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Effects of aging and weaning on mRNA expression of leptin and CCK receptors in the calf rumen and abomasum
Domestic Animal Endocrinology 22 (2002) 25–35
Effects of aging and weaning on mRNA expression of leptin and CCK receptors in the calf rumen and abomasum S. Yonekura∗ , K. Kitade, G. Furukawa, K. Takahashi, N. Katsumata, K. Katoh, Y. Obara Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Sendai 981-8555, Japan Received 2 April 2001; accepted 27 August 2001
Abstract In order to know the effects of weaning and volatile fatty acid feeding on gastric leptin expression, we investigated the expression of leptin and CCK receptor mRNA in the bovine rumen, abomasum and duodenum using RT-PCR in 3-week-old pre-weaning, 13-week-old post-weaning and adult animals. Leptin mRNA was expressed in the rumen and abomasum of 3-week-old pre-weaning animals, but it was abolished in 13-week-old and adult animals. In the duodenum, leptin expression was observed in the 3-, 13-week-old and adult animals. In the rumen, CCKA receptor mRNA was expressed in 3-week-old animals, but not in 13-week-old and adult animals. In the abomasum, CCKB receptor expression gradually decreased from 3-week-old to adult animals. Expression of CCKB receptor and of CCKA receptor was slight in the rumen and abomasum, respectively. In the next study, we examined the effect of weaning of 6 weeks or non-weaning (fed on milk replacer alone (milk) or milk replacer with volatile fatty acids (milk + VFA) until 13 weeks old) on leptin mRNA expression in the rumen and abomasum. In 13-week-old calf rumen and abomasum, leptin mRNA expression was detected in non-weaning milk-fed animals at 13 weeks old, although it was not observed in weaning and non-weaned milk + VFA-fed animals. The change in CCKA receptor expression in the rumen was similar to those of leptin mRNA expression. CCKB receptor transcription in the abomasum of milk-fed animals was higher than that of the weaning and milk + VFA-fed animals. These results indicate that leptin expression is coincident with CCK receptor expression in calf stomachs, and that leptin and CCK receptor mRNA expression are affected by the change in the physiological status brought about by weaning and VFA feeding. © 2002 Elsevier Science Inc. All rights reserved.
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Corresponding author. Tel.: +81-22-717-8700; fax: +81-22-717-8701. E-mail address: [email protected] (S. Yonekura).
0739-7240/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 0 1 ) 0 0 1 1 4 - X
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1. Introduction The physiological mechanisms by which satiety sensing occurs after food intake have not yet been defined. Several peptides secreted from the gastrointestinal tract during eating have been shown to suppress food intake [1,2]. One of them, cholecystokinin (CCK) is secreted from duodenal I cells after eating [3,4]. One of the physiological roles of peripheral CCK is as a short-term regulator of food intake via the receptors [5]. Binding sites for CCK have been identified in a variety of tissues, and two primary receptor subpopulations have been discerned on the basis of receptor binding and functional characteristics [6,7]. CCKA receptors have a high affinity for CCK but a low affinity for non-sulfated CCK and gastrin [7]. In contrast, CCKB receptors interact with gastrin and CCK at almost the same high affinity and poorly discriminate sulfated and non-sulfated peptide analogues [7]. Leptin, a 16 kDa protein product of the ob gene, was identified as an adipocyte-derived cytokine [8]. In contrast to CCK, the satiety effect induced by circulating leptin is thought to be only apparent in the long-term [9,10]. Leptin, which was initially thought to be exclusively expressed in adipocytes [8], has been shown to be expressed in gastric mucosa and fundic glands in rats [11] and humans [12]. In addition, the secretion of gastric leptin is caused by an increased CCK secretion stimulated by feeding [11]. It is also considered that there exists a functional synergistic interaction between leptin and CCK, leading to a short-term regulation of food intake [13]. With respect to ruminant animals, some studies on CCK indicate that CCK induces suppression of food intake similar to non-ruminant animals [14,15]. But, to our knowledge, there is no report of the existence of leptin in the reticulo-rumen, omasum or abomasums of ruminants. Ruminants are classically regarded as having four “stomachs,” with microbial fermentation occurring in the reticulo-rumen, resulting in the production of volatile fatty acids (VFA). The rumen of calves rapidly and drastically develops around the weaning period (about at age of 5–6 weeks) and becomes mature at 12 weeks of age. It is at 5–6 weeks of age when ruminant diets shifts from a milk-based to a solid food-based diet. Therefore, it is speculated that the regulatory mechanism of food intake may be drastically changed during this period. In this study, we investigated leptin expression in the rumen, abomasum and duodenum of developing calves. Furthermore, as the relationship between leptin and CCK is reported in rodents, we also studied CCK receptor expression in the rumen and abomasum.
2. Materials and methods 2.1. Chemicals All salts of VFA used for the experimental solutions were purchased from Sigma (St. Louis, MO). TRIzol reagent was purchased from Gibco BRL (Grand Island, NY). Isolation kit for mRNA was purchased from Miltenyi Biotec (Miltenyi Biotec, Bergisch Gladbach, Germany). T/A cloning kit was purchased from Invitrogen (San Diego, CA). All salts for the isolation of RNA were purchased from Nakarai (Tokyo, Japan).
