Tohoku
J. exp. Med.,
Production
1976, 119, 85-90
of Spontaneous
Repetition
of Selective
Diabetic
Rats
by
Breeding
YOSHIOGOTO, MASAEIKAKIZAKIand NAOYOSH1MASAKI The Third Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki
GOTO, Rats
by
Y.,
85-90 of
tolerance
from
were
By
increasing
experimental selective
urine
number
sugar results
normal diabetes;
rats
during
the
indicate by diabetes
etiology;
with
this
tolerance The
tolerance of of genetic
the
By
curve percentage
test
a
increases
production selective
Med.,
is the
from
211
the
with of
second
162
174
F3
more
cases the
of
of
number
spontaneous
Fl
18 rats
rats 215
diabetic a
the
Wistar
and
positive
(1),
glucose
report.
normal
breeding F2,
became of
119 that
impairment
This
204
Diabetic
1976,
hypothesis
slight
test
way,
Spontaneous
exp.
working
rats.
farm. in
of
J. a
tolerance
animal
possibility
repetition
rats
diabetes
glucose
generations.
the the
normal
glucose
glucose
Production
Tohoku under
breeding
mean of
of
oral
the
N.
conducted
spontaneous an
an
The
MASAKI, Breeding.
breeding
by
obtained.
and
was
produce
selected
obtained.
M. Selective
study
selective
would
were
obtained
The
of
This
repeating
rats
KAKIZAKI,
Repetition
were F4
with test
rats the for
of generation. diabetes
from
breeding.•\experimental
diabetes
Our experiment has been conducted under a working hypothesis that spontaneous diabetic rats would be produced from normal rats by a repetition of selective breeding. Namely, there is no diabetic rat in normal population but we can select animals with higher blood sugar levels among them. If such animals are mated, we can expect two possible results, i.e., 1) their offsprings have also normal blood sugar similar to their parents, or 2) their offsprings have more deviated blood sugar values than the parents. If the former is the case, the repetition of the selective breeding over numerous generations will yield only ones with normal blood sugar and diabetes will never occur unless a mutation does occur. On the other hand, if the latter is the case, the more the breeding is repeated, the more the blood sugar will become abnormal, and finally diabetic state will be provoked. A preliminary study on a small scale was made in 1969 and the study on a large scale was started in October, 1973. In proceeding with the experiment, we felt an omen suggesting a possibility to produce diabetic rats by the repetition of the selective breeding (Goto et al. 1975). In this report, the results obtained until the fifth generation are described. Received for publication, December 23, 1975. This work was supported by a scientific, research grant from the Ministry of Education of Japan (No. 044005). 85
86
Y. (Goto
et al.
METHODS
Two hundred and eleven normal rats of the Wistar strain obtained from Nogawa Experimental Animal Farm of Japan Clea Co. were used as the foundation stock. By the oral glucose tolerance test, 18 rats were selected for breeding (F). By thee breeding, 162 offsprings (Fr) yielded. 25 Fr rats were selected by the glucose tolerance test, elated and yielded 204 offsprings (F2). 28 F2 rats were selected for breeding and yielded 175 offsprings (F3). 31 F3 rats were selected for breeding yielded 215 offsprings (F4). In this experiment, inbreeding, especially sib-breeding was avoided. The oral glucose tolerance test (2 g/kg) was performed in the morning after 12 hr of fasting. Blood specimens were collected from tail vessels by cutting its end and the glucose content, was measured by the glucose oxidase method using Glucostat (Worthington Biochemical Co., Freehold. New Jersey) . Urine sugar was determined by Tes-Tape when urinated. The grade of glucose intolerance was expressed as the sum of blood glucose values obtained at five time periods , i.e., fasting, 30, 60, 90 and 120 min . The details of the experimental procedure were described in the previous report (Goto et al. 1975). RESULTS
The mean glucose tolerance tests of each generation are shown in Table 1 and depicted in Fig. 1. As shown in the figure , the glucose tolerance curve becomes more diabetic with the number of generation . The curve of F,, however , is almost the same as that of F3. The combination of crossing and the sums of blood glucose of F3 and F4 are shown in Tables 2 and 3 . Cases of a positive test for urine sugar during the glucose tolerance test were shown in bold type . The percentage of the cases of a positive test for urine sugar also increases with the number of genera tion.
There
is no tendency
to be obese in these
TABLE 1.
The
data,
are
mean•}S.D.
Mean
rats .
gleucose tolerance
text
Spontaneous
Fig. 1.
Diabetes
Mean glucose
in Rats
tolerance
87
curves.
