A disrupted cholecystokinin A receptor gene induces diabetes
in obese rats synergistically with ODB1 gene
Soichi
Takiguchi1,
Yutaka
Takata1,3,
Nobuhiko
Takahashi4,
Kazuhiro
Kataoka1,
Tsukasa
Hirashima5,
Kazuya
Kawano5,
Kyoko
Miyasaka3,
Akihiro
Funakoshi2, and
Akira
Kono1
Divisions of 1 Chemotherapy and
2 Gastroenterology, National
Kyushu Cancer Center, Fukuoka 815;
3 Department of Clinical
Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173;
4 Third Department of Internal
Medicine, Asahikawa Medical College, Asahikawa Hokkaido 078; and
5 Tokushima Research Institute,
Otsuka Pharmaceutical, Tokushima 771-01, Japan
 |
ABSTRACT |
Otsuka Long-Evans Tokushima fatty (OLETF) rats
develop hyperglycemia, hyperinsulinemia, and mild obesity, which are
characteristic of human non-insulin-dependent diabetes mellitus. We
have shown that two recessive genes, ODB1 mapped on the X chromosome
and ODB2 mapped on chromosome 14, are involved in the induction of the
diabetes in OLETF rats. Recently we found that OLETF rats are the naturally occurring cholecystokinin type A receptor (CCKAR) gene knockout rats. In this study, we focused on the
genotype of CCKAR gene and the ODB1 gene in regulation of glucose
homeostasis in the F2 cross of the OLETF rats. Relatively high plasma
glucose levels were observed in the F2 offspring with the homozygously disrupted CCKAR gene. A synergistic effect for increasing plasma glucose levels in F2 rats between disrupted CCKAR gene and the ODB1
gene was shown. The CCKAR gene was found to map very close to ODB2 by a
linkage analysis using microsatellite markers. These results suggest
that CCKAR gene maintains normoglycemia in rats.
Otsuka Long-Evans Tokushima fatty rats; non-insulin-dependent
diabetes mellitus model; ODB2 gene
 |
INTRODUCTION |
OTSUKA LONG-EVANS TOKUSHIMA fatty (OLETF) rats are an
inbred strain of spontaneous mutants, which develop persistent
hyperglycemia and mild obesity by 18 wk of age and insulin deficiency
by 65 wk of age (13). In this respect, these rats resemble
non-insulin-dependent diabetes mellitus (NIDDM) patients. Many genetic
and environmental factors are thought to be responsible for NIDDM (10).
OLETF rats are also thought to have various malfunctional genes, which account for their pathological states (13). Multiple recessive genes
such as ODB1 and ODB2, located on the X chromosome and chromosome 14, respectively, are reported to be involved in the induction of diabetes
mellitus in OLETF rats (10, 11). Recently, we demonstrated no
expression of cholecystokinin type A receptor (CCKAR) gene in OLETF,
although cholecystokinin type B receptor (CCKBR) mRNA was intact (5,
7). At present, disrupted CCKAR gene is the only gene that has been
cloned in OLETF rats (24). Our further studies (24) proved that OLETF
rats were homozygous deletion mutants of CCKAR gene. The DNA deletion,
which includes the promoter region and the first and second exons,
explained why CCKAR was not expressed in OLETF rats (24).