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2.2. Animals The treatment of the animals was according to the “Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences” (The Physiological Society of Japan) and the present experiment was approved by The Animal Care Committee of Tohoku University. Eleven male Holstein calves (TOHOKU UNIVERSITY FARM) were fed by their parents for 1 week after birth to ensure the consumption of colostrum before starting the experimental feeding. They were then divided into three groups and fed until 13 weeks of age: a weaning group (W), a milk replacer (milk)-fed group (M), and a milk + VFA-fed group (V). In W group, six calves were fed a commercial milk replacer and calf starter as described in Table 1, and three of them were sacrificed at 3 weeks old. Another three animals were fed on calf starter and hay until 13 weeks old and sacrificed. They were allowed free access to timothy hay from 5 weeks of age. Calves of M and V groups were fed milk replacer and injected daily with saline or VFA into the reticulo-rumen from 5 weeks of age, respectively, as described in Table 1, and sacrificed at 13 weeks old (n = 2 and 3, respectively). All calves were fed diets at 10:00 and 16:00 twice daily, and sacrificed 18 h after the last meal. Tissue samples from three adult male Holstein animals (1.5–2 years old) were obtained at a local abattoir (mean body weight: 316 ± 1.6 kg, n = 3). The gastrointestinal tract was removed approximately 15 min after death, and tissue samples were taken. The size of ventral sac of the rumen and fundus of the abomasums was approximately 2 cm × 2 cm, duodenum was 20 cm length from the pylorus. Tissue samples were immediately placed into polypropylene containers, frozen in liquid nitrogen, and stored at −80◦ C until RNA analysis. 2.3. Experimental diets and VFA administration Milk replacer and calf starter were purchased from Zenraku (Tokyo, Japan). The amounts of the experimental diets in W group were decided according to the Japanese Feeding Standard, Table 1 Weaning animals
Milk-fed animals
Milk + VFA-fed animals
Age (weeks)
Milk replacer (g per day)
Calf starter (g per day)
Milk replacer (g per day)
Milk replacer (g per day)
VFA (mol per day)
1 2 3 4 5 6 7 8 9 10 11 12 13
300 400 500 600 600 600 – – – – – – –
100 200 500 900 1200 1500 1700 1900 2100 2200 2300 2500 2500
300 400 500 600 700 800 950 1100 1250 1400 1550 1700 1850
300 400 500 600 700 800 950 1100 1250 1400 1550 1700 1850
– – – – 0.2 0.4 0.8 1.2 1.8 2.4 3.0 3.6 4.2
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and that of milk replacer in M and V groups were decided as reported in a previous study [16]. The milk replacer and calf starter consisted of 24.5 and 20.0% crude protein and 21.9 and 2.0% crude fat, respectively. The amount of the experimental diets given to animals is shown in Table 1. The calves of W group were allowed free access to timothy hay from 5 weeks of age. All calves had free access to water. The VFA solution (1.8 mol/L) consisted of equimolar amounts of acetic, propionic and butyric acids and pH was adjusted to pH 6.0 with sodium hydroxide and potassium hydroxide [17]. The intra-ruminal administration of VFA was carried out through polypropylane inserted into the reticulo-rumen through the esophagus every day from 5 to 13 weeks of age. The amount of VFA administrated was based on previous work [18]. The solution was administered into the reticulo-rumen of the animals of V group, and an equal volume of physiological saline was administrated to the M group through a tube after feeding of milk at 1600. All calves grow normally, and body weights were not different among three groups (data not shown). 2.4. RNA analysis by RT-PCR and semi-quantitative RT-PCR Total RNA was isolated from the tissues using the TRIzol extraction method according to the manufacturers instructions (Gibco BRL). Poly A+ mRNA was isolated from total RNA using a mRNA isolation kit (Miltenyi Biotec). The concentration of isolated mRNA was determined from the optical density at 260 nm and its purity from the wavelength ratio of 260/280 nm. PCR amplification of cDNA samples was carried out using the following primer pairs: bovine leptin sense (GTGCCCATCCGCAAGGTCCA-3 ) and antisense (TCAGCACCCGGGACTGAGGT-3 ) for the amplification of a 440 bp fragment; bovine CCKA receptor sense (CTGCTCAGCGTGCTGGGAAAC-3 ) and antisense (CGGGACTGTAAGGGTTTGCAAA-3 ) for the amplification of a 293 bp fragment [19]; bovine CCKB receptor (CGGGACACGAGAATTGGAGCTGG-3 ) and antisense (CCGTCAAAGCGAAGCCCTAAGTAG-3 ) for the amplification of a 618 bp fragment [19]. To provide an appropriate internal control, coamplification of a 450 bp fragment of G3PDH mRNA was carried out in each sample using the primer pair: G3PDH-sense (ACCACAGTCCATGCCATCAC-3 ) and antisense (TCCACCACCCTGTTGCTGTA-3 ). Forty cycles of PCR amplification were used for bovine leptin. The conditions used were 95◦ C for 1 min for denaturation, 64◦ C for 1 min for annealing, 72◦ C for 1 min for polymerization. The PCR product was run in a 2.0% agarose gel stained with ethidium bromide. PCR products were then ligated into the PCRII vector and used to transform one-shot Escherichia coli cells according to the protocol provided with the T/A cloning kit (Invitrogen, San Diego, CA). Colonies were screened by blue/white and ampicillin selection, and plasmids from resultant cultures were isolated and subjected to restriction analysis (with EcoRI) to check for the expected 440 bp insertion. The 440 bp product was then sequenced by use of the dideoxy method to confirm the encoded cDNA was same as the bovine leptin sequence. For quantification of the amount of mRNA by RT-PCR method, PCR amplification of bovine CCKA and CCKB receptor and G3PDH was carried out at different numbers of cycles. PCR amplifications were done with 95◦ C for 1 min for denaturation, 65◦ C (55◦ C for G3PDH) for 1 min for annealing, 72◦ C for 1 min 30 s for polymerization. An increase in the number of PCR cycles raised the products in an exponential manner. Different numbers of cycles were tested
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for CCKA and CCKB receptor (20–40 cycles) and G3PDH (14 and 30 cycles) and 33 and 26 cycles were employed for CCKA and CCKB receptors, and 22 cycles for G3PDH, respectively. In all assays, reactions with RT negative amplification were included (data not shown). The PCR products were separated in a 2% agarose gel, stained with ethidium bromide, and analyzed using Fluor-S MultiImager (Bio-Rad, Hercules, CA). The intensity of CCK receptor abundance was assigned as a ratio to that of G3PDH abundance. To minimize potential RT-PCR artifacts due to inherent reaction variability, all data points were repeated, for each target, at least three times using independent RNA samples. 2.5. Statistical analysis The results are expressed as means values ± SE. The data were analyzed by ANOVA, Student’s t-test and Bonferroni’s multiple range test with SAS. 3. Results 3.1. Effects of aging on leptin mRNA expression in various bovine tissues In RT-PCR analysis, a product of expected size (440 bp) was detected in fat, rumen, abomasum and duodenum, but not in liver tissues in cattle of 3 weeks of age. However, in 13-week-old and adult animals, leptin mRNA was not detected in the rumen and abomasums, although it was still found in the duodenum. Sequence analysis of 440 bp amplicon was confirmed 100% homology with the corresponding region of a published sequence for leptin cDNA (data not shown) (Fig. 1). 3.2. Effect of nutritional status on leptin mRNA expression in 13-week-old calf gut We examined whether the nutritional conditions affect leptin expression in bovine stomachs (rumen (A), abomasums (B) and duodenum (C)) by RT-PCR. In both rumen and abomasum, leptin mRNA was detected in the milk-fed animals. However, in the weaned and milk + VFA-fed animals, it was not detected in either the rumen or abomasum. However, leptin mRNA was found in the duodenum in all three groups (Fig. 2). 3.3. Effects of aging and nutritional status on CCKA receptor mRNA expression in bovine rumen We examined whether aging and nutritional conditions affect CCKA receptor expression in the bovine rumen. In the 3-week-old animals, CCKA receptor mRNA was detected. But it was not detected in the 13-week-old or adult animals (Fig. 3A). Furthermore, although CCKA receptor mRNA was detected in only the milk-fed animals, it was not found in the weaned and milk + VFA-fed animals (Fig. 3B). Even though 60 cycles of PCR amplification were employed, CCKA receptor expression was not detected in adult, weaned and milk + VFA-fed animals at 13 weeks old (data not shown). In addition, CCKB receptor mRNA expression was
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Fig. 1. RT-PCR analysis of the expression of leptin (upper panel) and G3PDH (lower panel) mRNA in various bovine tissues from 3-week-old (A), 13-week-old (B) and adult (C) animals. Mk, Molecular weight marker; lane 1, adipose tissue; lane 2, rumen; lane 3, abomasum; lane 4, duodenum; lane 5, liver.
