Obesity Research & Clinical Practice (2012) 6, e225—e232
ORIGINAL ARTICLE
Masked function of amino acid sensors on pancreatic hormone secretion in ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia Noriko Ishizuka a, Katsuaki Tanaka b, Yoko Suzuki a, Yuri Kintaka c, Ikiko Kinoshita d, Takeo Hashiguchi e, Hiroyuki Shimizu a, Akira Senoo a,∗, Nobuo Imazeki a, Yoko Kobayashi a, Katsumi Arai a, Ryota Haba a, Tosei Takahashi a, Kahoru Sasaki a, Masako Kako a, Kaori Hayashi a, Toshimasa Osaka f, Yuichi Suzuki g, Shuji Inoue a a
Faculty of Health Care, Kiryu University, 606-7 Azami, Kasakake-cho, Midori-shi, Gunma 379-2392, Japan b Yokohama City University Medical Center, 4-57 Urafunecho, Minami-ku, Yokohama-shi Kanagawa 232-0024, Japan c Faculty of Dairy Science, Rakuno Gakuen University, 582 Midorimachi, Bunkyodai, Ebetsu-shi, Hokkaido 069-8501, Japan d Faculty of Home Economics, Kyoritsu Women’s University, 2-2-1 Hitotsubashi, Chiyoda-ku, Tokyo 101-8433, Japan e Faculty of Science & Engineering, Teikyo University of Science & Technology, 2-2-1 Senjusakuragi, Adachi-ku, Tokyo 120-0045, Japan f National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan g University of Shizuoka, School of Food and Nutritional Sciences, 52-1 Yada Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan Received 3 October 2011 ; received in revised form 22 November 2011; accepted 25 November 2011
KEYWORDS Amino acid sensor; Arginine; Leucine; Alanine; Ventromedial hypothalamic lesion ∗
Summary In neural regulation of the endocrine pancreas, there is much evidence to suggest that vagal efferents alter insulin and glucagon secretion, but less information on the effects of vagal afferents. In this study, we investigated the role and function of afferent fibers of the vagus nerve in normal and ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia. In normal rats, hepatic vagotomy was associated with intraperitoneal (ip) arginine-induced enhancement of insulin and glucagon secretion without an accompanying change in blood glucose levels, ip leucine induced enhancement of insulin secretion accompanied by a
Corresponding author. Tel.: +81 277 48 9121; fax: +81 277 76 9454. E-mail addresses: [email protected], [email protected] (A. Senoo).
1871-403X/$ — see front matter © 2011 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.orcp.2011.11.008
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N. Ishizuka et al. decrease in blood glucose levels, and ip alanine-induced enhancement of glucagon secretion accompanied by an increase in blood glucose levels. In VMH lesioned rats with marked hyperinsulinemia, none of these amino acids caused significant changes in insulin and glucagon secretion. We conclude that amino acid sensors in normal rats inhibit excess release of pancreatic hormones induced directly by intake of amino acids, such as that in excess protein ingestion, and maintain blood glucose levels within the normal range. In contrast, in VMH lesioned rats with marked hyperinsulinemia, the function of the amino acid sensors is masked due to the marked hyperinsulinemia in these rats. © 2011 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction Pancreatic hormone secretion is regulated neurally via the vagus nerve [1]. This nerve includes efferent and afferent pathways, but most studies have been limited to the function of the efferent pathway, which exclusively stimulates pancreatic hormone secretion [2]. In contrast, little is known about the role and function of the afferent pathway. In 1963, Russek [3] proposed the presence of a glucose receptor (the so-called ‘‘glucoreceptor’’) that sends signals of elevation of serum glucose via carbohydrate ingestion to the central nervous system through the hepatic branch of the vagus nerve. The existence of this receptor was confirmed electrophysiologically by Niijima [4]. A further study by Niijima [5] showed that glucose administration into the portal vein elevated the electrical activity of the hepatic branch of the vagus nerve and reduced the electrical activity of the celiac branch via the central nervous system. In 1985, Lee and Miller [6] found that insulin secretion induced by intraperitoneal (ip) administration of glucose was reduced by sectioning the hepatic branch of the vagus nerve (hepatic vagotomy), and Nagase et al. [7] found that this effect was reversed by sectioning the celiac branch of the vagus nerve (celiac vagotomy), which innervates the pancreas. Based on these findings, it was concluded that the glucoreceptor inhibits glucose-induced hyperglycemia by stimulating insulin secretion, in addition to direct insulin secretion caused by glucose itself, through the liver glucoreceptor-afferent vagalbrainstem-efferent vagal axis, thereby contributing to maintenance of homeostasis of blood glucose. In 1986, we found that ip arginine administration significantly elevated the plasma insulin and glucagon levels in rats with hepatic vagotomy [8]. We also found electrophysiological evidence for the presence of an arginine-responsive nerve fiber in the hepatic branch of the vagus nerve [9]. These effects of arginine were inhibited by additional
celiac vagotomy or atropine administration [10]. We also found electrophysiological evidence for the presence of a nerve fiber that was responsive to alanine and leucine in the liver through the afferent vagal pathway [11]. In rats with hepatic vagotomy, glucagon secretion was significant enhanced by ip alanine administration and marked elevation of the plasma insulin level was induced by ip leucine administration, with reversal of these effects to the basal condition by additional celiac vagotomy [12]. These findings suggest that ‘‘amino acid sensors’’ are present in the hepatic branch of the vagus nerve in the liver. These sensors may detect amino acids and stimulate the afferent vagal nerve fiber under physiological conditions. In turn, this inhibits the efferent vagal nerve fiber through the central nervous system in a reflex manner [13,14], and subsequently suppresses arginine-induced insulin and glucagon secretion, leucine-induced insulin secretion, and alanine-induced glucagon secretion. This prevents overstimulation of insulin and glucagon and maintains homeostasis of blood glucose after massive ingestion of meat, which contains large amounts of these amino acids. Based on these findings, we proposed the presence of amino acid sensors in the liver that send inhibitory signals to efferent vagal nerve fiber through the afferent vagal pathway via the brainstem [9—12]. It is also of interest to determine the functions of these amino acid sensors under pathophysiological conditions. Destruction of the ventromedial hypothalamus (VMH) in animals produces autonomic derangements [15—18] in which vagal hyperactivity and sympathetic hypoactivity occur. These changes result in hyperinsulinemia in VMH lesioned animals through vagal efferents and sympathetic efferents [19,20]. In rats, the hepatic branch of the vagus nerve mostly comprises afferent nerve fiber and serves as the main afferent pathway connecting the liver and spinal cord [3,4]. The celiac branch of the vagus nerve serves as the vagal pathway to the pancreas and functionally is mostly
Amino acid sensors in ventromedial lesioned rats comprised of efferent nerve fiber [3—5]. The objective of this study was to investigate the functions of vagal afferents in secretion of insulin and glucagon in VMH lesioned rats, an animal model of pathological obesity with a marked increase in insulin secretion, in comparison with those in normal rats.