Receptors for CCK in the peripheral tissue and the central nervous
system have been classified pharmacologically into two subtypes, CCKAR
and CCKBR, based on their binding affinities for the CCK gastrin family
peptides. Molecular cloning analyses of both receptor types supported
the physiological observations. These findings have been extensively
reviewed in a recent article (26). CCK is known to stimulate the
release of insulin and other islet hormones (3, 15). Specific CCK
receptors have been found on rat pancreatic
-cells by light and
electron microscopic autoradiography (17) and by studying the effects
of specific antagonists on insulin release (25). An abnormal CCK
secretion occurs in NIDDM and may contribute to hyperglycemia, which is associated with this disease (16). CCKBR are widely distributed throughout the central nervous system, whereas CCKAR are found only in
certain regions such as the nucleus tractus solitarius, area postrema,
interpenduncular nucleus, posterior hypothalamus, and the nucleus
accumbens (2, 9). One function of CCKAR in the hypothalamus is to
regulate food intake (12, 14, 19-22), but the involvement of CCKBR
in this function is controversial (1, 4). Thus dysfunctional CCKAR may
cause disorders such as hyperglycemia, hyperphagia, and obesity, which
are characteristic of NIDDM. Indeed, neither endogenous nor exogenous
CCK stimulated insulin release in OLETF rats, but they did stimulate
its release in normal control Long-Evans Tokushima rats, even though
the insulin contents of both strains were not significantly different
(6). Administration of CCK into the lateral ventricles did not decrease food intake in OLETF rats, but it did in Long-Evans Tokushima rats
(14).
In this study, we focused on the genotypes of CCKAR gene and plasma
glucose levels in offspring of OLETF (OL) and Fisher 344 (F344)
and/or BN/crj (BN) rats. A synergistic relationship between CCKAR gene and ODB1 is shown.
 |
MATERIALS AND METHODS |
Animals.
OLETF rats of the 29th generation were supplied from Tokushima Research
Institute. F344 and BN rats were purchased from Charles River Japan. The animal facilities were free of specific pathogens. Temperature (23°C), humidity (55%), and lighting (0700-1900)
at the facilities were controlled. All rats were freely given a
standard diet (CRF-1; Oriental Yeast, Tokyo, Japan) and tap
water. Segregation studies were performed between OLETF
and F344 and/or BN rats (10, 11). A diagram of the studies is
shown in Fig.1.

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Fig. 1.
Breeding diagrams. A: diagram of
female (f) Otsuka Long-Evans Tokushima fatty (OLETF, OL) and male (m)
Fisher 344 (F344) rat crosses. B:
diagram of female OLETF and male BN/crj (BN) rat crosses. F2 rats were
produced by brother-sister matings of F1 rats.
C: diagram of backcross protocol.
Backcross progenies were obtained by mating male OLETF with female F1
rats produced by mating female OLETF with male BN rats.
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DNA from segregation studies.
As described by Sambrook et al. (18), the nuclei were separated from
rat liver tissues obtained from the offspring of the segregation
studies, and the DNA was purified by the standard phenol-chloroform
method. In the studies, F1 rats were obtained by mating female (f)
OLETF rats with male (m) F344 or BN rats. F2 rats were produced by
brother-sister matings of the F1 rats. Hyperglycemia was mostly
observed in males because of hormone dependence, although the genetic
background was the same for both sexes (13). Therefore, all male rat
progenies were killed and analyzed at 30 wk of age. Small samples of
the livers were subjected to DNA analysis. The DNA from the backcross
study, OLETF(m) × F1(f) [OLETF(f) × BN(m)]
(11), was also analyzed.
Southern blot analysis.
Genomic DNA (10 µg) was digested with a restriction enzyme
(BamH I), separated by 0.7% agarose
gel electrophoresis, and blotted onto Hybond-N nylon membranes
(Amersham). The blots were hybridized with a
32P-labeled, random-primed rat
CCKAR cDNA probe (a gift from Dr. S. A. Wank, National Institutes of
Health, Bethesda, MD) containing the full coding sequence (27). The
blots were washed once for 20 min in 2× saline-sodium citrate
(SSC; 1× SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.0)/0.1%
sodium dodecyl sulfate at room temperature and then twice for 20 min,
each time in 0.1× SSC/0.1% sodium dodecyl sulfate at 62°C.
Autoradiography was performed using X-ray film (X-OMAT, Kodak).
Oral glucose tolerance test.