COMMENT
This experiment demonstrated a possibility of the production of spontaneous diabetic rats by the repetition of the selective breeding. This indicates that the inheritance of diabetes mellitus is not determined by specific gene(s) but is influenced.by many genes relating carbohydrate metabolism. In other words, the diabetic state occurs as the result of an integration of factors such as the genes relating to endocrine function, those relating to enzymes regulating the metabolisms of carbohydrate, fat and protein, those relating to the formation of the islet cells, etc., when it exceeds a certain threshold. In the study on the offsprings of conjugal diabetes, we could demonstrate that the incidence of cases of a diabetic glucose tolerance test increases with age and all of them became diabetic when they were over 50 years old (Goto et al. 1971). This observation seems to be convenient evidence for the recessive theory of diabetes mellitus. However, this phenomenon can also be explained by the concept of multifactorial inheritance. Many kinds of spontaneous diabetes animals have been reported and they have been used as models of human diabetes (Meier and Yerganian 1959; Meier 1961; Nakaniura 1962; Schmidt-Nielsen et al. 1964; Hummel et al. 1966). Their diabetic state, however, was found accidentally. In this study, the diabetic state was provoked expectedly according to the working hypothesis in the Wistar strain. This has special meanings not only on the point that diabetic state can be made from the normal rats, but also on the point that this is a diabetic rat having normal control of the same strain. The former will cover an explanation for the existence of diabetic children without any family history of diabetes mellitus and the latter will be convenient for experimental studies of various purposes, especially in the experiment on implantation of the pancreatic tissue of normal rats to diabetic rats. A success of this experiment will indicate the possibility to produce other
88
Y.
TABLE2.
Bold type indicates retarded growth.
Onto
et
at.
Combination of mating in the F2 rats and sum of blood glucose of the offsprings (F3)
cases
of a positive
test
for urine
sugar.
Asterisk
represents
cases
of
metabolic disorders such as gout (hyperuricemia), atherosclerosis (hypercholester olemia), etc. by similar procedures. The present result showed clearly the inclination that the more numerous the generations, the more remarkable the glucose intolerance was. However , it is noticed that this aggravation of glucose intolerance became less remarkable
Spontaneous
TABLE 3.
Bold type indicates Asterisk represents
Diabetes
in Rats
Combination of mating in the F3 rats and sum of blood glucose of the offsprings (F4)
cases of a positive test for urine cases of retarded growth.
sugar.
89
90
Y. Goto et al.
between the F3 and the F4 rats. The purpose of this study is to obtain a severe diabetic rat. In this experiment, sib-breeding has been avoided. However, we learned that brother-sister breeding is a shorter way to condensate or intensify the nature of hyperglycemia in these rats. Now our experiment is continuing along this line. One of the most interesting themes in the further promotion of this study is to know whether a severe ketosis-prone juvenile-type diabetic state will be provoked by this procedure or not. If it is so, it could be concluded that the two types of diabetes, juvenile and adult , are essentially the same. If the severe diabetic state does not occur by the repetition of the crossing over numerous generations, juvenile-type insulin-deficient diabetes would be essentially different from the adult-type diabetic state. The sequel of this study may answer this question. References
1) 2)
3) 4) 5) 6) 7)
Hummel, K.P„ Dickie, M.M. & Coleman, D.L. (1966) Diabetes , a new mutation in the mouse. Science, 153, 1127-1128. Goto, Y., Toyota, T., Maruhama, Y., Fukuhara, N ., Sato, S., Chiba, M. & Sato, Y. (1971) Abnormalities in prediabetes. In: Diabetes, edited by R.R. Rodriguez, (Internat. Congr. Series No. 231). Excerpta Medica, Amsterdam, pp. 240-247. Goto, Y., Kakizaki, M. & Masaki, N. (1975) Spontaneous diabetes produced by selective breeding of normal Wistar rats. Proc. Jap . Acad., 51, 80-85. Meier, H. (1961) Comparative aspects of spontaneous diabetes mellitus in animals . Amer. J. Med., 31, 868-873. Meier, H. & Yerganian, G.A. (1959) Diabetes mellitus in Chinese hamster (Cricetulus griseus). I. Pathological findings. Proc. Soc. exp. Biol. Med. (N.Y.), 100, 810-815. Nakamura, M. (1962) A diabetic strain of the mouse . Proc. Jap. Acad., 38, 348-352. Schmidt-Nielsen, K., Haines, H.B. & Rackel , D.B, (1964) Diabetes mellitus in the sand rat induced by standard laboratory diets . Science, 143, 689-690.
