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Central thyrotropin-releasing hormone stimulates hepatic DNA synthesis in rats
Central Thyrotropin-Releasing Hormone Stimulates Hepatic DNA Synthesis in Rats MASASHI YONEDA, KEISUKE TAMORI, YOICHI SATO, SHIRO YOKOHAMA, KIMIHIDE NAKAMURA, TORU KONO,
Central neuropeptides play a role as physiological regulators in the autonomic nervous system. One of these neuropeptides, thyrotropin-releasing hormone (TRH), is distributed throughout the central nervous system (CNS) and acts as a neurotransmitter to regulate gastric functions through the vagus nerve. However, the autonomic nervous system is also involved in hepatic regeneration, but the effect of TRH is unknown. Therefore, the CNS’s effect of TRH on hepatic DNA synthesis was studied in rats. Hepatic DNA synthesis was assessed by [Methyl-3H]thymidine incorporation 6, 12, 24, 48, and 72 hours after intracisternal injection of the TRH analog, RX 77368 (1, 5, 10, and 100 ng), and by 5-bromo-2*deoxyuridine (BrdU) labeling of the liver section. Hepatic DNA synthesis was stimulated by intracisternal TRH analog (10 ng), with a peak response at 24 hours after peptide injection, and returned to baseline by 72 hours. This stimulatory effect by central TRH analog on hepatic DNA synthesis was dose-related, ranging from 1 ng to 10 ng (dpm/mg DNA at 24 hours [mean { SE]: saline, 95 { 6; 1 ng, 114 { 14; 5 ng, 318 { 57; 10 ng, 693 { 78; 100 ng, 710 { 135). Hepatocytes were randomly labeled by BrdU 24 hours after intracisternal TRH analog (10 ng). Intravenous TRH analog (10 ng) did not influence hepatic DNA synthesis. The stimulatory effect of TRH analog was blocked by hepatic branch vagotomy and atropine, but not by hepatic sympathectomy, 6-hydroxydopamine, insulin antibody, or hypophysectomy. These results indicate that TRH acts in the CNS to stimulate hepatic DNA synthesis through vagal and cholinergic mechanisms, and that TRH may be the chemical messenger involved in brain regulation of hepatic proliferation. (HEPATOLOGY 1997;26:12031208.) Although abundant anatomical, physiological, and pharmacological evidence suggests that the autonomic nervous system plays an important role in regulating hepatic functions,1,2 the transmitters in the central nervous system (CNS) mediating these effects are not well characterized. In addition to the classical neurotransmitters, such as acetylcholine and
Abbreviations: CNS, central nervous system; TRH, thyrotropin-releasing hormone; VMH, ventromedial hypothalamus; BrdU, 5-bromo-2*-deoxyuridine. From the Second Department of Medicine and Second Department of Surgery, Asahikawa Medical College, Asahikawa, Japan. Received February 20, 1997; accepted July 11, 1997. Supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan (0767554 and 09670503). Address reprint requests to: Masashi Yoneda, M.D., Second Department of Medicine, Asahikawa Medical College, Nishikagura 4-5-3, Asahikawa 078, Japan. Fax: 81166-65-1182. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2605-0019$3.00/0
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noradrenaline, peptides have also been identified as possible neurotransmitters in the central and peripheral nervous system.3 These neuropeptides have been shown to act centrally to regulate many physiological functions, including behavior, glucose metabolism, body temperature, and cardiovascular and gastrointestinal functions.4-7 In particular, thyrotropinreleasing hormone (TRH) in the medulla plays a physiological role in the vagal regulation of the digestive system.6,8-10 The liver is richly innervated,11 and the autonomic nervous system has an important role in the process of hepatic proliferation after partial hepatectomy and experimental liver necrosis.12-14 Recent experiments have shown that vagal activation by lesioning of the ventromedial hypothalamus (VMH) stimulates hepatic proliferation in rats, and that cholinergic stimulation results in an increase of hepatic DNA synthesis after carbon tetrachloride–induced hepatic damage.15,16 These studies have prompted us to examine a possible role for TRH as a centrally acting chemical messenger involved in the CNS regulation of hepatic proliferation. We report here that TRH acts in the CNS to elicit a vagal-dependent stimulation of hepatic DNA synthesis as a prerequisite to hepatic proliferation. MATERIALS AND METHODS Animals. Male Wistar rats weighing 200 to 220 g (Charles River Japan Inc., Yokohama, Japan) were used. Rats were housed in group cages under controlled conditions of temperature and illumination, and fed ad libitum for at least 7 days before experiments. Protocols describing the use of rats were approved by the Animal Care Committee of Asahikawa Medical College, and in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. All experiments were performed according to the same time schedule, starting at 8 AM in 24-hour food-deprived rats with free access to water up to the beginning of the study. Chemicals. The following substances were used: the stable TRH analog, RX 77368; p-Glu-His-(3,3*-dimethyl)-Pro-NH 2 (Reckitt and Colman, Kingdom-upon-Hill, England); atropine methyl nitrate (Sigma Chemical, St. Louis, MO); 5-bromo-2*-deoxyuridine (BrdU) (Sigma); 6-hydroxydopamine (Sigma); and anti-insulin antibody (IgG class, Linco Research, Inc., St. Louis, MO). RX 77368 has similar binding characteristics as natural TRH on rat brain membranes but is more stable compared with natural TRH.17,18 RX 77368 was aliquoted in 0.5% bovine serum albumin and 0.9% saline (pH 7.0) at a concentration of 1.5 nmol/mL and kept frozen at 0207C. The stock solution was diluted in 0.9% saline before the experiment and injected intracisternally in 10-mL volume. Other chemicals were freshly dissolved in 0.9% saline before use and injected intravenously in 0.2-mL or intraperitoneally in 0.4-mL volume. Effect of Intracisternal Injection of TRH Analog on Hepatic DNA Content and Synthesis. Under light ether anesthesia, rats were mounted on