An oral glucose tolerance test (OGTT) was performed for 30-wk-old rats
being fasted for 16 h before the test. Glucose (2 g/kg) solution was
given per os, and plasma levels of glucose were measured before and 30, 60, 90, and 120 min after its administration (10, 11). Rats were
classified into three types on the basis of the results by the OGTT:
1) diabetic, with a peak level of
plasma glucose >300 mg/dl and a level of plasma glucose at 120 min
>200 mg/dl, and only rats showing both conditions by OGTT were
diagnosed with clinical diabetes mellitus (DM);
2) impaired glucose tolerance (IGT)
in that either one of the two conditions was observed in rats; and
3) normal, in which neither DM nor
IGT was present (13).
Polymerase chain reaction amplification.
We designed a polymerase chain reaction (PCR) method to analyze whether
the F1 and F2 rats have the OLETF-type deleted CCKAR allele or the F344
and/or BN-type normal allele. Two pairs of primers
were made, one pair (primer A = 5'-AGGAGAGAGACAGGAATGAGC-3' and primer B = 5'-AAGGTTGAGGTTGATCCAAGC-3') was designed to
amplify 165 base pairs (bp) of exon 1 in which the homozygously deleted DNA was not amplified, and the other pair (primer C = 5'-AACACTCACCATGGCAAGG-3' and primer D = 5'-GTCACTTGGCAACAGGAAGC-3') was designed to identify the
deletion. Sequences of ~200 bases distal to the deletion break point,
from which 453 bases were amplified in the deleted DNA (24), gave no
amplified product with the normal DNA (23) because the sequence was too
long (ca. 6.9 kilobase) to be amplified. When these four primers were
mixed together in a PCR system, only a 165-bp fragment of exon 1 was
amplified in the homozygote with the normal allele. A 453-bp DNA
fragment was amplified from DNA with the homozygously deleted allele.
Both the 165-bp and 453-bp DNA fragments were found in the PCR mixture,
in which the heterozygous CCKAR gene was used as the template DNA (Fig.
1, A and
B). The parameters for DNA
amplification were 42 cycles of denaturation (1 min at 94°C),
annealing (1 min at 58°C), and extension (3 min at 72°C), with
a final extension period of 10 min.
Classification of F2 rats by genotypes of CCKAR and ODB1.
The CCKAR genotypes of all F2 rats (Fig.
1A) were determined, and the rats
were classified as CCKAR(
/
), CCKAR(+/
), or CCKAR(+/+). Then
each group was further classified by the genotypes of PRPSII, a
microsatellite marker closely linked with ODB1 on chromosome X,
obtained previously (12) as OLETF-type PRPSII [(
/
)OL,
(+/
)OL, (+/+)OL] and F344-type PRPSII
[(
/
)F344, (+/
)F344, (+/+)F344]. The blood glucose
level was compared between one group and another.
Classification of F2 and backcross rats by genotypes of CCKAR and
ODB2.
The CCKAR genotypes of all F2 (Fig.
1B) and backcross (Fig.
1C) rats were determined, and the
rats were classified as CCKAR(
/
), CCKAR(+/
), or
CCKAR(+/+). Then each group was further classified by the
genotypes of D14Mit4, a microsatellite marker of chromosome 14, obtained previously (13) as OLETF-type D14Mit4 [(
/
)OL, (+/
)OL, (+/+)OL] and BN-type D14Mit4 [(
/
)BN,
(+/
)BN, (+/+)BN]. The blood glucose level was
compared between one group and another.
Statistical analysis.
Values are expressed as means ± SE. Determination of plasma glucose
levels was performed by the multiple analysis of variance with repeated
measure and analysis of variance followed by Fischer's protected
least-significant difference test, respectively.
Segregation of various genotypes was examined by the
2 test. Incidence of diabetic
syndrome in each genotype was analyzed by Fisher's exact test.
Statistical significance was assumed with a
P value < 0.05.
 |
RESULTS |
Identification of CCKAR genotypes.