Methods
e227 Anterior trunk Posterior trunk
Hepac branch
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Stomach Pancreas
Animals Thirteen-week-old female Sprague-Dawley (SD) rats weighing about 260—290 g were purchased from Charles River Japan. The animals were maintained in a room controlled at a constant temperature (23 ± 2 ◦ C) under a 12-h lighting cycle (light on: AM 8:00—PM 8:00) and given free access to food (normal pellets, Oriental Yeast) and water. The animals were subjected to experiments after 7 days of acclimation. All procedures were performed according to the Japanese Physiological Society’s guidelines for animal care. This protocol was approved by the Animal Research Committee of the Faculty of Health Care, Kiryu University.
Experimental procedures Two experiments were performed. In the first experiment, rats were divided into two groups with sham vagotomy or hepatic vagotomy. In the second experiment, rats were also divided into two groups that were sham-VMH lesioned or VMH lesioned, both with hepatic vagotomy. In both experiments a catheter was installed in the jugular vein at the same time. Seven days later each group was divided into three subgroups and body weight was measured. Intraperitoneal amino acid (arginine, leucine, and alanine) load tests were performed under pentobarbital (50 mg/kg) anesthesia in an overnight fasted condition.
Preparation of VMH lesioned rats VMH lesioned rats were prepared according to the method of Inoue et al. [21] under isoflurane inhalation anesthesia (Forane, Dainippon Pharmaceutical Co., Japan). The head was fixed in a brain fixation device (S-5, Narishige, Japan) and an electrode exposed only at the tip was inserted at the stereotactic coordinates of the VMH (anteroposterior position: 2.8 mm posterior to the bregma, lateral position: 0.7 mm from the sagittal line on the left and right sides, vertical position: 0.5 mm
Figure 1 Schematic diagram of the abdominal subdiaphragmatic vagal system in rat.
above the skull floor) using the coordinates of De Groot [22]. The VMH was destroyed by passing current at 2.0 mA for 20 s. Rats with sham-VMH lesions were prepared using the same procedure, but without passing electric current.
Hepatic vagotomy and catheter placement through the jugular vein Hepatic vagotomy was performed as reported by Tanaka et al. [10]. Under hexobarbital anesthesia (50 mg/kg), the hepatic branch of the vagus nerve branching from the anterior root of the vagus nerve located 2—3 mm proximal to the cardiac part of the stomach was exposed and the hepatic branch coming out from the anterior root was completely sectioned at the closest site under a binocular microscope (Fig. 1). In sham vagotomy, the same surgical procedure was performed, except for sectioning of the hepatic branch of vagus nerve. Immediately after hepatic vagotomy, the tip of a polyethylene catheter (Clay Adams Co., Parsippany, NJ, USA) was inserted close to the right atria through the jugular vein. The catheter stump was passed through the subcutaneous tissue, exposed out of the posterior neck, and fixed [23]. Rats with installed jugular vein catheters were returned to individual cages. The catheter was washed with heparin-containing saline once a day to prevent obstruction of the catheter by thrombosis until the ip amino acid tests were performed.
Ip amino acids tests After overnight fasting (18 h), 3 amino acids tests were performed without anesthesia and restraint [23,24]. Arginine (10 mM, 1 g/kg in water), a leucine suspension in 10% arabic gum (0.3 g/kg), and alanine (0.5 g/kg in water) were warmed to 36—37 ◦ C and injected ip to three different groups of rats (n = 7 each). At baseline and 5, 10, 15, 30 and 60 min
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after administration, 0.9 ml of blood was collected through the jugular vein catheter using a heparinized syringe and the blood was transferred into a cooled tube containing 1000 U aprotinin (Bayer Yakuhin, Japan). After centrifugation, serum was stored at −80 ◦ C until measurement. After blood sampling, an equal volume of heparinized blood was administered to minimize the influence of blood volume depletion [24].
Confirmation of VMH lesions and hepatic vagotomy At the end of each experiment, the brains of VMH lesioned rats were removed and HE staining was used to verify formation of VMH lesions. To confirm the success of hepatic and celiac vagotomy, rats were laparotomized under isoflurane inhalation anesthesia, adhered tissue in the vagotomized region was dissected, and the region was observed with a binocular microscope.
Measurements Plasma glucose was measured with a Beckman glucose analyzer using the glucose oxidase method. Plasma insulin and glucagon were measured using an immunological Insulin Assay Kit (Amersham, Japan) and a Glucagon Assay Kit (Daiichi Radioisotope Labs, Japan), respectively. Rat insulin and glucagon were used for preparation of standard curves.
Statistical analysis All results are presented as the mean ± standard error. Duncan’s multiple comparison was used for comparison of body weight changes in each group and a t-test was applied for comparison between two groups. The significance level was set at P < 0.05.
Results Confirmation of destruction of the VMH Brain sections of the VMH region were prepared to confirm the presence of VMH lesions. Only data from rats in which the VMH was entirely destroyed were included in the analysis. A typical image showing complete destruction of the bilateral VMH is shown in Fig. 2.
Figure 2 An illustration of a typical ventromedial hypothalamic (VMH) lesion in rat.
Body weight In normal rats, there was no significant difference in body weight before and after sham or hepatic vagotomy with jugular vein catheterization. In all VMH lesioned rats, body weight markedly increased one week after VMH lesioning, but with no significant difference between animals that underwent sham or hepatic vagotomy (Table 1).
Ip amino acid tests in normal rats After ip injection of arginine, plasma glucose levels slowly rose and reached a peak within 10—15 min in normal rats with sham or hepatic vagotomy. These levels then slowly decreased until 30 min and subsequently remained constant until 60 min. There were no significant differences between the two groups (Fig. 3A). Plasma insulin levels rose relatively sharply until 10 min in both groups, sharply decreased until 30 min, and then remained constant until 60 min. The increase was particularly sharp in normal rats with hepatic vagotomy and the levels in this group were significantly higher than those in the sham vagotomy group at 5, 10, and 15 min. Plasma glucagon levels rose sharply until 5 min, maintained peak levels until 15 min, and then decreased in both groups. The rise was particularly sharp in the hepatic vagotomy group and the levels in this group were significantly higher than those in the sham vagotomy group at 5, 10, 15, and 30 min. After ip injection of leucine, plasma glucose levels rose slowly in normal rats with sham vagotomy (Fig. 3B), but transiently decreased in those with hepatic vagotomy and were significantly lower than the levels in the sham vagotomy group at 5, 10, and
Amino acid sensors in ventromedial lesioned rats Table 1
Body weights of sham VMH lesioned rats and VMH lesioned rats with sham vagotomy or hepatic vagotomy.