Fig. 2. Effect of nutritional conditions on leptin expression in 13-week-old calf rumen (A), abomasum (B) and duodenum (C). Mk, molecular weight marker; W, weaning animals; M, milk-fed animals; V, milk + VFA-fed animals, upper panel, leptin band; lower panel, G3PDH band.
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Fig. 3. Effects of age and nutritional conditions on CCKA receptor expression in the calf rumen. (A) Expression in 3-, 13-week-old and adult animals. Mk, molecular weight marker; lane 1, 3-week-old animals; lane 2, 13-week-old animals; lane 3, adult animals. (B) Expression in 13-week-old animals. Mk, molecular weight marker; W, weaning animals; M, milk-fed animals; V, milk + VFA-fed animals, upper panel, leptin band; lower panel, G3PDH band.
Fig. 4. Effects of age and nutritional conditions on CCKB receptor expression in the calf abomasum analyzed by the semi-quantitative RT-PCR. (A) Expression in 3-, 13-week-old and adult animals. Mk, molecular weight marker; lane 1, 3-week-old animals; lane 2, 13-week-old animals; lane 3, adult animals. (B) Expression in 13-week-old animals in different nutritional conditions. Mk, molecular weight marker; W, weaned animals; M, milk-fed animals; V, milk + VFA-fed animals. Upper panel: representative ethidium bromide-stained gels (out of triplicates) of target products as well as amplified signals for G3PDH in the same sample (internal control). The molecular sizes of the generated products were calculated by comparison with the mobility of a 100 bp DNA step ladder (left side). Lower panel: CCKB receptor mRNA abundance assigned as a ratio to G3PDH mRNA abundance. The results are shown as the means ± SEM (n = 3). Means with different letters are significantly different (P < 0.05).
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at a very low level, because it could only be finally detected in all animals only when 60 cycles of PCR amplifications were employed (data not shown). 3.4. Effects of aging and nutritional conditions on CCKB receptor mRNA expression in the bovine abomasum We examined whether aging and nutritional conditions affect CCKB receptor expression in the bovine abomasum by semi-quantitative RT-PCR. The ratio of CCKB receptor mRNA abundance against G3PDH abundance in the 3-week-old animals was 100.0 ± 4.5, which was significantly reduced to 29.9 ± 4.53 and 0.06 ± 0.01 in the 13-week-old and adult animals, respectively (Fig. 4A). In 13-week-old animals, CCKB receptor transcription of milk-fed animals (95.4, 104.6) was higher than that of the weaning (37.0 ± 1.2) and milk + VFA-fed animals (33.1 ± 2.7) (Fig. 4B). However, CCKA receptor mRNA expression level was very low in the abomasum (data not shown).
4. Discussion Our present findings demonstrated for the first time that the mRNA of the leptin and CCKA receptor are expressed in the calf rumen and abomasum at 3 but not 13 weeks of age, and that the expression was detected even at 13 weeks old when the animals were fed on milk alone. In addition, the expression in the duodenum was not changed by aging. To our knowledge, although leptin has also been reported to be expressed in the human and rodent stomachs, there are no reports of leptin expression in the duodenum of any other animal species. The development of the stomach is intimately related with the food that is ingested, and the function of the rumen is dramatically changed during the weaning period. In the ruminant, once forage enters into the rumen, it results in an increased activity of microbes and the development of the forestomachs [20]. Disappearance of leptin expression in the upper alimentary tract at 13 weeks old may be involved in gastric development due to the changes in nutritional conditions around weaning. Fig. 2 shows that in 13-week-old animals, leptin expression was found only in the rumen and abomasum only when animals were fed on milk alone. Ingested milk is known to by-pass the reticulo-rumen, and directly enter the abomasum through the esophageal groove, and is digested in a similar manner to non-ruminant animals. Thus, milk-fed animals maintain the forestomachs at a pre-ruminant stage. In fact, the papillary length of the rumen of the milk-fed animals was much shorter than that of the weaning animals, although body growth rate was scarcely different in the present study (data not shown). Furthermore, in the milk + VFA-fed animals, leptin expression in the stomachs was not observed, which was clearly different from that in the milk-fed animals at 13 weeks old. The fermentation of carbohydrate by ruminal microorganisms results in VFA production [21]. Previous studies showed that solid feed consumption [22] or intra-ruminal VFA administration stimulated the growth of rumen epithelium in milk-fed animals [18,23]. Although, rumen epithelial cells oxidize glucose and butyrate at similar rates at birth [24], the rate of glucose oxidation decreases and butyrate becomes the preferred oxidative substrate at weaning [24–26]. Also, at weaning, the production of the ketone body such as β-hydroxybutyrate from butyrate
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increases six-fold in rumen epithelial cells. Previous findings suggest that VFA might stimulate metabolic development of the rumen in milk-fed lambs [23], indicating that disappearance of leptin expression in the stomachs resulted from changes in the function and morphology caused by weaning rather than aging. The present study also showed that leptin mRNA was expressed in the calf duodenum at all ages, although it has not been shown to be expressed in the duodenum of non-ruminant animal species. Furthermore, it was found even in the duodenum of milk-fed and milk + VFA-fed calves. Therefore, it is possible that duodenal leptin in ruminant animals, while gastric leptin in non-ruminant animals, may play a role in the short-term regulation of food intake [27]. Digestive mechanisms greatly differ between non-ruminant and ruminant animals. Although further detail studies are required, it is plausible that gastric leptin in non-weaning pre-ruminant animals plays a role as a satiety factor as in non-ruminant animals. But, when the rumen developed and animals showed the characteristic digestive system specific for the ruminants, the gastric leptin expression disappeared in the present study. It is, therefore, assumed that the satiety mechanism changes during the weaning period of the calf. Gastric leptin is thought to be modulated by CCK in the rodent [11]. In order to ascertain the relationship between leptin and CCK expression in the stomachs, we examined CCK receptor mRNA expression as well as leptin expression in the present study. In the rumen, CCKA receptor mRNA expression was detected in 3-week-old animals, but not in 13-week-old or adult animals (Fig. 3). Furthermore, although CCKA receptor mRNA expression was found in milk-fed animals even at 13 weeks of age, it was not observed in either weaned or milk + VFA-fed animals (Fig. 3). To our knowledge, there has been no report of CCK receptor expression in the rumen. Although further detailed studies are required, the present study implies that CCKA receptor mRNA expression was affected by changes in the gut function and morphology caused by weaning in a similar way to leptin expression. CCKB receptor mRNA was scarcely expressed in the rumen of 3- and 13-week-old animals (data not shown). Therefore, it is clear that CCKA receptor expression is dominant in the rumen. In the abomasum, CCKB receptor transcription gradually decreased between 3 weeks of age and the adult stages, but CCKA receptor mRNA was expressed at very low level. These results are consistent with those by Toullec et al. [28]. In 13-week-old animals, CCKB receptor transcription of the milk-fed animals was higher than that of the weaned and milk + VFA-fed animals (Fig. 4). Therefore, it is suggested that CCKB receptor mRNA expression is affected by changes in stomach function and morphology occasioned by weaning, in a similar way to the CCKA receptor mRNA expression in the rumen. In pre-ruminant calves, the plasma CCK concentration increased significantly after milk ingestion [28]. By contrast, in adult ruminant animals, feeding had no effect on the plasma CCK concentration [28]. Therefore, the present findings suggest that differences in CCK receptor expression may be involved in the difference in post-prandial changes in plasma CCK concentrations between pre-ruminant and ruminant animals. The finding that mRNA expression of leptin and CCK receptors were similar in the stomachs leads us to the speculation that CCK may modulate gastric leptin expression and secretion in pre-weaning calves. Although further detailed studies remain to be done, these findings imply that the mechanism of synergistic interaction between CCK and gastric leptin on the regulation of food intake is changed in calves by the nutritional conditions brought about by weaning.