ear bars of a stereotaxic apparatus (Kopf model 900, David Kopf Instruments, Tujunga, CA), and RX 77368 (1, 5, 10, and 100 ng) or saline vehicle was injected intracisternally. The doses of TRH
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analog were selected based on previous studies that indicated the central effects of RX 77368 on gastric functions.7,10,19 Rats regained light reflex within 7 minutes and returned to their home cages. The accuracy of the intracisternal injection was ascertained by the aspiration of cerebrospinal fluid before and after injection of the peptide. Hepatic DNA synthesis was assessed by [methyl-3H]thymidine incorporation. Rats were intraperitoneally injected with [methyl-3H]thymidine (20 mCi/100 g) (Amersham, Tokyo, Japan) 6, 12, 24, 48, or 72 hours after the peptide injection, and then were killed 2 hours later. The right lateral lobe of the liver was removed and then homogenized in 10% ice-cold trichloroacetic acid with a Polytron homogenizer. DNA was extracted with hot 5% trichloroacetic acid according to Schneider’s method.20 DNA content was determined by Burton’s method,21 and radioactivity of the solubilized DNA was measured in a liquid scintillation counter. In another experiment, RX 77368 (10 ng) or saline vehicle was injected through the jugular vein under identical conditions. To exclude the possibility that food intake was affected by either intracisternal or intravenous peptide injection, rats were pair-fed with respective vehicle-treated rats after the peptide injection. Effect of Intracisternal TRH Analog on Hepatic BrdU Labeling. Twentyfour hours after intracisternal injection of TRH analog (10 ng), BrdU (50 mg/kg) was injected intraperitoneally, and rats were killed 1 hour later. The liver was removed, fixed in cold 70% ethanol for 12 hours, embedded in paraffin, and then cut in 6-mm sections. After deparaffinization of liver sections, endogenous peroxidase was inactivated in 0.3% H2O2 in absolute methanol. The sections were washed in 0.5% Tween 20 in phosphate-buffered saline, and DNA was denatured in 2 N HCl for 1 hour. After neutralization in 0.1 mol/L borate buffer (pH 8.5), the liver sections were incubated with mouse monoclonal antibody to BrdU (Becton Dickinson, San Jose, CA) overnight at 47C. After treating with normal horse serum and washing with phosphate-buffered saline, the sections were stained by the avidin-biotin-peroxidase method using a commercially available kit (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA). The labeling index of hepatocytes was determined by counting more than 2,000 nuclei of three randomly selected fields under a light microscope. Effect of Atropine, 6-Hydroxydopamine, Hepatic Vagotomy, Hepatic Sympathectomy, Hypophysectomy, and Anti-Insulin Antibody on Intracisternal TRH Analog-Induced Modulation of Hepatic DNA Synthesis. Atropine
methyl nitrate (0.15 mg/kg) dissolved in saline was injected intraperitoneally 60 minutes before the peptide injection. 6-Hydroxydopamine dissolved in saline was intraperitoneally injected twice (100 mg/kg on the first day, 80 mg/kg on the fourth day), and intracisternal injection of TRH analog was performed on the seventh day.22 Either hepatic branch vagotomy or hepatic sympathectomy, or the respective sham operation, was performed 72 hours before the peptide injection. Hepatic branch vagotomy was achieved under a dissection microscope by selective section of the hepatic branch of the vagus nerve branching off from the left main vagal trunk a few millimeters proximal to the cardia.14 Hepatic sympathectomy was performed with the use of a dissection microscope by resecting the hepatic branch of the splanchnic nerves around the proper hepatic artery.23 Hypophysectomy was performed transaurally by Japan SLC, Inc. (Hamamatsu, Japan) 7 days before the intracisternal injection of TRH analog. The hypophysectomized rats were fed with standard laboratory chow supplemented with 0.5% glucose solution. Only animals showing no weight gain during a 1-week stabilization period were used. The completeness of the removal of the gland was checked by testis size and visual inspection at autopsy. Anti-insulin antibody was injected 30 minutes before intracisternal TRH analog injection and once a day at 8 AM through the tail vein. The amount of anti-insulin antibody used in this study had the ability to block the effect of insulin action with a plasma insulin level up to at least 120 ng/mL.16,24,25 To exclude the effect of atropine, 6hydroxydopamine, hepatic vagotomy, hepatic sympathectomy, hypophysectomy, and insulin antibody on food intake, rats were pairfed with respective vehicle-treated or sham-operated rats.