Normal rats Sham vagotomy Hepatic vagotomy VMH lesioned rats Sham vagotomy Hepatic vagotomy **
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Before VMH lesions (g)
1 week after VMH lesioning with vagotomy and jugular cannulation (g)
276.85 ± 2.8 276.03 ± 4.2
275.45 ± 3.1 272.42 ± 2.5
283.05 ± 2.8 281.03 ± 3.9
328.95 ± 2.8** 330.42 ± 4.1**
P < 0.01 vs. baseline.
15 min. Plasma insulin levels rose sharply in normal rats with hepatic vagotomy and were significantly higher than those with sham vagotomy at 5, 10, 15, and 30 min. The plasma glucagon level did not differ significantly between the two groups. After ip injection of alanine, plasma glucose levels rose until 30 min in normal rats with sham vagotomy and then slowly decreased or remained constant thereafter (Fig. 3C). The hepatic vagotomy group showed a sharp increase and the levels in this group were significantly higher than those in the sham vagotomy group at 5, 10, 15, and 30 min. Plasma insulin levels rose a little until 30 min and then slowly decreased in both groups, with no significant difference between the two groups at any time point. Plasma glucagon levels rose sharply in the hepatic vagotomy group and were significantly higher than those in the sham vagotomy group at 5, 10, 15, and 30 min.
Ip amino acid tests in VMH lesioned rats with marked hyperinsulinemia After ip injection of arginine, plasma glucose levels rose relatively sharply in VMH lesioned rats with sham or hepatic vagotomy, reached a peak within 5—15 min, and then slowly decreased (Fig. 4A). There was no significant difference between the two groups at any time point. Plasma insulin levels rose sharply in both groups (Fig. 4B), again with no significant difference between the two groups. Plasma glucagon levels rose relatively sharply in both groups, reached a peak within 5—15 min, and then decreased, with no significant difference between the two groups at any time point. After ip injection of leucine, plasma glucose levels rose relatively sharply in VMH lesioned rats with sham or hepatic vagotomy, reached a peak within 5—15 min, and then slowly decreased (Fig. 4B), with no significant difference between the groups at any time point. Plasma insulin levels also rose relatively sharply in both groups, reached a peak within
10 min, and then decreased, again with no significant difference between the two groups. Plasma glucagon levels did not differ significantly between the two groups at any time point. After ip injection of alanine, plasma glucose levels rose slowly in both groups, reached a peak within 30 min, and remained constant thereafter (Fig. 4C), with no significant difference between the two groups at any time point. Plasma insulin levels rose a little in both groups, with no significant differences between the groups. Plasma glucagon levels rose sharply, reached a peak within 10 min, in the both groups and decreased relatively sharply thereafter, with no significant difference between the two groups at any time point.
Discussion In normal rats, ip arginine administration enhanced insulin and glucagon secretion without causing a significant change in blood glucose levels, ip leucine enhanced insulin secretion in association with a significant decrease in blood glucose levels, and ip alanine enhanced glucagon secretion in association with a significant rise in blood glucose levels after hepatic vagotomy. These findings are consistent with previous results [13,15]. Arginine, leucine, and alanine strongly stimulate secretion of pancreatic hormones, insulin and glucagon [25,26]. After excessive ingestion of protein, many amino acids enter the circulation via the portal vein and reach the pancreas, which may result in excess insulin or glucagon secretion that disturbs blood glucose homeostasis through hypoglycemia or hyperglycemia. Thus, we hypothesize that amino acid sensors are present in the portal vein, and that when these sensors detect an excess of amino acids, they stimulate afferent vagal nerve fiber and inhibit efferent vagal nerve fiber via reflex through the brainstem. This, in turn, regulates inhibition of excess pancreatic hormone secretion by amino
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Figure 3 Changes of plasma glucose, insulin and glucagon levels in response to ip injections of arginine (A), leucine (B), and alanine (C) in normal rats after sham hepatic vagotomy or hepatic vagotomy. *P < 0.05, **P < 0.01 for hepatic vagotomy vs. sham hepatic vagotomy.
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acids themselves to maintain glucose homeostasis by keeping blood glucose within the normal range. In this context, we have also investigated the properties of other amino acids, such as glutamate, isoleucine and phenylalanine, and found that they do not induce an equivalent ‘‘amino acid sensor’’ function in the regulation of pancreatic hormones [12]. In VMH lesioned rats with marked hyperinsulinemia, administration of ip arginine, leucine, and alanine did not significantly change blood glucose levels or secretion of insulin and glucagon in rats with hepatic vagotomy compared to those with sham hepatic vagotomy. These results suggest that the function of amino acid sensors was masked in these rats. It is well known that insulin secretion is markedly increased in VMH lesioned animals [16,27,28], and we found that the increased insulin secretion is induced by autonomic neural derangements with elevated efferent vagus nerve activity in the previous study using celiac vagotomy [29] and reduced sympathetic nerve activity using administration of sympathetic antagonists in VMH lesioned rats [20]. Hepatic vagotomy did not affect insulin secretion in VMH lesioned rats because of the masked function of amino acid sensors with the marked hyperinsulinemia in these rats. Hepatic vagotomy in VMH lesioned rats did not significantly alter glucagon secretion induced by ip administration of any amino acids, which may be attributable to inhibition of glucagon secretion by excess insulin
secretion [30,31]. Overall, the function of amino acid sensors on pancreatic hormone secretion were masked in VMH lesioned rats with marked hyperinsulinemia. This is also consistent with the proposal that neural regulation plays only a small role in glucagon secretion [25,30,32]. In a previous study, we found that the function of glucoreceptors in the liver was disturbed, but that of an arginine sensor was retained, in carbon tetrachloride (CCl4 )induced cirrhotic rats after hepatic vagotomy [33]. In conclusion, in normal rats with hepatic vagotomy, ip arginine enhanced insulin and glucagon secretion without a change in blood glucose levels, ip leucine enhanced insulin secretion in association with a decrease in blood glucose levels, and ip alanine enhanced glucagon secretion in association with an increase in blood glucose levels. These phenomena can be explained by the presence of amino acid sensors in the hepatic portal vein that prevent direct induction of excess pancreatic hormone secretion following ingestion of excessive amino acids. In general practice, we should be careful to take too much meat, which provide excessive amino acids in the circulation and break the function of amino acid sensors, resulting in hypoglycemic attack. In VMH lesioned rats with hepatic vagotomy, insulin and glucagon secretion did not change significantly after administration of any amino acids. These results suggest that the functions of amino acid sensors are masked due to marked hyperinsulinemia in these rats.