J. exp. Med.,
Production
1976, 119, 85-90
of Spontaneous
Repetition
of Selective
Diabetic
Rats
by
Breeding
YOSHIOGOTO, MASAEIKAKIZAKIand NAOYOSH1MASAKI The Third Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki
GOTO, Rats
by
Y.,
85-90 of
tolerance
from
were
By
increasing
experimental selective
urine
number
sugar results
normal diabetes;
rats
during
the
indicate by diabetes
etiology;
with
this
tolerance The
tolerance of of genetic
the
By
curve percentage
test
a
increases
production selective
Med.,
is the
from
211
the
with of
second
162
174
F3
more
cases the
of
of
number
spontaneous
Fl
18 rats
rats 215
diabetic a
the
Wistar
and
positive
(1),
glucose
report.
normal
breeding F2,
became of
119 that
impairment
This
204
Diabetic
1976,
hypothesis
slight
test
way,
Spontaneous
exp.
working
rats.
farm. in
of
J. a
tolerance
animal
possibility
repetition
rats
diabetes
glucose
generations.
the the
normal
glucose
glucose
Production
Tohoku under
breeding
mean of
of
oral
the
N.
conducted
spontaneous an
an
The
MASAKI, Breeding.
breeding
by
obtained.
and
was
produce
selected
obtained.
M. Selective
study
selective
would
were
obtained
The
of
This
repeating
rats
KAKIZAKI,
Repetition
were F4
with test
rats the for
of generation. diabetes
from
breeding.•\experimental
diabetes
Our experiment has been conducted under a working hypothesis that spontaneous diabetic rats would be produced from normal rats by a repetition of selective breeding. Namely, there is no diabetic rat in normal population but we can select animals with higher blood sugar levels among them. If such animals are mated, we can expect two possible results, i.e., 1) their offsprings have also normal blood sugar similar to their parents, or 2) their offsprings have more deviated blood sugar values than the parents. If the former is the case, the repetition of the selective breeding over numerous generations will yield only ones with normal blood sugar and diabetes will never occur unless a mutation does occur. On the other hand, if the latter is the case, the more the breeding is repeated, the more the blood sugar will become abnormal, and finally diabetic state will be provoked. A preliminary study on a small scale was made in 1969 and the study on a large scale was started in October, 1973. In proceeding with the experiment, we felt an omen suggesting a possibility to produce diabetic rats by the repetition of the selective breeding (Goto et al. 1975). In this report, the results obtained until the fifth generation are described. Received for publication, December 23, 1975. This work was supported by a scientific, research grant from the Ministry of Education of Japan (No. 044005). 85
86
Y. (Goto
et al.
METHODS
Two hundred and eleven normal rats of the Wistar strain obtained from Nogawa Experimental Animal Farm of Japan Clea Co. were used as the foundation stock. By the oral glucose tolerance test, 18 rats were selected for breeding (F). By thee breeding, 162 offsprings (Fr) yielded. 25 Fr rats were selected by the glucose tolerance test, elated and yielded 204 offsprings (F2). 28 F2 rats were selected for breeding and yielded 175 offsprings (F3). 31 F3 rats were selected for breeding yielded 215 offsprings (F4). In this experiment, inbreeding, especially sib-breeding was avoided. The oral glucose tolerance test (2 g/kg) was performed in the morning after 12 hr of fasting. Blood specimens were collected from tail vessels by cutting its end and the glucose content, was measured by the glucose oxidase method using Glucostat (Worthington Biochemical Co., Freehold. New Jersey) . Urine sugar was determined by Tes-Tape when urinated. The grade of glucose intolerance was expressed as the sum of blood glucose values obtained at five time periods , i.e., fasting, 30, 60, 90 and 120 min . The details of the experimental procedure were described in the previous report (Goto et al. 1975). RESULTS
The mean glucose tolerance tests of each generation are shown in Table 1 and depicted in Fig. 1. As shown in the figure , the glucose tolerance curve becomes more diabetic with the number of generation . The curve of F,, however , is almost the same as that of F3. The combination of crossing and the sums of blood glucose of F3 and F4 are shown in Tables 2 and 3 . Cases of a positive test for urine sugar during the glucose tolerance test were shown in bold type . The percentage of the cases of a positive test for urine sugar also increases with the number of genera tion.
There
is no tendency
to be obese in these
TABLE 1.
The
data,
are
mean•}S.D.
Mean
rats .
gleucose tolerance
text
Spontaneous
Fig. 1.
Diabetes
Mean glucose
in Rats
tolerance
87
curves.