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In conclusion, we have demonstrated (1) leptin mRNA was expressed in the rumen and abomasum of 3-week-old calves, but not after 13 weeks of age, (2) leptin mRNA was expressed in the duodenum of 3-, 13-week-old and adult animals, although it was not reported to be expressed in the non-ruminant duodenum, (3) in 13-week-old calves, rumen and abomasum leptin mRNA was expressed in the milk-fed animals, but not in weaning and milk + VFA-fed animals, and (4) in the rumen and abomasum, the changes in leptin and CCK receptor mRNA expression coincided with aging and change in nutritional conditions. It is speculated that gastric leptin may be modulated by CCK and that gastric leptin and CCK receptor expression may be changed by nutritional conditions caused by weaning in calves. The detailed interaction among gastric leptin, CCK receptors, food intake remains to be clarified. Acknowledgments We gratefully acknowledge Dr. Michael T. Rose (Institute of Rural Studies, Aberystwyth, UK) for his contribution in the preparation of manuscript. The authors are indebted to Mr. Ohtomo (Tohoku University, Japan) for care of the animals. This work was supported by the Association of Livestock Technology, Japan. References [1] Smith GP, Gibbs J. Brain-gut peptides and the control of food intake. In: Martin JB, Reichlin S, Bick KL, editors. Neorosecretion and brain peptides. New York: Raven, 1981. p. 389–95. [2] Gibbs J, Smith GP. Satiety: the roles of peptides from the stomach and the intestine. Fed Proc 1986;45:1391–5. [3] Gibbs J, Young C, Smith GP. Cholecystokinin elicits satiety in rats with open gastric fistulas. Nature 1973;245:323–5. [4] Polak JM, Bloom SR, Rayford PL, Pearse AGE, Buchan AMJ, Thompson JC. Identification of cholecystokinin-secreting cells. Lancet 1975;22:1016–8. [5] Gibbs J, Smith GP, Greenberg D. Cholecystokinin: a neuroendocrine key to feeding behavior. In: Schulkin J, editor. Hormonally-induced changes in mind and brain. New York: Academic Press, 1993, p. 51–69. [6] Innis RB, Snyder SH. Distinct cholecystokinin receptors in brain and pancreas. Proc Natl Acad Sci USA 1980;77:6917–21. [7] Moran TH, Robinson PH, Goldrich MS, McHugh PR. Two brain cholecystokinin receptors: implications for behavioral actions. Brain Res 1986;362:175–9. [8] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse ob gene and its human homologue. Nature 1996;372:425–33. [9] Scholz GH, Englaro P, Thiele I, Scholz M, Klusmann T, Kellner K, Rascher W, Blum WF. Dissociation of serum leptin concentration and body fat content during long-term dietary intervention in obese individuals. Horm Metab Res 1996;28:718–23. [10] Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci USA 1997;94:8878– 83. [11] Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, Bortoluzzi MN, Moizo L, Lehy T, Guerre-Millo M, Le Marchan-Brustel Y, Lewin MJ. The stomach is a source of leptin. Nature 1998;394:790–3. [12] Sobhani I, Bado A, Vissuzaine C, Buyse M, Kermorgant S, Laigneau JP, Attoub S, Lehy T, Henin D, Mignon M, Lewin MJ. Leptin secretion and leptin receptor in the human stomach. Gut 2000;47:178–83. [13] Barrachina MD, Martinez V, Wang L, Wei LW, Tache Y. Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice. Proc Natl Acad Sci USA 1997;94:10455– 60.