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Conflict of interest None.
References [1] Ahren B. Autonomic regulation of islet hormone secretionimplications for health and disease. Diabetologia 2000;43:393—410. [2] Miller RE. Pancreatic neuroendocrinology: peripheral neural mechanisms in the regulation of the islets of Langerhans. Endocr Rev 1981;2:471—94. [3] Russek M. A hypothesis on the participation of hepatic glucoreceptors in the control of food intake. Nature 1963;197:1089—95. [4] Niijima A. Afferent impulse discharges from glucoreceptors in the liver of the guinea pig. Ann NY Acad Sci 1969;157:690—700. [5] Niijima A. Nervous regulatory mechanism of blood glucose levels. In: Katsuki Y, et al., editors. Food intake and chemical senses. Tokyo, Japan: Scientific Societies Press; 1977. p. 413—26. [6] Lee KC, Miller RE. The hepatic vagus nerve and the neural regulation of insulin secretion. Endocrinology 1985;117:307—14. [7] Nagase H, Inoue S, Tanaka K, Takamura Y, Niijima A. Hepatic glucose-sensitive unit regulation of glucoseinduced insulin secretion in rats. Physiol Behav 1993;53: 139—43. [8] Tanaka K, Inoue S, Fujii T, Takamura Y. Enhancement of insulin and glucagon secretion by arginine after hepatic vagotomy. Neurosci Lett 1986;72:74—8. [9] Tanaka K, Inoue S, Takamura Y, Jiang ZY, Niijima A. Arginine sensors in the hepato-portal system and their reflex effects on pancreatic efferents in the rat. Neurosci Lett 1986;72:69—73. [10] Tanaka K, Inoue S, Nagase H, Takamura Y. Modulation of arginine-induced insulin and glucagon secretion by the hepatic vagus nerve in the rat: effects of celiac vagotomy and administration of atropine. Endocrinology 1990;127:2017—23. [11] Tanaka K, Inoue S, Nagase H, Takamura Y, Niijima A. Amino acid sensors sensitive to alanine and leucine exist in the hepato-portal system in the rat. J Auton Nerv Syst 1990;31:41—6. [12] Tanaka K, Inoue S, Saito S, Nagase H, Takamura Y. Hepatic vagal amino acid sensors modulate amino acid induced insulin and glucagon secretion in the rat. J Auton Nerv Syst 1993;42:225—32. [13] Adachi A, Niijima A, Jacobs HL. An hepatic osmoreceptor mechanism in the electrophysiological and behavioral studies. Am J Physiol 1976;231:1043—9. [14] Magni F, Carobi C. The afferent and preganglionic parasympathetic innervation of the rat demonstrated by the retrograde transport of horseradish peroxidase. J Auton Nerv Syst 1983;8:237—60.
[15] Inoue S, Bray G. The effects of subdiaphragmatic vagotomy in rat with ventromedial hypothalamic obesity. Endocrinology 1977;100:108—14. [16] Inoue S, Bray GA, Mullen Y. Effect of transplantation of pancreas on development of hypothalamic obesity. Nature 1977;226:742—4. [17] Yoshimatsu H, Niijima A, Oomura Y, Yamabe K, Katafuchi T. Effects of hypothalamic lesion on pancreatic autonomic nerve activity in the rat. Brain Res 1984;303:147—52. [18] Bray GA, Nishizawa Y. Ventromedial hypothalamus modulates fat mobilisation during fasting. Nature 1978;274:900—2. [19] Inoue S, Bray GA. An autonomic hypothesis for hypothalamic obesity. Life Sci 1979;25:561—6. [20] Inoue S, Mullen Y, Bray GA. Hyperinsulinemia in rats with hypothalamic obesity: effects of autonomic drugs and glucose. Am J Physiol 1983;245:372—8. [21] Inoue S, Camplifield LA, Bray GA. Comparison of metabolic alterations in hypothalamic and high fat diet induced obesity. Am J Physiol 1977;233:162—8. [22] De Groot J. The rat forebrain in stereotaxic coordinates. Amsterdam: N.V. Noord-Hollandsch Uitgevers Maatschappij; 1959. [23] Shah J, Wongsurawat N, Aran P, Motto G, Bowser E. A method for studying acute insulin secretion and glucose tolerance in unanesthetized and unrestrained rats. Diabetes 1977;26:1—6. [24] Berthoud H, Laughton W, Powley T. A method for large volume blood sampling and transfusion in rats. Am J Physiol 1986;250:331—7. [25] Quesada I, Tudurí E, Ripoll C, Nadal A. Physiology of the pancreatic alpha-cell and glucagon secretion: role in glucose homeostasis and diabetes. J Endocrinol 2008;199:5—19. [26] Floyds Jr JC, Fajans S, Conn JW, Knopf RF, Rull J. Stimulation of insulin secretion by amino acids. J Clin Invest 1966;45:1487—502. [27] Bray GA, York DA. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol Rev 1979;59:719—809. [28] King BM. The rise, fall, and resurrection of the ventromedial hypothalamus in the regulation of feeding behavior and body weight. Physiol Behav 2006;87:221—44. [29] Inoue S, Nagase H, Satoh S, Egawa M, Tanaka K, Takamura Y. Role of the efferent and afferent vagus nerve in the development of ventromedial hypothalamic (VMH) obesity. Brain Res Bull 1991;27:511—5. [30] MacDonald PE, Zhang Y, Ramracheta R, Salehi A, Ma X, Johnson PR, et al. AKATP channel-dependent pathway within ␣ cells regulates glucagon release from both rodent and human islets of Langerhans. PLoS Biol 2007;5:1236—47. [31] Bansal P, Wang Q. Insulin as a physiological modulator of glucagon secretion. Am J Physiol Endocrinol Metab 2008;295:751—61. [32] Gromada J, Franklin I, Wollheim CB. ␣-Cells of the endocrine pancreas: 35 years of research but the enigma remains. Endocr Rev 2007;28:84—116. [33] Okazaki H, Tanaka K, Nagase H, Inoue S. Modulation of insulin secretion by hepatic vagotomy in cirrhotic rats. Physiol Behav 1993;53:521—5.
