly with insulin but both destroy the mechanism of insulin release in the beta-cell leading to large amounts of
95
stored insulin entering the blood. Whether insulin synthesis is deranged solely by the drugs or partly by exhaustion secondary to the destruction of the insulin releasing mechanism is not clear. Preliminary results (Rerup, unpublished) obtained in alloxan poisoned mice have shown that insulin does not disappear from the body in a single exponential clearance. Lack of insulin is the main cause of the chronic diabetic state in both alloxan and streptozotocin treated animals. A possible peripheral insensitivity to insulin is unlikely in view of the finding that 0.8 milliunits of insulin per 20 g mouse evoked a clear blood sugar lowering response. This is a dose which in normal mice lowers the blood glucose level somewhat less. In normal mice, however, insulin treatment produces hypoglycaemia and activation of counteracting mechanisms suggesting that the sensitivity to exogenous insulin was not different in normal, streptozotocin- and alloxan-diabetic mice. The two drugs appear to have basically different actions in the chronic diabetic state. Streptozotocindiabetic mice were in a poor general condition, their body weight never resumed the initial level, and their blood glucose levels stayed practically unchanged. Alloxan-diabetic animals, on the other hand, do resume and surpass their preinjection body weight and reach the weight of normals after 3 weeks. Their hyperglycaemia is reduced and they are finally able to produce - and upon challenge with injected corticotropin to increase - insulin secretion again (Rerup, to be published). There are species differences in the response to alloxan and streptozotocin. In rats, Arison et al. (1967) found a lower mortality (8 per cent) following streptozotocin than after alloxan (37 per cent), which is in contrast with the present findings in mice. The above authors consider the possibility of alpha-cell damage by streptozotocin to explain the low toxicity and suggest a possible application of streptozotocin for the investigation of regeneration of beta-cells. Here again the findings in rats are in contrast to the above results in mice. Finally, the data of Mansford and Opie (1968) give support to the view that streptozotocin and alloxan do produce different types of diabetes. These authors found in rats ketosis and abnormally high glycolytic intermediates during alloxan-, but not streptozotocin-diabetes.
96
C.RERUP and F.TARD1NG
REFERENCES Arison, R.N., E.I.Ciaccio, M.S.Glitzer, J.A.Cassaro and M.P. Pruss, 1967, Light and electron microscopy of lesions in rats rendered diabetic with streptozotocin, Diabetes 16, 51. Arison, R.N. and E.L.Feudale, 1967, Induction of renal tumour by streptozotocin in rats, Nature 214, 1254. Evans, J.S., G.C.Gerritsen, K.M.Mann and S.P.Owen, 1965, Antitumour and hyperglycemic activity of streptozotocin (NSC 37917) and its cofactor U-15774, Cancer Chemotherapy Rept. 48, 1-6. Heding, L., 1966, A simplified insulin radioimmunoassay method, in "Labelled proteins in tracer studies", L.Donato et al. (eds.), Euratom, Brussels, 345. Herr, R.R., H.K.Jahnke and A.D.Argoudelis, 1967, The structure of streptozotocin, J. Amer. Chem. Soc. 89, 4808. Junod, A., A.E.Lambert, L.Orci, R.Pictet, A.E.Gonet and A.E.Renold, 1967, Studies of the diabetogenic action of streptozotocin, Proc. Soc. Exptl. Biol. Med. 126, 201. kundquist, 1. and C.Rerup, 1967, On the development of alloxan-diabetes in mice, Europ. J. Pliarmacol. 2, 35.
Mansford, K.R.L. and L.Opie, 1968, Comparison of metabolic abnormalities in diabetes mellitus induced by streptozotocin or by alloxan, Lancet 7554, 670. Marks, V., 1959, An improved glucose oxidase method for determining blood, CSF, and urine glucose levels, Clin. Chim. Acta 4, 395. Rakieten, N., M.L.Rakieten and M.V.Nadkarni, 1963, Studies on the diabetogenic actions of streptozotocin (NSC37917), Cancer Chemotherapy Rept. 29. 91. Rerup, C. and l.Lundquist, 1900, Blood glucose level in mice, 1. Evaluation of a new technique of multiple serial sampling, Acta Endocr. 52, 357. Rerup, C. and l.Lundquist, 1967, Non-specific reaction of current glucose oxidase preparations with glycogen and its application for glycogen determination in tissues, Acta Pharmacol. et Toxicol. 25, 41. Schein, P.S., D.A.Cooney and M.L.Vernon, 1967, The use of nicotinamide to modify the toxicity of streptozotocindiabetes without loss of antitumour activity, Cancer Re~ 27, 2324. Schein, P.S. and S.Loftus, 1968, Streptozotocin: Depression of mouse liver pyridine nucleotides, Cancer Research 28, 1501.