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[14] Della-Fera MA, Baile CA. Cholecystokinin octapeptide: continuous picomole injections into the cerebral ventricles of sheep suppress feeding. Science 1979;206:471–3. [15] Della-Fera MA, Baile CA. Control of feed intake in sheep. J Anim Sci 1984;59:1362–8. [16] Le Drean G, Le Huerou-Luron I, Chayvialle JA, Philouze-Rome V, Gestin M, Bernard C, Toullec R, Guilloteau P. Kinetics of pancreatic exocrine secretion and plasma gut regulatory peptide release in response to feeding in preruminant and ruminant calves. Comp Biochem Physiol A Physiol 1997;117:245–55. [17] Sasaki Y. Effect of the intra-ruminal administration of volatile fatty acids (VFA) on the VFA consumption by the calf rumen epithelium. Jpn J Zootechnol Sci 1969;40:266–70. [18] Tamate H, McGilliard AD, Jacobson NL, Getty R. Effect of various dietaries on the anatomical development of the stomach in the calf. J Dairy Sci 1962;45:408–19. [19] Desbois C, Clerc P, Le Huerou-Luron I, Le Drean G, Gestin M, Dufresne M, Fourmy D, Guilloteau P. Differential tissue expression of the CCK(A) and CCK(B) gastrin receptor genes during postnatal development in the calf. Life Sci 1998;63:2059–70. [20] Thivend P, Toullec R, Guilloteau P. Digestive adaptation in the preruminant. In: Ruckebusch Y, Thivend P, editors. Digestive physiology and metabolism in ruminants. Lancaster: MTP Press, 1980, p. 513–37. [21] van Houtert MFJ. The production and metabolism of volatile fatty acids by ruminans fed roughages. Anim Feed Sci Technol 1993;43:189–255. [22] Warner RG, Flatt WP, Loosli JK. Dietary factors influencing the development of the ruminant stomach. Agric Food Chem 1956;4:788–92. [23] Lane MA, Jesse BW. Effect of volatile fatty acid infusion on development of neonatal sheep rumen epithelium. J Dairy Sci 1997;80:740–6. [24] Baldwin VI RL, Jesse BW. Developmental changes in glucose and butyrate metabolism by isolated sheep rumen epithelial cells. J Nutr 1992;122:1149–53. [25] Giesecke D, Beck U, Wiesmayr S, Stangassinger M. The effect of rumen epithelial development on metabolic activities and ketogenesis by tissue in vitro. Com Biochem Physiol 1979;62B:459–63. [26] Bush RS. Effect of age and diet on in vitro metabolism in rumen epithelium from Holstein calves. Can J Anim Sci 1988;62:429–38. [27] Cinti S, Matteis RD, Pico C, Ceresi E, Obrador A, Maffeis C, Oliver J, Palou A. Secretory granules of endocrine and chief cells of human stomach mucosa contain leptin. Int J Obes Relat Metab Disord 2000;24:789–93. [28] Toullec R, Chayvialle JA, Guilloteau P, Bernard C. Early-life patterns of plasma gut regulatory peptide levels in calves: effects of age. Comp Biochem Physiol 1992;102:203–9.
Effects of aging and weaning on mRNA expression of leptin and CCK receptors in the calf rumen and abomasum S. Yonekura∗ , K. Kitade, G. Furukawa, K. Takahashi, N. Katsumata, K. Katoh, Y. Obara Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Sendai 981-8555, Japan Received 2 April 2001; accepted 27 August 2001
Abstract In order to know the effects of weaning and volatile fatty acid feeding on gastric leptin expression, we investigated the expression of leptin and CCK receptor mRNA in the bovine rumen, abomasum and duodenum using RT-PCR in 3-week-old pre-weaning, 13-week-old post-weaning and adult animals. Leptin mRNA was expressed in the rumen and abomasum of 3-week-old pre-weaning animals, but it was abolished in 13-week-old and adult animals. In the duodenum, leptin expression was observed in the 3-, 13-week-old and adult animals. In the rumen, CCKA receptor mRNA was expressed in 3-week-old animals, but not in 13-week-old and adult animals. In the abomasum, CCKB receptor expression gradually decreased from 3-week-old to adult animals. Expression of CCKB receptor and of CCKA receptor was slight in the rumen and abomasum, respectively. In the next study, we examined the effect of weaning of 6 weeks or non-weaning (fed on milk replacer alone (milk) or milk replacer with volatile fatty acids (milk + VFA) until 13 weeks old) on leptin mRNA expression in the rumen and abomasum. In 13-week-old calf rumen and abomasum, leptin mRNA expression was detected in non-weaning milk-fed animals at 13 weeks old, although it was not observed in weaning and non-weaned milk + VFA-fed animals. The change in CCKA receptor expression in the rumen was similar to those of leptin mRNA expression. CCKB receptor transcription in the abomasum of milk-fed animals was higher than that of the weaning and milk + VFA-fed animals. These results indicate that leptin expression is coincident with CCK receptor expression in calf stomachs, and that leptin and CCK receptor mRNA expression are affected by the change in the physiological status brought about by weaning and VFA feeding. © 2002 Elsevier Science Inc. All rights reserved.
∗
Corresponding author. Tel.: +81-22-717-8700; fax: +81-22-717-8701. E-mail address: [email protected] (S. Yonekura).
0739-7240/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 0 1 ) 0 0 1 1 4 - X
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1. Introduction The physiological mechanisms by which satiety sensing occurs after food intake have not yet been defined. Several peptides secreted from the gastrointestinal tract during eating have been shown to suppress food intake [1,2]. One of them, cholecystokinin (CCK) is secreted from duodenal I cells after eating [3,4]. One of the physiological roles of peripheral CCK is as a short-term regulator of food intake via the receptors [5]. Binding sites for CCK have been identified in a variety of tissues, and two primary receptor subpopulations have been discerned on the basis of receptor binding and functional characteristics [6,7]. CCKA receptors have a high affinity for CCK but a low affinity for non-sulfated CCK and gastrin [7]. In contrast, CCKB receptors interact with gastrin and CCK at almost the same high affinity and poorly discriminate sulfated and non-sulfated peptide analogues [7]. Leptin, a 16 kDa protein product of the ob gene, was identified as an adipocyte-derived cytokine [8]. In contrast to CCK, the satiety effect induced by circulating leptin is thought to be only apparent in the long-term [9,10]. Leptin, which was initially thought to be exclusively expressed in adipocytes [8], has been shown to be expressed in gastric mucosa and fundic glands in rats [11] and humans [12]. In addition, the secretion of gastric leptin is caused by an increased CCK secretion stimulated by feeding [11]. It is also considered that there exists a functional synergistic interaction between leptin and CCK, leading to a short-term regulation of food intake [13]. With respect to ruminant animals, some studies on CCK indicate that CCK induces suppression of food intake similar to non-ruminant animals [14,15]. But, to our knowledge, there is no report of the existence of leptin in the reticulo-rumen, omasum or abomasums of ruminants. Ruminants are classically regarded as having four “stomachs,” with microbial fermentation occurring in the reticulo-rumen, resulting in the production of volatile fatty acids (VFA). The rumen of calves rapidly and drastically develops around the weaning period (about at age of 5–6 weeks) and becomes mature at 12 weeks of age. It is at 5–6 weeks of age when ruminant diets shifts from a milk-based to a solid food-based diet. Therefore, it is speculated that the regulatory mechanism of food intake may be drastically changed during this period. In this study, we investigated leptin expression in the rumen, abomasum and duodenum of developing calves. Furthermore, as the relationship between leptin and CCK is reported in rodents, we also studied CCK receptor expression in the rumen and abomasum.
2. Materials and methods 2.1. Chemicals All salts of VFA used for the experimental solutions were purchased from Sigma (St. Louis, MO). TRIzol reagent was purchased from Gibco BRL (Grand Island, NY). Isolation kit for mRNA was purchased from Miltenyi Biotec (Miltenyi Biotec, Bergisch Gladbach, Germany). T/A cloning kit was purchased from Invitrogen (San Diego, CA). All salts for the isolation of RNA were purchased from Nakarai (Tokyo, Japan).