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ORIGINAL ARTICLE
Masked function of amino acid sensors on pancreatic hormone secretion in ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia Noriko Ishizuka a, Katsuaki Tanaka b, Yoko Suzuki a, Yuri Kintaka c, Ikiko Kinoshita d, Takeo Hashiguchi e, Hiroyuki Shimizu a, Akira Senoo a,∗, Nobuo Imazeki a, Yoko Kobayashi a, Katsumi Arai a, Ryota Haba a, Tosei Takahashi a, Kahoru Sasaki a, Masako Kako a, Kaori Hayashi a, Toshimasa Osaka f, Yuichi Suzuki g, Shuji Inoue a a
Faculty of Health Care, Kiryu University, 606-7 Azami, Kasakake-cho, Midori-shi, Gunma 379-2392, Japan b Yokohama City University Medical Center, 4-57 Urafunecho, Minami-ku, Yokohama-shi Kanagawa 232-0024, Japan c Faculty of Dairy Science, Rakuno Gakuen University, 582 Midorimachi, Bunkyodai, Ebetsu-shi, Hokkaido 069-8501, Japan d Faculty of Home Economics, Kyoritsu Women’s University, 2-2-1 Hitotsubashi, Chiyoda-ku, Tokyo 101-8433, Japan e Faculty of Science & Engineering, Teikyo University of Science & Technology, 2-2-1 Senjusakuragi, Adachi-ku, Tokyo 120-0045, Japan f National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan g University of Shizuoka, School of Food and Nutritional Sciences, 52-1 Yada Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan Received 3 October 2011 ; received in revised form 22 November 2011; accepted 25 November 2011
KEYWORDS Amino acid sensor; Arginine; Leucine; Alanine; Ventromedial hypothalamic lesion ∗
Summary In neural regulation of the endocrine pancreas, there is much evidence to suggest that vagal efferents alter insulin and glucagon secretion, but less information on the effects of vagal afferents. In this study, we investigated the role and function of afferent fibers of the vagus nerve in normal and ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia. In normal rats, hepatic vagotomy was associated with intraperitoneal (ip) arginine-induced enhancement of insulin and glucagon secretion without an accompanying change in blood glucose levels, ip leucine induced enhancement of insulin secretion accompanied by a
Corresponding author. Tel.: +81 277 48 9121; fax: +81 277 76 9454. E-mail addresses: [email protected], [email protected] (A. Senoo).
1871-403X/$ — see front matter © 2011 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.orcp.2011.11.008
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N. Ishizuka et al. decrease in blood glucose levels, and ip alanine-induced enhancement of glucagon secretion accompanied by an increase in blood glucose levels. In VMH lesioned rats with marked hyperinsulinemia, none of these amino acids caused significant changes in insulin and glucagon secretion. We conclude that amino acid sensors in normal rats inhibit excess release of pancreatic hormones induced directly by intake of amino acids, such as that in excess protein ingestion, and maintain blood glucose levels within the normal range. In contrast, in VMH lesioned rats with marked hyperinsulinemia, the function of the amino acid sensors is masked due to the marked hyperinsulinemia in these rats. © 2011 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction Pancreatic hormone secretion is regulated neurally via the vagus nerve [1]. This nerve includes efferent and afferent pathways, but most studies have been limited to the function of the efferent pathway, which exclusively stimulates pancreatic hormone secretion [2]. In contrast, little is known about the role and function of the afferent pathway. In 1963, Russek [3] proposed the presence of a glucose receptor (the so-called ‘‘glucoreceptor’’) that sends signals of elevation of serum glucose via carbohydrate ingestion to the central nervous system through the hepatic branch of the vagus nerve. The existence of this receptor was confirmed electrophysiologically by Niijima [4]. A further study by Niijima [5] showed that glucose administration into the portal vein elevated the electrical activity of the hepatic branch of the vagus nerve and reduced the electrical activity of the celiac branch via the central nervous system. In 1985, Lee and Miller [6] found that insulin secretion induced by intraperitoneal (ip) administration of glucose was reduced by sectioning the hepatic branch of the vagus nerve (hepatic vagotomy), and Nagase et al. [7] found that this effect was reversed by sectioning the celiac branch of the vagus nerve (celiac vagotomy), which innervates the pancreas. Based on these findings, it was concluded that the glucoreceptor inhibits glucose-induced hyperglycemia by stimulating insulin secretion, in addition to direct insulin secretion caused by glucose itself, through the liver glucoreceptor-afferent vagalbrainstem-efferent vagal axis, thereby contributing to maintenance of homeostasis of blood glucose. In 1986, we found that ip arginine administration significantly elevated the plasma insulin and glucagon levels in rats with hepatic vagotomy [8]. We also found electrophysiological evidence for the presence of an arginine-responsive nerve fiber in the hepatic branch of the vagus nerve [9]. These effects of arginine were inhibited by additional
celiac vagotomy or atropine administration [10]. We also found electrophysiological evidence for the presence of a nerve fiber that was responsive to alanine and leucine in the liver through the afferent vagal pathway [11]. In rats with hepatic vagotomy, glucagon secretion was significant enhanced by ip alanine administration and marked elevation of the plasma insulin level was induced by ip leucine administration, with reversal of these effects to the basal condition by additional celiac vagotomy [12]. These findings suggest that ‘‘amino acid sensors’’ are present in the hepatic branch of the vagus nerve in the liver. These sensors may detect amino acids and stimulate the afferent vagal nerve fiber under physiological conditions. In turn, this inhibits the efferent vagal nerve fiber through the central nervous system in a reflex manner [13,14], and subsequently suppresses arginine-induced insulin and glucagon secretion, leucine-induced insulin secretion, and alanine-induced glucagon secretion. This prevents overstimulation of insulin and glucagon and maintains homeostasis of blood glucose after massive ingestion of meat, which contains large amounts of these amino acids. Based on these findings, we proposed the presence of amino acid sensors in the liver that send inhibitory signals to efferent vagal nerve fiber through the afferent vagal pathway via the brainstem [9—12]. It is also of interest to determine the functions of these amino acid sensors under pathophysiological conditions. Destruction of the ventromedial hypothalamus (VMH) in animals produces autonomic derangements [15—18] in which vagal hyperactivity and sympathetic hypoactivity occur. These changes result in hyperinsulinemia in VMH lesioned animals through vagal efferents and sympathetic efferents [19,20]. In rats, the hepatic branch of the vagus nerve mostly comprises afferent nerve fiber and serves as the main afferent pathway connecting the liver and spinal cord [3,4]. The celiac branch of the vagus nerve serves as the vagal pathway to the pancreas and functionally is mostly
Amino acid sensors in ventromedial lesioned rats comprised of efferent nerve fiber [3—5]. The objective of this study was to investigate the functions of vagal afferents in secretion of insulin and glucagon in VMH lesioned rats, an animal model of pathological obesity with a marked increase in insulin secretion, in comparison with those in normal rats.