COMMENT
This experiment demonstrated a possibility of the production of spontaneous diabetic rats by the repetition of the selective breeding. This indicates that the inheritance of diabetes mellitus is not determined by specific gene(s) but is influenced.by many genes relating carbohydrate metabolism. In other words, the diabetic state occurs as the result of an integration of factors such as the genes relating to endocrine function, those relating to enzymes regulating the metabolisms of carbohydrate, fat and protein, those relating to the formation of the islet cells, etc., when it exceeds a certain threshold. In the study on the offsprings of conjugal diabetes, we could demonstrate that the incidence of cases of a diabetic glucose tolerance test increases with age and all of them became diabetic when they were over 50 years old (Goto et al. 1971). This observation seems to be convenient evidence for the recessive theory of diabetes mellitus. However, this phenomenon can also be explained by the concept of multifactorial inheritance. Many kinds of spontaneous diabetes animals have been reported and they have been used as models of human diabetes (Meier and Yerganian 1959; Meier 1961; Nakaniura 1962; Schmidt-Nielsen et al. 1964; Hummel et al. 1966). Their diabetic state, however, was found accidentally. In this study, the diabetic state was provoked expectedly according to the working hypothesis in the Wistar strain. This has special meanings not only on the point that diabetic state can be made from the normal rats, but also on the point that this is a diabetic rat having normal control of the same strain. The former will cover an explanation for the existence of diabetic children without any family history of diabetes mellitus and the latter will be convenient for experimental studies of various purposes, especially in the experiment on implantation of the pancreatic tissue of normal rats to diabetic rats. A success of this experiment will indicate the possibility to produce other
88
Y.
TABLE2.
Bold type indicates retarded growth.
Onto
et
at.
Combination of mating in the F2 rats and sum of blood glucose of the offsprings (F3)
cases
of a positive
test
for urine
sugar.
Asterisk
represents
cases
of
metabolic disorders such as gout (hyperuricemia), atherosclerosis (hypercholester olemia), etc. by similar procedures. The present result showed clearly the inclination that the more numerous the generations, the more remarkable the glucose intolerance was. However , it is noticed that this aggravation of glucose intolerance became less remarkable
Spontaneous
TABLE 3.
Bold type indicates Asterisk represents
Diabetes
in Rats
Combination of mating in the F3 rats and sum of blood glucose of the offsprings (F4)
cases of a positive test for urine cases of retarded growth.
sugar.
89
90
Y. Goto et al.
between the F3 and the F4 rats. The purpose of this study is to obtain a severe diabetic rat. In this experiment, sib-breeding has been avoided. However, we learned that brother-sister breeding is a shorter way to condensate or intensify the nature of hyperglycemia in these rats. Now our experiment is continuing along this line. One of the most interesting themes in the further promotion of this study is to know whether a severe ketosis-prone juvenile-type diabetic state will be provoked by this procedure or not. If it is so, it could be concluded that the two types of diabetes, juvenile and adult , are essentially the same. If the severe diabetic state does not occur by the repetition of the crossing over numerous generations, juvenile-type insulin-deficient diabetes would be essentially different from the adult-type diabetic state. The sequel of this study may answer this question. References
1) 2)
3) 4) 5) 6) 7)
Hummel, K.P„ Dickie, M.M. & Coleman, D.L. (1966) Diabetes , a new mutation in the mouse. Science, 153, 1127-1128. Goto, Y., Toyota, T., Maruhama, Y., Fukuhara, N ., Sato, S., Chiba, M. & Sato, Y. (1971) Abnormalities in prediabetes. In: Diabetes, edited by R.R. Rodriguez, (Internat. Congr. Series No. 231). Excerpta Medica, Amsterdam, pp. 240-247. Goto, Y., Kakizaki, M. & Masaki, N. (1975) Spontaneous diabetes produced by selective breeding of normal Wistar rats. Proc. Jap . Acad., 51, 80-85. Meier, H. (1961) Comparative aspects of spontaneous diabetes mellitus in animals . Amer. J. Med., 31, 868-873. Meier, H. & Yerganian, G.A. (1959) Diabetes mellitus in Chinese hamster (Cricetulus griseus). I. Pathological findings. Proc. Soc. exp. Biol. Med. (N.Y.), 100, 810-815. Nakamura, M. (1962) A diabetic strain of the mouse . Proc. Jap. Acad., 38, 348-352. Schmidt-Nielsen, K., Haines, H.B. & Rackel , D.B, (1964) Diabetes mellitus in the sand rat induced by standard laboratory diets . Science, 143, 689-690.
