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*
Department of Clinical Physiology and
Nutrition, Tokyo Metropolitan Institute of Gerontology, Tokyo-l73–0015, Japan;
**
Third Department of Internal Medicine, Kobe University School of Medicine, Kobe-650, Japan;
Department of Molecular Genetics, Tohoku University School of Medicine, Sendai-980–8575, Japan; and
Research Institute and
Department of Gastroenterology, National Kyushu Cancer Center, Fukuoka-811–1395, Japan
2To whom correspondence should be addressed. E-mail: miyasaka{at}tmig.or.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • energy expenditure • energy intake • energy metabolism • knockout mice • cholecystokinin-receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Two types of CCK receptors (types A and B) have been identified. These receptors have highly homologous amino acid sequences but are derived from different genes. Several pharmacologic studies using the appropriate antagonists have shown that the CCK-B receptor (CCK-BR) is involved in anxiety and the CCK-A receptor (CCK-AR) has been implicated in satiety and behavior. However, the distributions of these receptors overlap in tissues and cross-reactivity of each antagonist could not be excluded.
Homozygous CCK-BR gene deficient mice (-/-) have been developed (2
). We recently cloned the genomic structures of CCK-AR in rats (3
), mice (4
) and humans (5
), and generated the CCK-AR gene knockout mice (6
). Then, we produced CCK-AR(-/-)BR(-/-) mice. Because these mice were viable and fertile into adulthood, we were able to examine their daily energy metabolism and energy turnover.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The protocol was reviewed and approved by the appropriate committee of the Tokyo Metropolitan Institute of Gerontology.
Three male CCK-AR(-/-) mice were mated with 12 female CCK-BR(-/-) mice; the F1 progeny exhibited genotype CCK-AR(+/-) BR(+/-). Then, male F1 mice were mated with female F1 mice, and the progeny showed nine kinds of genotypes: CCK-AR(+/+)BR(+/+), CCK-AR(+/+)BR(+/-), CCK-AR(+/+)BR(-/-), CCK-AR(+/-)BR(+/+), CCK-AR(+/-)BR(+/-), CCK-AR(+/-)BR(-/-), CCK-AR(-/-)BR(+/+), CCK-AR(-/-)BR(+/-), CCK-AR(-/-)BR(-/-). Then, male CCK-AR(-/-)BR(-/-) mice were mated with female CCK-AR(-/-)BR(-/-) mice to obtain double knockout mice.
CCK-AR(-/-) and CCK-BR(-/-) mice were selected from their respective lines and wild-type mice [CCK-AR(+/+)BR(+/+)] were selected at random from CCK-AR and CCK-BR lines. Exactly age-matched male progeny were used for experiments. Mice were fed commercial nonpurified diet (CRF-1; Charles River Japan, Yokohama, Japan). Three separate studies were conducted using different mice.
Daily energy intake study.
We estimated daily food intake over 3 d using age-matched (36 wk) wild-type [CCK-AR(+/+)BR(+/+)] (n = 7), CCK-AR(-/-) (n = 8), CCK-BR(-/-) (n = 4) and CCK-AR(-/-)BR(-/-) (n = 9) mice. They were kept individually in metabolic cages. Food (110–120 g), which was previously weighed, was offered to mice for 3 d. Body weight was measured before the test. After the 3-d period, the remaining food including crumbs was weighed, and the daily energy intake [kJ/(d · kg)] was estimated.
Energy metabolism study.
Mice of the 4 different genotypes, wild-type [CCK-AR(+/+)BR(+/+)] (n = 13), CCK-AR(-/-) (n = 8), CCK-BR(-/-) (n = 5) and CCK-AR(-/-)BR(-/-)(n = 6) (36 wk old) were kept for 3 d in individual metabolic cages for simultaneous measurements of energy metabolism (7
,8
). Oxygen consumption and carbon dioxide production in expired air were measured continuously with an automatic O2-CO2 analyzer (NEC Medical Systems, Model IH26, Tokyo, Japan). Energy expenditure per hour and per day was calculated. The basal metabolic rate was calculated on the basis of the lowest daily energy expenditure value per hour.
Organ weight study.
Age-matched wild-type [CCK-AR(+/+)BR(+/+)] (n = 4) and CCK-AR(-/-)BR(-/-) (n = 5) mice were killed and the wet weights of liver, kidneys, pancreas, and the sums of epididymal and perinephric fat, were measured.
Statistical analysis.
Values are expressed as means ± SEM. The results were analyzed by one-way ANOVA with respect to strain, followed by Fisher’s Protected Least Significant Difference tests. Student’s t test was used for the analysis of data in Table 1
. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Body weights of CCK-AR(+/+)BR(+/+), CCK-AR(-/-), and CCK-BR(-/-) mice were greater than CCK-AR(-/-)BR(-/-) mice (P < 0.01), i.e., 33.7 ± 0.8 g for wild-type [CCK-AR(+/+)BR(+/+)], 32.7 ± 0.7 for CCK-AR(-/-), 35.6 ± 0.3 for CCK-BR(-/-) and 30.5 ± 0.4 for CCK-AR(-/-)BR(-/-) mice.
Energy intake was significantly greater in CCK-BR(-/-) and CCK-AR(-/-)BR(-/-) mice than in CCK-AR(-/-) and wild-type [CCK-AR(+/+)BR(+/+)] mice (P < 0.001; Fig. 1
). CCK-BR(-/-) and CCK-AR(-/-)BR(-/-) mice did not differ from one another nor did CCK-AR(-/-) and wild-type mice.