Methods
e227 Anterior trunk Posterior trunk
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Animals Thirteen-week-old female Sprague-Dawley (SD) rats weighing about 260—290 g were purchased from Charles River Japan. The animals were maintained in a room controlled at a constant temperature (23 ± 2 ◦ C) under a 12-h lighting cycle (light on: AM 8:00—PM 8:00) and given free access to food (normal pellets, Oriental Yeast) and water. The animals were subjected to experiments after 7 days of acclimation. All procedures were performed according to the Japanese Physiological Society’s guidelines for animal care. This protocol was approved by the Animal Research Committee of the Faculty of Health Care, Kiryu University.
Experimental procedures Two experiments were performed. In the first experiment, rats were divided into two groups with sham vagotomy or hepatic vagotomy. In the second experiment, rats were also divided into two groups that were sham-VMH lesioned or VMH lesioned, both with hepatic vagotomy. In both experiments a catheter was installed in the jugular vein at the same time. Seven days later each group was divided into three subgroups and body weight was measured. Intraperitoneal amino acid (arginine, leucine, and alanine) load tests were performed under pentobarbital (50 mg/kg) anesthesia in an overnight fasted condition.
Preparation of VMH lesioned rats VMH lesioned rats were prepared according to the method of Inoue et al. [21] under isoflurane inhalation anesthesia (Forane, Dainippon Pharmaceutical Co., Japan). The head was fixed in a brain fixation device (S-5, Narishige, Japan) and an electrode exposed only at the tip was inserted at the stereotactic coordinates of the VMH (anteroposterior position: 2.8 mm posterior to the bregma, lateral position: 0.7 mm from the sagittal line on the left and right sides, vertical position: 0.5 mm
Figure 1 Schematic diagram of the abdominal subdiaphragmatic vagal system in rat.
above the skull floor) using the coordinates of De Groot [22]. The VMH was destroyed by passing current at 2.0 mA for 20 s. Rats with sham-VMH lesions were prepared using the same procedure, but without passing electric current.
Hepatic vagotomy and catheter placement through the jugular vein Hepatic vagotomy was performed as reported by Tanaka et al. [10]. Under hexobarbital anesthesia (50 mg/kg), the hepatic branch of the vagus nerve branching from the anterior root of the vagus nerve located 2—3 mm proximal to the cardiac part of the stomach was exposed and the hepatic branch coming out from the anterior root was completely sectioned at the closest site under a binocular microscope (Fig. 1). In sham vagotomy, the same surgical procedure was performed, except for sectioning of the hepatic branch of vagus nerve. Immediately after hepatic vagotomy, the tip of a polyethylene catheter (Clay Adams Co., Parsippany, NJ, USA) was inserted close to the right atria through the jugular vein. The catheter stump was passed through the subcutaneous tissue, exposed out of the posterior neck, and fixed [23]. Rats with installed jugular vein catheters were returned to individual cages. The catheter was washed with heparin-containing saline once a day to prevent obstruction of the catheter by thrombosis until the ip amino acid tests were performed.
Ip amino acids tests After overnight fasting (18 h), 3 amino acids tests were performed without anesthesia and restraint [23,24]. Arginine (10 mM, 1 g/kg in water), a leucine suspension in 10% arabic gum (0.3 g/kg), and alanine (0.5 g/kg in water) were warmed to 36—37 ◦ C and injected ip to three different groups of rats (n = 7 each). At baseline and 5, 10, 15, 30 and 60 min
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after administration, 0.9 ml of blood was collected through the jugular vein catheter using a heparinized syringe and the blood was transferred into a cooled tube containing 1000 U aprotinin (Bayer Yakuhin, Japan). After centrifugation, serum was stored at −80 ◦ C until measurement. After blood sampling, an equal volume of heparinized blood was administered to minimize the influence of blood volume depletion [24].
Confirmation of VMH lesions and hepatic vagotomy At the end of each experiment, the brains of VMH lesioned rats were removed and HE staining was used to verify formation of VMH lesions. To confirm the success of hepatic and celiac vagotomy, rats were laparotomized under isoflurane inhalation anesthesia, adhered tissue in the vagotomized region was dissected, and the region was observed with a binocular microscope.
Measurements Plasma glucose was measured with a Beckman glucose analyzer using the glucose oxidase method. Plasma insulin and glucagon were measured using an immunological Insulin Assay Kit (Amersham, Japan) and a Glucagon Assay Kit (Daiichi Radioisotope Labs, Japan), respectively. Rat insulin and glucagon were used for preparation of standard curves.
Statistical analysis All results are presented as the mean ± standard error. Duncan’s multiple comparison was used for comparison of body weight changes in each group and a t-test was applied for comparison between two groups. The significance level was set at P < 0.05.
Results Confirmation of destruction of the VMH Brain sections of the VMH region were prepared to confirm the presence of VMH lesions. Only data from rats in which the VMH was entirely destroyed were included in the analysis. A typical image showing complete destruction of the bilateral VMH is shown in Fig. 2.
Figure 2 An illustration of a typical ventromedial hypothalamic (VMH) lesion in rat.
Body weight In normal rats, there was no significant difference in body weight before and after sham or hepatic vagotomy with jugular vein catheterization. In all VMH lesioned rats, body weight markedly increased one week after VMH lesioning, but with no significant difference between animals that underwent sham or hepatic vagotomy (Table 1).
Ip amino acid tests in normal rats After ip injection of arginine, plasma glucose levels slowly rose and reached a peak within 10—15 min in normal rats with sham or hepatic vagotomy. These levels then slowly decreased until 30 min and subsequently remained constant until 60 min. There were no significant differences between the two groups (Fig. 3A). Plasma insulin levels rose relatively sharply until 10 min in both groups, sharply decreased until 30 min, and then remained constant until 60 min. The increase was particularly sharp in normal rats with hepatic vagotomy and the levels in this group were significantly higher than those in the sham vagotomy group at 5, 10, and 15 min. Plasma glucagon levels rose sharply until 5 min, maintained peak levels until 15 min, and then decreased in both groups. The rise was particularly sharp in the hepatic vagotomy group and the levels in this group were significantly higher than those in the sham vagotomy group at 5, 10, 15, and 30 min. After ip injection of leucine, plasma glucose levels rose slowly in normal rats with sham vagotomy (Fig. 3B), but transiently decreased in those with hepatic vagotomy and were significantly lower than the levels in the sham vagotomy group at 5, 10, and
Amino acid sensors in ventromedial lesioned rats Table 1
Body weights of sham VMH lesioned rats and VMH lesioned rats with sham vagotomy or hepatic vagotomy.