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2.2. Animals The treatment of the animals was according to the “Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences” (The Physiological Society of Japan) and the present experiment was approved by The Animal Care Committee of Tohoku University. Eleven male Holstein calves (TOHOKU UNIVERSITY FARM) were fed by their parents for 1 week after birth to ensure the consumption of colostrum before starting the experimental feeding. They were then divided into three groups and fed until 13 weeks of age: a weaning group (W), a milk replacer (milk)-fed group (M), and a milk + VFA-fed group (V). In W group, six calves were fed a commercial milk replacer and calf starter as described in Table 1, and three of them were sacrificed at 3 weeks old. Another three animals were fed on calf starter and hay until 13 weeks old and sacrificed. They were allowed free access to timothy hay from 5 weeks of age. Calves of M and V groups were fed milk replacer and injected daily with saline or VFA into the reticulo-rumen from 5 weeks of age, respectively, as described in Table 1, and sacrificed at 13 weeks old (n = 2 and 3, respectively). All calves were fed diets at 10:00 and 16:00 twice daily, and sacrificed 18 h after the last meal. Tissue samples from three adult male Holstein animals (1.5–2 years old) were obtained at a local abattoir (mean body weight: 316 ± 1.6 kg, n = 3). The gastrointestinal tract was removed approximately 15 min after death, and tissue samples were taken. The size of ventral sac of the rumen and fundus of the abomasums was approximately 2 cm × 2 cm, duodenum was 20 cm length from the pylorus. Tissue samples were immediately placed into polypropylene containers, frozen in liquid nitrogen, and stored at −80◦ C until RNA analysis. 2.3. Experimental diets and VFA administration Milk replacer and calf starter were purchased from Zenraku (Tokyo, Japan). The amounts of the experimental diets in W group were decided according to the Japanese Feeding Standard, Table 1 Weaning animals
Milk-fed animals
Milk + VFA-fed animals
Age (weeks)
Milk replacer (g per day)
Calf starter (g per day)
Milk replacer (g per day)
Milk replacer (g per day)
VFA (mol per day)
1 2 3 4 5 6 7 8 9 10 11 12 13
300 400 500 600 600 600 – – – – – – –
100 200 500 900 1200 1500 1700 1900 2100 2200 2300 2500 2500
300 400 500 600 700 800 950 1100 1250 1400 1550 1700 1850
300 400 500 600 700 800 950 1100 1250 1400 1550 1700 1850
– – – – 0.2 0.4 0.8 1.2 1.8 2.4 3.0 3.6 4.2
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and that of milk replacer in M and V groups were decided as reported in a previous study [16]. The milk replacer and calf starter consisted of 24.5 and 20.0% crude protein and 21.9 and 2.0% crude fat, respectively. The amount of the experimental diets given to animals is shown in Table 1. The calves of W group were allowed free access to timothy hay from 5 weeks of age. All calves had free access to water. The VFA solution (1.8 mol/L) consisted of equimolar amounts of acetic, propionic and butyric acids and pH was adjusted to pH 6.0 with sodium hydroxide and potassium hydroxide [17]. The intra-ruminal administration of VFA was carried out through polypropylane inserted into the reticulo-rumen through the esophagus every day from 5 to 13 weeks of age. The amount of VFA administrated was based on previous work [18]. The solution was administered into the reticulo-rumen of the animals of V group, and an equal volume of physiological saline was administrated to the M group through a tube after feeding of milk at 1600. All calves grow normally, and body weights were not different among three groups (data not shown). 2.4. RNA analysis by RT-PCR and semi-quantitative RT-PCR Total RNA was isolated from the tissues using the TRIzol extraction method according to the manufacturers instructions (Gibco BRL). Poly A+ mRNA was isolated from total RNA using a mRNA isolation kit (Miltenyi Biotec). The concentration of isolated mRNA was determined from the optical density at 260 nm and its purity from the wavelength ratio of 260/280 nm. PCR amplification of cDNA samples was carried out using the following primer pairs: bovine leptin sense (GTGCCCATCCGCAAGGTCCA-3 ) and antisense (TCAGCACCCGGGACTGAGGT-3 ) for the amplification of a 440 bp fragment; bovine CCKA receptor sense (CTGCTCAGCGTGCTGGGAAAC-3 ) and antisense (CGGGACTGTAAGGGTTTGCAAA-3 ) for the amplification of a 293 bp fragment [19]; bovine CCKB receptor (CGGGACACGAGAATTGGAGCTGG-3 ) and antisense (CCGTCAAAGCGAAGCCCTAAGTAG-3 ) for the amplification of a 618 bp fragment [19]. To provide an appropriate internal control, coamplification of a 450 bp fragment of G3PDH mRNA was carried out in each sample using the primer pair: G3PDH-sense (ACCACAGTCCATGCCATCAC-3 ) and antisense (TCCACCACCCTGTTGCTGTA-3 ). Forty cycles of PCR amplification were used for bovine leptin. The conditions used were 95◦ C for 1 min for denaturation, 64◦ C for 1 min for annealing, 72◦ C for 1 min for polymerization. The PCR product was run in a 2.0% agarose gel stained with ethidium bromide. PCR products were then ligated into the PCRII vector and used to transform one-shot Escherichia coli cells according to the protocol provided with the T/A cloning kit (Invitrogen, San Diego, CA). Colonies were screened by blue/white and ampicillin selection, and plasmids from resultant cultures were isolated and subjected to restriction analysis (with EcoRI) to check for the expected 440 bp insertion. The 440 bp product was then sequenced by use of the dideoxy method to confirm the encoded cDNA was same as the bovine leptin sequence. For quantification of the amount of mRNA by RT-PCR method, PCR amplification of bovine CCKA and CCKB receptor and G3PDH was carried out at different numbers of cycles. PCR amplifications were done with 95◦ C for 1 min for denaturation, 65◦ C (55◦ C for G3PDH) for 1 min for annealing, 72◦ C for 1 min 30 s for polymerization. An increase in the number of PCR cycles raised the products in an exponential manner. Different numbers of cycles were tested