A PCR method was designed (Fig.
2A) for the rapid identification of
the CCKAR genotypes [the homozygously disrupted allele (
/
),
the normal allele (+/+), and the heterozygously disrupted allele
(+/
)] of progeny rats in the segregation study (see
MATERIALS AND METHODS). The normal
CCKAR allele was detected in F344 rats and the disrupted CCKAR allele
(
/
) in OLETF rats. When the F1 progenies, obtained by mating
female OLETF and male F344 rats, were analyzed, both the 165-bp and
453-bp DNA fragments were amplified in the PCR mixture (Fig.
2B). The results showed that all the F1 progenies had both a disrupted and normal CCKAR gene. Southern blotting results supported this (Fig.
2C). Expression of the CCKAR gene in
the pancreas was almost the same in the F1 rats as in F344 rats,
irrespective of age or sex, as shown by Northern blot analysis
(unpublished results).

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Fig. 2.
Determination of the genotypes of cholecystokinin type A receptor
(CCKAR) gene in each rat. A:
polymerase chain reaction (PCR) strategy used to detect genotype.
Positions of primers are represented by closed triangles under gene
structures. Each PCR product derived from normal or disrupted allele is
represented by a 165 or 453 bp closed box. Restriction enzyme sites are
shown as B, BamH I; E,
EcoR I; H,
Hind III; S,
Sal I; and X,
Xba I. B: PCR results. Each genomic DNA was
amplified by 4 primers shown in A.
Lane 1, male F344;
lane 2, female OLETF;
lane 3, male F1; lane
4, male F2(+/+); lane
5, male F2(+/ ); lane
6, male F2( / ).
C: Southern blotting results. Each DNA
(10 µg) was digested with a restriction endonuclease
(BamH I), separated by 0.7% agarose
gel electrophoresis, blotted onto a nylon membrane, and hybridized with
radiolabeled rat CCKAR cDNA (see MATERIALS AND
METHODS). Lane 1,
male F344; lane 2, female OLETF;
lane 3, male F1; lane
4, male F2(+/+); lane
5, male F2(+/ ); lane
6, male F2( / ).
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Plasma glucose levels of F2 rats and backcross progenies:
synergistic action of disrupted CCKAR gene and ODB1.
All F1 progenies obtained from OLETF and F344 and/or BN rats
were heterozygotes of the normal and disrupted CCKAR genes. One hundred
sixty male F2 (OLETF × F344) rats (Fig.
1A), which were obtained from
brother-sister matings of the F1 rats, were also analyzed by PCR, and
43 had a homozygous disrupted CCKAR gene (
/
), 80 had a
heterozygous CCKAR gene (+/
), and 37 had a homozygously normal CCKAR
gene (+/+). The frequency of F2 rats with different CCKAR genotypes was
roughly distributed according to Mendel's law. The relationship
between the CCKAR genotypes and the OGTT results was analyzed for all
F2 rats, and plasma glucose in rats with the CCKAR gene (
/
) was
found to be at a relatively higher level than in rats of the (+/
) or
the (+/+) genotype on average (Fig. 3). The
difference of plasma glucose levels at 30, 60, and 90 min was
significant among the CCKAR genotypes [(
/
) vs. (+/
) or
(+/+)]. Rats with a heterozygously disrupted CCKAR(+/
) gene seemed to have similar plasma glucose levels to rats with the normal gene (+/+) (Fig. 3).

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Fig. 3.
Plasma glucose levels as determined by oral glucose tolerance test
(OGTT) on F2 rats with different CCKAR genotype. Rats were analyzed
based on CCKAR genotype ( / ), (+/ ), and (+/+). Numbers for F2
rats [OLETF(f) × F344(m)] were 43 ( / ), 80 (+/ ), and 37 (+/+), respectively. Changes in blood glucose levels
were significantly different with respect to the various genotypes
(F = 5.96, P < 0.005) and time
(F = 520.8, P < 0.0001). * Significantly
higher than corresponding F2(+/+) and F2(+/ ) values.