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Body weights were not different among genotypes (P = 0.08), i.e., 31.8 ± 0.7 g for wild-type [CCK-AR(+/+)BR(+/+)], 36.7 ± 2.0 for CCK-AR(-/-), 37.1 ± 2.4 for CCK-BR(-/-) and 34.3 ± 1.9 for CCK-AR(-/-)BR(-/-) mice.
Daily energy expenditure was greater in CCK-BR(-/-) and CCK-AR(-/-)BR(-/-) mice than in CCK-AR(-/-) and wild-type [CCK-AR(+/+)BR(+/+)] mice (P < 0.0001; Fig. 2
). The lowest energy expenditures were observed during the light period (1200–1400 h). These lowest values were used to estimate the basal metabolic rate. The basal metabolic rate was also greater in CCK-BR(-/-) [1416 ± 125 kJ/(d · kg)] and CCK-AR(-/-)BR(-/-) [1558 ± 34 kJ/(d · kg)] mice than in CCK-AR(-/-) [1027 ± 108 kJ/(d · kg)] and wild-type [CCK-AR(+/+)BR(+/+)] [1133 ± 82 kJ/(d · kg)] mice (P < 0.01). CCK-BR(-/-) and CCK-AR(-/-)BR(-/-) mice, and CCK-AR(-/-) and wild-type mice did not differ from one another.
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Relative weights of liver and kidneys (g/kg body) were greater in CCK-AR(-/-)BR(-/-) mice than in wild-type CCK-AR(+/+)BR(+/+) mice, whereas those of pancreas and the sums of epididymal fat and perinephric fat were not (Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Therefore, metabolic turnover was apparently enhanced by the lack of CCK-BR in mice.
The relative wet weights of liver and kidneys were significantly greater in CCK-AR(-/-)BR(-/-) mice than in wild-type [CCK-AR(+/+)BR(+/+)] mice, whereas those of pancreas and visceral fat (epididymal and perinephric fat) were comparable. No abnormality was visible in these organs. The greater relative weights of these organs might relate to the enhanced energy metabolism with the energy turnover in mice lacking CCK-BR, although the underlying mechanism has not been clarified. Neither CCK-AR nor BR was expressed in the liver, and CCK-AR, but not BR, was expressed in the kidney of mice (1
). Thus, the reasons for the greater relative weights of liver and kidneys in CCK-AR(-/-)BR(-/-) mice are unknown.
On the other hand, CCK-BR has been reported to be involved in anxiety (1
), and the possibility that some of the increase in energy expenditure was due to increased activity could not be excluded. Moreover, we have not examined the wet weight of brown adipose tissue, which is responsible for thermogenesis. An examination of behavioral abnormalities and the regulation of body temperature in these mice is now in progress in our laboratory.
In conclusion, energy metabolism and energy turnover were increased in mice with a disruption of the CCK-BR gene, although the underlying mechanisms for these alterations are unknown.
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FOOTNOTES |
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3 Abbreviations used: CCK, cholecystokinin; CCK-AR, CCK-A receptor; CCK-BR, CCK-B receptor.
Manuscript received 11 June 2001. Initial review completed 19 July 2001. Revision accepted 18 January 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1.
Wank, S. A. (1995) Cholecystokinin receptors. A review article. Am. J. Physiol. 269:G628-G646.
2.
Nagata, A., Ito, M., Iwata, N., Kuno, J., Takano, H., Minowa, O., Chihara, K., Matsui, T. & Noda, T. (1996) G protein-coupled cholecystokinin-B/gastrin receptors are responsible for physiological cell growth of the stomach mucosa in vivo. Proc. Natl. Acad. Sci. U.S.A. 93:11825-11830.
3. Takata, Y., Takiguchi, S., Funakoshi, A. & Kono, A. (1995) Gene structure of rat cholecystokinin type-A receptor. Biochem. Biophys. Res. Commun. 213:958-966.[Medline]
4. Takata, Y., Takiguchi, S., Kataoka, K., Funakoshi, A., Miyasaka, K. & Kono, A. (1997) Mouse cholecystokinin type-A receptor gene: Alternative splice acceptor site in exon 2. Gene 187:267-271.[Medline]
5. Funakoshi, A., Miyasaka, K., Yamamori, S., Takata, Y., Kataoka, K., Takiguchi, S., Kono, A. & Shimokata, H. (2000) Body fat content is related to cholecystokinin A receptor gene promoter polymorphism. FEBS Lett. 466:264-266.[Medline]
6. Takiguchi, S., Suzuki, S., Sato, Y., Kanai, S., Miyasaka, K., Jimi, A., Shinozaki, H., Takata, Y., Funakoshi, A., Kono, A., Minowa, O., Kobayashi, T. & Noda, T. (2002) Role of CCK-A receptor for pancreatic function in mice: A study in CCK-A receptor knockout mice. Pancreas (in press).
7. Ichikawa, M. & Fujita, Y. (1987) Effects of nitrogen and energy metabolism on body weight in later life of male Wistar rats consuming a constant amount of food. J. Nutr. 117:1751-1758.
8.
Ichikawa, M., Kanai, S., Ichimaru, Y., Funakoshi, A. & Miyasaka, K. (2000) The diurnal rhythm of energy expenditure differs between obese and glucose-intolerant rats and streptozotocin-induced diabetic rats. J. Nutr. 130:2562-2567.
9. Kopin, A. S., Mathes, W. F., McBride, E. W., Nguyen, M., Al-Haider, W., Schmitz, F., Bonner-Weir, S., Kanarek, R. & Beinborn, M. (1999) The cholecystokinin-A receptor mediates inhibition of food intake yet is not essential for the maintenance of body weight. J. Clin. Investig. 103:383-391.[Medline]
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