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FIG. 1. Effect of intracisternal injection of TRH analog, RX 77368 (10 ng), on hepatic DNA synthesis. Hepatic DNA was assessed by [methyl3 H]thymidine uptake. Rats fasted for 24 hours were injected intracisternally with saline or TRH analog (10 ng). [Methyl-3H]thymidine (20 mCi/100 g) was injected intraperitoneally 6, 12, 24, 48, or 72 hours after peptide injection; rats were killed 2 hours later, and the liver was removed for determination of [methyl-3H]thymidine incorporation into hepatic DNA. Each point represents the mean { SE. **P õ .01 as compared with the saline-treated group decapitated at the same time postinjection.
Statistical Analysis. Data were expressed as means { SE. Comparison between two groups was performed by the Student’s t test, and multiple comparison was calculated by ANOVA, followed by Duncan’s contrast. P õ .05 was considered statistically significant.
RESULTS Effect of Intracisternal TRH Analog on Hepatic DNA Synthesis and Content, and Liver Weight. Intracisternal injection of saline
vehicle did not influence hepatic DNA synthesis as assessed by [Methyl-3H]thymidine incorporation into hepatic DNA in rats (Fig. 1). Intracisternal injection of RX 77368 at 10 ng caused an increase in [Methyl-3H]thymidine incorporation into hepatic DNA (Fig. 1). The response reached a maximum within 24 hours of peptide injection, and thereafter returned to the basal level by 72 hours (dpm/mg DNA: 0 hours, 92 { 6; 6 hours, 131 { 11; 12 hours, 195 { 42; 24 hours, 693 { 78; 48 hours, 378 { 81; 72 hours, 120 { 27). A significant dose-related stimulatory effect was observed following intracisternal injection of RX 77368 in doses ranging from 1 to 10 ng, and reached a plateau at 10 ng as measured 24 hours after peptide injection (dpm/mg DNA at 24 hours: saline, 95 { 6; 1 ng, 114 { 14; 5 ng, 318 { 57; 10 ng, 693 { 78; 100 ng, 710 { 135) (Fig. 2). Intracisternal injection of RX 77368 (10 ng) increased hepatic DNA content at 24 hours after the peptide injection, and this stimulatory effect was maintained over the 72-hour observation period after the peptide (Fig. 3). In contrast, although intracisternal RX 77368 (10 ng) slightly increased the liver weight at 48 and 72 hours after the peptide, this change did not reach a statistically significant level (Fig. 3). RX 77368 injected intravenously at 10 ng did not alter hepatic DNA synthesis (Table 1). Effect of Intracisternal TRH Analog on Hepatic BrdU Labeling. Although the labeled hepatocytes undergoing DNA syn-
thesis in intracisternal saline-injected rats was 0.25 { 0.32% (n Å 5), that in intracisternal RX 77368 (10 ng)-injected rats was significantly elevated 24 hours after the peptide (8.9 { 2.6%; n Å 5; P õ .01) (Fig. 4A and 4B). The labeled hepato-
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FIG. 2. Dose-dependent stimulatory effect of intracisternal injection of RX 77368 on hepatic DNA synthesis. Hepatic DNA synthesis was assessed by [Methyl-3H]thymidine uptake. Rats fasted for 24 hours were injected intracisternally with saline or TRH analog (1-100 ng). [Methyl-3H]thymidine (20 mCi/100 g) was injected intraperitoneally 24 hours after peptide injection; rats were decapitated 2 hours later, and the liver was removed for determination of [Methyl-3H]thymidine incorporation into hepatic DNA. Each column represents the mean { SE. *P õ .05; **P õ .01 as compared with the saline-trated group.