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When the male F2 [OLETF(f) × F344(m)] rats (Fig.
1A) were analyzed according to
CCKAR genotypes and PRPSII, the highest incidence rates (35%) of DM
(20%) and IGT (15%) were found in the (
/
)OL genotype of F2
rats; each genotype of the F2 rats was analyzed in relation to their
OGTT results (
2 = 15.97, P = 0.0069; Table
1). When the
incidence of diabetic syndrome in each genotype was analyzed by
Fisher's exact test, (
/
)OL vs. (+/+)F344, (+/+)OL vs. (+/+)F344,
and (
/
)F344 vs. (+/+)F344 were P = 0.005, P = 0.230, and
P = 0.15, respectively. Similar
results were obtained by the analysis of the F2 offspring of OLETF and
BN rats (data not shown).
Backcross male offspring obtained by mating male OLETF and female F1
[OLETF(f) × BN(m)] rats were analyzed for their
plasma glucose level at OGTT. The difference in plasma glucose levels at 30, 60, and 90 min was significant among the two groups of disrupted
CCKAR gene homozygotes F2 rats [OLETF(f) × BN(m)]
(Fig. 1B) and the backcross rats on
{F1(f) [OLETF(f) × BN(m)] × OLETF(m)]} (Figs. 1C and 4).
CCKAR gene and ODB2 on chromosome 14.
D14Mit4 was the microsatellite marker, closely linked to ODB2 on
chromosome 14 (11). The F2 rats from female OLETF and male BN rats
(Fig. 1B) were analyzed by the types
of CCKAR gene and D14Mit4. Fifty-four F2 rats were homozygotes of the
disrupted CCKAR gene, and 50 of these were homozygotes of OLETF type
D14Mit4, localizing within 3.1 cM of CCKAR gene on rat chromosome 14 (Table 2). In the backcross male offspring of male OLETF
and female F1 [OLETF(f) × BN(m)] rats (Fig.
1C), 20 of the 59 progenies were homozygotes of the disrupted CCKAR gene and the other 39 were heterozygotes of the gene. All homozygotes with the disrupted CCKAR
gene were homozygotes of OLETF type D14Mit4. The results showed that
CCKAR gene localized within 1.7 cM of D14Mit4 on rat chromosome 14 (Table 2).
 |
DISCUSSION |
In the mating study F344 and/or BN rats were used as partners
to the OLETF rats because both strains are normoglycemic and are
thought to have no abnormal genes increasing the plasma glucose level.
We suspected that the disrupted CCKAR gene is segregated in a group of
F2 progenies in the study. The PCR method was useful for analysis of
the types of CCKAR gene in the F1 and F2 progenies. The distribution of
the disrupted CCKAR gene was elucidated in F1 progenies, with both
males and females heterozygous (+/
) for the CCKAR gene (Fig. 2,
B and
C). This result indicates that the CCKAR gene is located on an autosomal chromosome in rats, consistent with the result of our fluorescence in situ hybridization analysis (24). When we classified the CCKAR genotypes of 160 F2
male rats and analyzed the relationship with their OGTT values at 30 wk
of age, we found that 43 F2 rats with the CCKAR(
/
) genotype had
significantly high blood glucose levels. However, approximately one-half the F2 rats with the disrupted CCKAR(
/
) gene were
normoglycemic, and two F2 of 37 with the wild type gene (+/+) had
relatively high plasma glucose levels. Therefore, factor(s) other than
disrupted CCKAR gene act(s) to elevate blood glucose levels in F2 rats, and these factors likely act together. The lack of CCKAR may partly account for the rise in blood glucose levels of some F2 rats. Then, the
plasma glucose levels were compared in backcross offspring with
CCKAR(
/
) genes and (
/
)F2 rats of OLETF and BN rats, and a
higher plasma glucose level was recognized in the former rats than in
the latter (Fig. 4). These results suggested that in addition to the
disrupted CCKAR gene, other factor(s) act to elevate the plasma glucose
level, that is, hyperglycemia in OLETF rats is a polygenic event.