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for CCKA and CCKB receptor (20–40 cycles) and G3PDH (14 and 30 cycles) and 33 and 26 cycles were employed for CCKA and CCKB receptors, and 22 cycles for G3PDH, respectively. In all assays, reactions with RT negative amplification were included (data not shown). The PCR products were separated in a 2% agarose gel, stained with ethidium bromide, and analyzed using Fluor-S MultiImager (Bio-Rad, Hercules, CA). The intensity of CCK receptor abundance was assigned as a ratio to that of G3PDH abundance. To minimize potential RT-PCR artifacts due to inherent reaction variability, all data points were repeated, for each target, at least three times using independent RNA samples. 2.5. Statistical analysis The results are expressed as means values ± SE. The data were analyzed by ANOVA, Student’s t-test and Bonferroni’s multiple range test with SAS. 3. Results 3.1. Effects of aging on leptin mRNA expression in various bovine tissues In RT-PCR analysis, a product of expected size (440 bp) was detected in fat, rumen, abomasum and duodenum, but not in liver tissues in cattle of 3 weeks of age. However, in 13-week-old and adult animals, leptin mRNA was not detected in the rumen and abomasums, although it was still found in the duodenum. Sequence analysis of 440 bp amplicon was confirmed 100% homology with the corresponding region of a published sequence for leptin cDNA (data not shown) (Fig. 1). 3.2. Effect of nutritional status on leptin mRNA expression in 13-week-old calf gut We examined whether the nutritional conditions affect leptin expression in bovine stomachs (rumen (A), abomasums (B) and duodenum (C)) by RT-PCR. In both rumen and abomasum, leptin mRNA was detected in the milk-fed animals. However, in the weaned and milk + VFA-fed animals, it was not detected in either the rumen or abomasum. However, leptin mRNA was found in the duodenum in all three groups (Fig. 2). 3.3. Effects of aging and nutritional status on CCKA receptor mRNA expression in bovine rumen We examined whether aging and nutritional conditions affect CCKA receptor expression in the bovine rumen. In the 3-week-old animals, CCKA receptor mRNA was detected. But it was not detected in the 13-week-old or adult animals (Fig. 3A). Furthermore, although CCKA receptor mRNA was detected in only the milk-fed animals, it was not found in the weaned and milk + VFA-fed animals (Fig. 3B). Even though 60 cycles of PCR amplification were employed, CCKA receptor expression was not detected in adult, weaned and milk + VFA-fed animals at 13 weeks old (data not shown). In addition, CCKB receptor mRNA expression was
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Fig. 1. RT-PCR analysis of the expression of leptin (upper panel) and G3PDH (lower panel) mRNA in various bovine tissues from 3-week-old (A), 13-week-old (B) and adult (C) animals. Mk, Molecular weight marker; lane 1, adipose tissue; lane 2, rumen; lane 3, abomasum; lane 4, duodenum; lane 5, liver.
Fig. 2. Effect of nutritional conditions on leptin expression in 13-week-old calf rumen (A), abomasum (B) and duodenum (C). Mk, molecular weight marker; W, weaning animals; M, milk-fed animals; V, milk + VFA-fed animals, upper panel, leptin band; lower panel, G3PDH band.
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Fig. 3. Effects of age and nutritional conditions on CCKA receptor expression in the calf rumen. (A) Expression in 3-, 13-week-old and adult animals. Mk, molecular weight marker; lane 1, 3-week-old animals; lane 2, 13-week-old animals; lane 3, adult animals. (B) Expression in 13-week-old animals. Mk, molecular weight marker; W, weaning animals; M, milk-fed animals; V, milk + VFA-fed animals, upper panel, leptin band; lower panel, G3PDH band.
Fig. 4. Effects of age and nutritional conditions on CCKB receptor expression in the calf abomasum analyzed by the semi-quantitative RT-PCR. (A) Expression in 3-, 13-week-old and adult animals. Mk, molecular weight marker; lane 1, 3-week-old animals; lane 2, 13-week-old animals; lane 3, adult animals. (B) Expression in 13-week-old animals in different nutritional conditions. Mk, molecular weight marker; W, weaned animals; M, milk-fed animals; V, milk + VFA-fed animals. Upper panel: representative ethidium bromide-stained gels (out of triplicates) of target products as well as amplified signals for G3PDH in the same sample (internal control). The molecular sizes of the generated products were calculated by comparison with the mobility of a 100 bp DNA step ladder (left side). Lower panel: CCKB receptor mRNA abundance assigned as a ratio to G3PDH mRNA abundance. The results are shown as the means ± SEM (n = 3). Means with different letters are significantly different (P < 0.05).
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at a very low level, because it could only be finally detected in all animals only when 60 cycles of PCR amplifications were employed (data not shown). 3.4. Effects of aging and nutritional conditions on CCKB receptor mRNA expression in the bovine abomasum We examined whether aging and nutritional conditions affect CCKB receptor expression in the bovine abomasum by semi-quantitative RT-PCR. The ratio of CCKB receptor mRNA abundance against G3PDH abundance in the 3-week-old animals was 100.0 ± 4.5, which was significantly reduced to 29.9 ± 4.53 and 0.06 ± 0.01 in the 13-week-old and adult animals, respectively (Fig. 4A). In 13-week-old animals, CCKB receptor transcription of milk-fed animals (95.4, 104.6) was higher than that of the weaning (37.0 ± 1.2) and milk + VFA-fed animals (33.1 ± 2.7) (Fig. 4B). However, CCKA receptor mRNA expression level was very low in the abomasum (data not shown).