Normal rats Sham vagotomy Hepatic vagotomy VMH lesioned rats Sham vagotomy Hepatic vagotomy **
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Before VMH lesions (g)
1 week after VMH lesioning with vagotomy and jugular cannulation (g)
276.85 ± 2.8 276.03 ± 4.2
275.45 ± 3.1 272.42 ± 2.5
283.05 ± 2.8 281.03 ± 3.9
328.95 ± 2.8** 330.42 ± 4.1**
P < 0.01 vs. baseline.
15 min. Plasma insulin levels rose sharply in normal rats with hepatic vagotomy and were significantly higher than those with sham vagotomy at 5, 10, 15, and 30 min. The plasma glucagon level did not differ significantly between the two groups. After ip injection of alanine, plasma glucose levels rose until 30 min in normal rats with sham vagotomy and then slowly decreased or remained constant thereafter (Fig. 3C). The hepatic vagotomy group showed a sharp increase and the levels in this group were significantly higher than those in the sham vagotomy group at 5, 10, 15, and 30 min. Plasma insulin levels rose a little until 30 min and then slowly decreased in both groups, with no significant difference between the two groups at any time point. Plasma glucagon levels rose sharply in the hepatic vagotomy group and were significantly higher than those in the sham vagotomy group at 5, 10, 15, and 30 min.
Ip amino acid tests in VMH lesioned rats with marked hyperinsulinemia After ip injection of arginine, plasma glucose levels rose relatively sharply in VMH lesioned rats with sham or hepatic vagotomy, reached a peak within 5—15 min, and then slowly decreased (Fig. 4A). There was no significant difference between the two groups at any time point. Plasma insulin levels rose sharply in both groups (Fig. 4B), again with no significant difference between the two groups. Plasma glucagon levels rose relatively sharply in both groups, reached a peak within 5—15 min, and then decreased, with no significant difference between the two groups at any time point. After ip injection of leucine, plasma glucose levels rose relatively sharply in VMH lesioned rats with sham or hepatic vagotomy, reached a peak within 5—15 min, and then slowly decreased (Fig. 4B), with no significant difference between the groups at any time point. Plasma insulin levels also rose relatively sharply in both groups, reached a peak within
10 min, and then decreased, again with no significant difference between the two groups. Plasma glucagon levels did not differ significantly between the two groups at any time point. After ip injection of alanine, plasma glucose levels rose slowly in both groups, reached a peak within 30 min, and remained constant thereafter (Fig. 4C), with no significant difference between the two groups at any time point. Plasma insulin levels rose a little in both groups, with no significant differences between the groups. Plasma glucagon levels rose sharply, reached a peak within 10 min, in the both groups and decreased relatively sharply thereafter, with no significant difference between the two groups at any time point.
Discussion In normal rats, ip arginine administration enhanced insulin and glucagon secretion without causing a significant change in blood glucose levels, ip leucine enhanced insulin secretion in association with a significant decrease in blood glucose levels, and ip alanine enhanced glucagon secretion in association with a significant rise in blood glucose levels after hepatic vagotomy. These findings are consistent with previous results [13,15]. Arginine, leucine, and alanine strongly stimulate secretion of pancreatic hormones, insulin and glucagon [25,26]. After excessive ingestion of protein, many amino acids enter the circulation via the portal vein and reach the pancreas, which may result in excess insulin or glucagon secretion that disturbs blood glucose homeostasis through hypoglycemia or hyperglycemia. Thus, we hypothesize that amino acid sensors are present in the portal vein, and that when these sensors detect an excess of amino acids, they stimulate afferent vagal nerve fiber and inhibit efferent vagal nerve fiber via reflex through the brainstem. This, in turn, regulates inhibition of excess pancreatic hormone secretion by amino
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Figure 3 Changes of plasma glucose, insulin and glucagon levels in response to ip injections of arginine (A), leucine (B), and alanine (C) in normal rats after sham hepatic vagotomy or hepatic vagotomy. *P < 0.05, **P < 0.01 for hepatic vagotomy vs. sham hepatic vagotomy.
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acids themselves to maintain glucose homeostasis by keeping blood glucose within the normal range. In this context, we have also investigated the properties of other amino acids, such as glutamate, isoleucine and phenylalanine, and found that they do not induce an equivalent ‘‘amino acid sensor’’ function in the regulation of pancreatic hormones [12]. In VMH lesioned rats with marked hyperinsulinemia, administration of ip arginine, leucine, and alanine did not significantly change blood glucose levels or secretion of insulin and glucagon in rats with hepatic vagotomy compared to those with sham hepatic vagotomy. These results suggest that the function of amino acid sensors was masked in these rats. It is well known that insulin secretion is markedly increased in VMH lesioned animals [16,27,28], and we found that the increased insulin secretion is induced by autonomic neural derangements with elevated efferent vagus nerve activity in the previous study using celiac vagotomy [29] and reduced sympathetic nerve activity using administration of sympathetic antagonists in VMH lesioned rats [20]. Hepatic vagotomy did not affect insulin secretion in VMH lesioned rats because of the masked function of amino acid sensors with the marked hyperinsulinemia in these rats. Hepatic vagotomy in VMH lesioned rats did not significantly alter glucagon secretion induced by ip administration of any amino acids, which may be attributable to inhibition of glucagon secretion by excess insulin
secretion [30,31]. Overall, the function of amino acid sensors on pancreatic hormone secretion were masked in VMH lesioned rats with marked hyperinsulinemia. This is also consistent with the proposal that neural regulation plays only a small role in glucagon secretion [25,30,32]. In a previous study, we found that the function of glucoreceptors in the liver was disturbed, but that of an arginine sensor was retained, in carbon tetrachloride (CCl4 )induced cirrhotic rats after hepatic vagotomy [33]. In conclusion, in normal rats with hepatic vagotomy, ip arginine enhanced insulin and glucagon secretion without a change in blood glucose levels, ip leucine enhanced insulin secretion in association with a decrease in blood glucose levels, and ip alanine enhanced glucagon secretion in association with an increase in blood glucose levels. These phenomena can be explained by the presence of amino acid sensors in the hepatic portal vein that prevent direct induction of excess pancreatic hormone secretion following ingestion of excessive amino acids. In general practice, we should be careful to take too much meat, which provide excessive amino acids in the circulation and break the function of amino acid sensors, resulting in hypoglycemic attack. In VMH lesioned rats with hepatic vagotomy, insulin and glucagon secretion did not change significantly after administration of any amino acids. These results suggest that the functions of amino acid sensors are masked due to marked hyperinsulinemia in these rats.