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Fig. 4.
Plasma glucose levels as determined by OGTT on backcross and F2
progenies with disrupted CCKAR genotype. Homozygotes with disrupted
CCKAR gene, F2 rats [OLETF(f) × BN(m)] and the
backcross rats {F1(f) [OLETF(f) × BN(m)] × OLETF(m)}, were compared. Numbers of F2 rats
[OLETF(f) × BN(m)] and the backcross rats
{F1(f) [OLETF(f) × BN(m)] × OLETF(m)} were 54 and 19, respectively. Changes in blood glucose
levels were significantly different with respect to the various
genotypes (F = 45.94, P < 0.0001) and time
(F = 219.58, P < 0.0001).
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When the male F2 [OLETF(f) × F344(m)] rats were
analyzed according to CCKAR genotype and PRPSII (a microsatellite
marker closely linked with ODB1 gene), the highest incidence of DM and
IGT was found in the (
/
)OL genotype, as analyzed in relation to
OGTT results (Table 1). According to the analysis with
2 tests, the disrupted CCKAR
and OLETF type ODB1 seem to act synergistically to elevate the plasma
glucose level in F2 rats. Similar results were obtained on the analysis
of the F2 offspring of OLETF and BN rats (data not shown). These
findings suggested that the CCKAR and the ODB1 genes act together to
induce hyperglycemia in F2 rats. Recently, ODB2 was suggested as a gene
responsible for hyperglycemia in OLETF rats and mapped to chromosome 14 (11), on which CCKAR gene is also located (24). The CCKAR gene and
D14Mit4, the microsatellite marker of ODB2, were found to be very
closely linked in the F2 rats and in the backcross offsprings. These
analytic results suggested that the CCKAR and ODB2 genes are the same
gene or very closely located on chromosome 14.
At present, disrupted CCKAR gene is the only gene that has been cloned
and analyzed in correlation with hyperglycemia in OLETF rats. The
disruption of the CCKAR gene was recessive as for the hyperglycemic
phenotype, which is consistent with the Northern blotting results
obtained from rats with heterozygously disrupted CCKAR gene (+/
),
which produces an average amount of CCKAR mRNA in the pancreas
(unpublished result).
The genetic background of the NIDDM of OLETF rats is very complicated
(8, 10, 11, 13). According to the results of this study, a null
mutation in the CCKAR gene seems to elevate the plasma glucose level in
OLETF rats in correlation with other factors. The mechanism behind this
is likely to be the lack of the "incretin" effect of CCK (6),
rather than a reduction in the effectiveness of insulin in the muscle,
caused by obesity. In the F2 rats with homozygously disrupted CCKAR
gene, the insulin response after the oral ingestion of glucose might be
reduced. In this respect, the insulin levels in the F2 rats needed to
be determined, but unfortunately it was not measured in the segregation study because it was difficult to gather enough blood to follow the
time course.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a grant from the Tokyo
Metropolitan Institute of Gerontology, Short-Term Project, and by the
Research Grant for Longevity Sciences (9C-03) from the Ministry of
Health and Welfare. This work was also supported in part by a
grant-in-aid for the 2nd-Term Comprehensive 10-Year Strategy for Cancer
Control from the Ministry of Health and Welfare. K. Kataoka is an
Awardee of a Research Resident Fellowship from the Research Foundation
for Promotion of Cancer Research, Japan.
 |
FOOTNOTES |
Address for reprint requests: A. Kono, Div. of Chemotherapy, National
Kyushu Cancer Center, 3-1-1 Notame, Minami-ku, Fukuoka, 815 Japan.
Received 14 July 1997; accepted in final form 9 October 1997.
 |
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