4. Discussion Our present findings demonstrated for the first time that the mRNA of the leptin and CCKA receptor are expressed in the calf rumen and abomasum at 3 but not 13 weeks of age, and that the expression was detected even at 13 weeks old when the animals were fed on milk alone. In addition, the expression in the duodenum was not changed by aging. To our knowledge, although leptin has also been reported to be expressed in the human and rodent stomachs, there are no reports of leptin expression in the duodenum of any other animal species. The development of the stomach is intimately related with the food that is ingested, and the function of the rumen is dramatically changed during the weaning period. In the ruminant, once forage enters into the rumen, it results in an increased activity of microbes and the development of the forestomachs [20]. Disappearance of leptin expression in the upper alimentary tract at 13 weeks old may be involved in gastric development due to the changes in nutritional conditions around weaning. Fig. 2 shows that in 13-week-old animals, leptin expression was found only in the rumen and abomasum only when animals were fed on milk alone. Ingested milk is known to by-pass the reticulo-rumen, and directly enter the abomasum through the esophageal groove, and is digested in a similar manner to non-ruminant animals. Thus, milk-fed animals maintain the forestomachs at a pre-ruminant stage. In fact, the papillary length of the rumen of the milk-fed animals was much shorter than that of the weaning animals, although body growth rate was scarcely different in the present study (data not shown). Furthermore, in the milk + VFA-fed animals, leptin expression in the stomachs was not observed, which was clearly different from that in the milk-fed animals at 13 weeks old. The fermentation of carbohydrate by ruminal microorganisms results in VFA production [21]. Previous studies showed that solid feed consumption [22] or intra-ruminal VFA administration stimulated the growth of rumen epithelium in milk-fed animals [18,23]. Although, rumen epithelial cells oxidize glucose and butyrate at similar rates at birth [24], the rate of glucose oxidation decreases and butyrate becomes the preferred oxidative substrate at weaning [24–26]. Also, at weaning, the production of the ketone body such as β-hydroxybutyrate from butyrate
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increases six-fold in rumen epithelial cells. Previous findings suggest that VFA might stimulate metabolic development of the rumen in milk-fed lambs [23], indicating that disappearance of leptin expression in the stomachs resulted from changes in the function and morphology caused by weaning rather than aging. The present study also showed that leptin mRNA was expressed in the calf duodenum at all ages, although it has not been shown to be expressed in the duodenum of non-ruminant animal species. Furthermore, it was found even in the duodenum of milk-fed and milk + VFA-fed calves. Therefore, it is possible that duodenal leptin in ruminant animals, while gastric leptin in non-ruminant animals, may play a role in the short-term regulation of food intake [27]. Digestive mechanisms greatly differ between non-ruminant and ruminant animals. Although further detail studies are required, it is plausible that gastric leptin in non-weaning pre-ruminant animals plays a role as a satiety factor as in non-ruminant animals. But, when the rumen developed and animals showed the characteristic digestive system specific for the ruminants, the gastric leptin expression disappeared in the present study. It is, therefore, assumed that the satiety mechanism changes during the weaning period of the calf. Gastric leptin is thought to be modulated by CCK in the rodent [11]. In order to ascertain the relationship between leptin and CCK expression in the stomachs, we examined CCK receptor mRNA expression as well as leptin expression in the present study. In the rumen, CCKA receptor mRNA expression was detected in 3-week-old animals, but not in 13-week-old or adult animals (Fig. 3). Furthermore, although CCKA receptor mRNA expression was found in milk-fed animals even at 13 weeks of age, it was not observed in either weaned or milk + VFA-fed animals (Fig. 3). To our knowledge, there has been no report of CCK receptor expression in the rumen. Although further detailed studies are required, the present study implies that CCKA receptor mRNA expression was affected by changes in the gut function and morphology caused by weaning in a similar way to leptin expression. CCKB receptor mRNA was scarcely expressed in the rumen of 3- and 13-week-old animals (data not shown). Therefore, it is clear that CCKA receptor expression is dominant in the rumen. In the abomasum, CCKB receptor transcription gradually decreased between 3 weeks of age and the adult stages, but CCKA receptor mRNA was expressed at very low level. These results are consistent with those by Toullec et al. [28]. In 13-week-old animals, CCKB receptor transcription of the milk-fed animals was higher than that of the weaned and milk + VFA-fed animals (Fig. 4). Therefore, it is suggested that CCKB receptor mRNA expression is affected by changes in stomach function and morphology occasioned by weaning, in a similar way to the CCKA receptor mRNA expression in the rumen. In pre-ruminant calves, the plasma CCK concentration increased significantly after milk ingestion [28]. By contrast, in adult ruminant animals, feeding had no effect on the plasma CCK concentration [28]. Therefore, the present findings suggest that differences in CCK receptor expression may be involved in the difference in post-prandial changes in plasma CCK concentrations between pre-ruminant and ruminant animals. The finding that mRNA expression of leptin and CCK receptors were similar in the stomachs leads us to the speculation that CCK may modulate gastric leptin expression and secretion in pre-weaning calves. Although further detailed studies remain to be done, these findings imply that the mechanism of synergistic interaction between CCK and gastric leptin on the regulation of food intake is changed in calves by the nutritional conditions brought about by weaning.
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In conclusion, we have demonstrated (1) leptin mRNA was expressed in the rumen and abomasum of 3-week-old calves, but not after 13 weeks of age, (2) leptin mRNA was expressed in the duodenum of 3-, 13-week-old and adult animals, although it was not reported to be expressed in the non-ruminant duodenum, (3) in 13-week-old calves, rumen and abomasum leptin mRNA was expressed in the milk-fed animals, but not in weaning and milk + VFA-fed animals, and (4) in the rumen and abomasum, the changes in leptin and CCK receptor mRNA expression coincided with aging and change in nutritional conditions. It is speculated that gastric leptin may be modulated by CCK and that gastric leptin and CCK receptor expression may be changed by nutritional conditions caused by weaning in calves. The detailed interaction among gastric leptin, CCK receptors, food intake remains to be clarified. Acknowledgments We gratefully acknowledge Dr. Michael T. Rose (Institute of Rural Studies, Aberystwyth, UK) for his contribution in the preparation of manuscript. The authors are indebted to Mr. Ohtomo (Tohoku University, Japan) for care of the animals. This work was supported by the Association of Livestock Technology, Japan. References [1] Smith GP, Gibbs J. Brain-gut peptides and the control of food intake. In: Martin JB, Reichlin S, Bick KL, editors. Neorosecretion and brain peptides. New York: Raven, 1981. p. 389–95. [2] Gibbs J, Smith GP. Satiety: the roles of peptides from the stomach and the intestine. Fed Proc 1986;45:1391–5. [3] Gibbs J, Young C, Smith GP. Cholecystokinin elicits satiety in rats with open gastric fistulas. Nature 1973;245:323–5. [4] Polak JM, Bloom SR, Rayford PL, Pearse AGE, Buchan AMJ, Thompson JC. Identification of cholecystokinin-secreting cells. Lancet 1975;22:1016–8. [5] Gibbs J, Smith GP, Greenberg D. Cholecystokinin: a neuroendocrine key to feeding behavior. In: Schulkin J, editor. Hormonally-induced changes in mind and brain. New York: Academic Press, 1993, p. 51–69. [6] Innis RB, Snyder SH. Distinct cholecystokinin receptors in brain and pancreas. Proc Natl Acad Sci USA 1980;77:6917–21. [7] Moran TH, Robinson PH, Goldrich MS, McHugh PR. Two brain cholecystokinin receptors: implications for behavioral actions. Brain Res 1986;362:175–9. [8] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse ob gene and its human homologue. Nature 1996;372:425–33. [9] Scholz GH, Englaro P, Thiele I, Scholz M, Klusmann T, Kellner K, Rascher W, Blum WF. Dissociation of serum leptin concentration and body fat content during long-term dietary intervention in obese individuals. Horm Metab Res 1996;28:718–23. [10] Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci USA 1997;94:8878– 83. [11] Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, Bortoluzzi MN, Moizo L, Lehy T, Guerre-Millo M, Le Marchan-Brustel Y, Lewin MJ. The stomach is a source of leptin. Nature 1998;394:790–3. [12] Sobhani I, Bado A, Vissuzaine C, Buyse M, Kermorgant S, Laigneau JP, Attoub S, Lehy T, Henin D, Mignon M, Lewin MJ. Leptin secretion and leptin receptor in the human stomach. Gut 2000;47:178–83. [13] Barrachina MD, Martinez V, Wang L, Wei LW, Tache Y. Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice. Proc Natl Acad Sci USA 1997;94:10455– 60.
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