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Conflict of interest None.
References [1] Ahren B. Autonomic regulation of islet hormone secretionimplications for health and disease. Diabetologia 2000;43:393—410. [2] Miller RE. Pancreatic neuroendocrinology: peripheral neural mechanisms in the regulation of the islets of Langerhans. Endocr Rev 1981;2:471—94. [3] Russek M. A hypothesis on the participation of hepatic glucoreceptors in the control of food intake. Nature 1963;197:1089—95. [4] Niijima A. Afferent impulse discharges from glucoreceptors in the liver of the guinea pig. Ann NY Acad Sci 1969;157:690—700. [5] Niijima A. Nervous regulatory mechanism of blood glucose levels. In: Katsuki Y, et al., editors. Food intake and chemical senses. Tokyo, Japan: Scientific Societies Press; 1977. p. 413—26. [6] Lee KC, Miller RE. The hepatic vagus nerve and the neural regulation of insulin secretion. Endocrinology 1985;117:307—14. [7] Nagase H, Inoue S, Tanaka K, Takamura Y, Niijima A. Hepatic glucose-sensitive unit regulation of glucoseinduced insulin secretion in rats. Physiol Behav 1993;53: 139—43. [8] Tanaka K, Inoue S, Fujii T, Takamura Y. Enhancement of insulin and glucagon secretion by arginine after hepatic vagotomy. Neurosci Lett 1986;72:74—8. [9] Tanaka K, Inoue S, Takamura Y, Jiang ZY, Niijima A. Arginine sensors in the hepato-portal system and their reflex effects on pancreatic efferents in the rat. Neurosci Lett 1986;72:69—73. [10] Tanaka K, Inoue S, Nagase H, Takamura Y. Modulation of arginine-induced insulin and glucagon secretion by the hepatic vagus nerve in the rat: effects of celiac vagotomy and administration of atropine. Endocrinology 1990;127:2017—23. [11] Tanaka K, Inoue S, Nagase H, Takamura Y, Niijima A. Amino acid sensors sensitive to alanine and leucine exist in the hepato-portal system in the rat. J Auton Nerv Syst 1990;31:41—6. [12] Tanaka K, Inoue S, Saito S, Nagase H, Takamura Y. Hepatic vagal amino acid sensors modulate amino acid induced insulin and glucagon secretion in the rat. J Auton Nerv Syst 1993;42:225—32. [13] Adachi A, Niijima A, Jacobs HL. An hepatic osmoreceptor mechanism in the electrophysiological and behavioral studies. Am J Physiol 1976;231:1043—9. [14] Magni F, Carobi C. The afferent and preganglionic parasympathetic innervation of the rat demonstrated by the retrograde transport of horseradish peroxidase. J Auton Nerv Syst 1983;8:237—60.
[15] Inoue S, Bray G. The effects of subdiaphragmatic vagotomy in rat with ventromedial hypothalamic obesity. Endocrinology 1977;100:108—14. [16] Inoue S, Bray GA, Mullen Y. Effect of transplantation of pancreas on development of hypothalamic obesity. Nature 1977;226:742—4. [17] Yoshimatsu H, Niijima A, Oomura Y, Yamabe K, Katafuchi T. Effects of hypothalamic lesion on pancreatic autonomic nerve activity in the rat. Brain Res 1984;303:147—52. [18] Bray GA, Nishizawa Y. Ventromedial hypothalamus modulates fat mobilisation during fasting. Nature 1978;274:900—2. [19] Inoue S, Bray GA. An autonomic hypothesis for hypothalamic obesity. Life Sci 1979;25:561—6. [20] Inoue S, Mullen Y, Bray GA. Hyperinsulinemia in rats with hypothalamic obesity: effects of autonomic drugs and glucose. Am J Physiol 1983;245:372—8. [21] Inoue S, Camplifield LA, Bray GA. Comparison of metabolic alterations in hypothalamic and high fat diet induced obesity. Am J Physiol 1977;233:162—8. [22] De Groot J. The rat forebrain in stereotaxic coordinates. Amsterdam: N.V. Noord-Hollandsch Uitgevers Maatschappij; 1959. [23] Shah J, Wongsurawat N, Aran P, Motto G, Bowser E. A method for studying acute insulin secretion and glucose tolerance in unanesthetized and unrestrained rats. Diabetes 1977;26:1—6. [24] Berthoud H, Laughton W, Powley T. A method for large volume blood sampling and transfusion in rats. Am J Physiol 1986;250:331—7. [25] Quesada I, Tudurí E, Ripoll C, Nadal A. Physiology of the pancreatic alpha-cell and glucagon secretion: role in glucose homeostasis and diabetes. J Endocrinol 2008;199:5—19. [26] Floyds Jr JC, Fajans S, Conn JW, Knopf RF, Rull J. Stimulation of insulin secretion by amino acids. J Clin Invest 1966;45:1487—502. [27] Bray GA, York DA. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol Rev 1979;59:719—809. [28] King BM. The rise, fall, and resurrection of the ventromedial hypothalamus in the regulation of feeding behavior and body weight. Physiol Behav 2006;87:221—44. [29] Inoue S, Nagase H, Satoh S, Egawa M, Tanaka K, Takamura Y. Role of the efferent and afferent vagus nerve in the development of ventromedial hypothalamic (VMH) obesity. Brain Res Bull 1991;27:511—5. [30] MacDonald PE, Zhang Y, Ramracheta R, Salehi A, Ma X, Johnson PR, et al. AKATP channel-dependent pathway within ␣ cells regulates glucagon release from both rodent and human islets of Langerhans. PLoS Biol 2007;5:1236—47. [31] Bansal P, Wang Q. Insulin as a physiological modulator of glucagon secretion. Am J Physiol Endocrinol Metab 2008;295:751—61. [32] Gromada J, Franklin I, Wollheim CB. ␣-Cells of the endocrine pancreas: 35 years of research but the enigma remains. Endocr Rev 2007;28:84—116. [33] Okazaki H, Tanaka K, Nagase H, Inoue S. Modulation of insulin secretion by hepatic vagotomy in cirrhotic rats. Physiol Behav 1993;53:521—5.
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