cytes were located randomly in the lobules in intracisternal RX 77368–injected rats (Fig. 4B). Effect of Atropine, 6-Hydroxydopamine, Hepatic Vagotomy, Hepatic Sympathectomy, Hypophysectomy, and Anti-Insulin Antibody on the Intracisternal TRH Analog-Induced Stimulation of Hepatic DNA Synthesis. Hepatic branch vagotomy performed 72 hours
before, or atropine methyl nitrate (0.15 mg/kg) injected intraperitoneally 60 minutes before, the peptide injection completely abolished intracisternal TRH analog (10 ng)-induced stimulation of hepatic DNA synthesis (Figs. 5 and 6). Hepatic sympathectomy 72 hours before and 6-hydroxydopamine treatment 7 days before the peptide injection did not modify the stimulation of hepatic DNA synthesis induced by intracisternal injection of TRH analog (10 ng) (Table 2). Intracisternal TRH analog (10 ng) stimulated hepatic DNA synthesis even in hypophysectomized rats and anti-insulin–treated rats (Table 2). DISCUSSION
In the present study, we demonstrate that the TRH analog, RX 77368 (1-100 ng), injected intracisternally stimulates hepatic DNA synthesis in conscious rats as assessed by [Methyl3 H]thymidine incorporation. Hepatic BrdU labeling reveals that DNA synthesis in hepatocytes is stimulated by central TRH analog. The stimulatory effect of intracisternal injection of TRH analog is dose-related in doses ranging from 1 to 10 ng. Intracisternal TRH analog (RX 77368, 10 ng) injection significantly increases hepatic DNA synthesis, with a peak response at 24 hours, and thereafter the enhanced hepatic DNA synthesis gradually returns to the baseline by 72 hours. The dose of 10 ng induces 693 { 78 dpm/mg DNA of hepatic DNA synthesis, which represents the maximal response because 100 ng does not further stimulate hepatic DNA synthesis (710 { 135 dpm/mg DNA). In contrast, TRH analog in-
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FIG. 3. Effect of intracisternal injection of RX 77368 (10 ng) on hepatic DNA content and hepatic weight. Hepatic DNA was extracted by Schneider’s method, and the DNA content was determined by Burton’s method. Rats were killed 6, 12, 24, 36, 48, and 72 hours after intracisternal injection of TRH analog. Each point represents the mean { SE. **P õ .01 as compared with the saline-treated group decapitated at the same time postinjection.
jected intravenously at the maximal effective dose, given intracisternally, does not influence hepatic DNA synthesis under identical conditions. These results indicate that TRH analog, injected into the cisternal magna, acts in the CNS to stimulate hepatic DNA synthesis and not through leakage into the peripheral circulation. The pathways through which central TRH analog stimulates hepatic DNA synthesis were investigated. The neurohumoral mechanisms through which intracisternal injection of TRH analog stimulates hepatic DNA synthesis are unrelated to its regulatory action on the pituitary-thyroidal axis,26 because intracisternal injection of TRH analog-induced [Methyl-3H]thymidine uptake persists in hypophysectomized rats, and intravenous TRH analog did not stimulate hepatic DNA synthesis. TRH has already been shown to have various CNS-mediated actions unrelated to its neuro-endocrine activity, including modification of behavior27; alteration
TABLE 1. Effect of Intravenous Injection of RX 77368 on Hepatic DNA Synthesis in Rats 3
Treatment
H-Thymidine Incorporation Into Hepatic DNA 24 Hours After TRH Analog Injection (dpm/mg DNA)
Saline (intravenously) (n Å 6) RX 77368 (10 ng intravenously) (n Å 7)
89 { 12 92 { 10
NOTE. Rats were injected intravenously (i.v.) with RX 77368 (10 ng) through the jugular vein under light ether anesthesia; 24 hours later, the animals were decapitated, and the liver was removed for determination of thymidine incorporation into hepatic DNA.
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FIG. 4. Distribution of cells undergoing DNA synthesis in the livers of rats 24 hours after intracisternal injection of RX 77368 (10 ng) or saline vehicle. (A) Rats intracisternally injected with saline. (B) Rats intracisternally injected with TRH analog. DNA synthesis was detected by BrdU uptake and following immunochemical staining using monoclonal antibody to BrdU. (Original magnification 140.)
of thermoregulation28; blood pressure5; food intake29; and cardiac,5 respiratory,30 and gastric7,31,32 functions. These central actions of TRH are mediated through the sympathetic or parasympathetic nervous systems.31-33 Central TRH analoginduced stimulation of hepatic DNA synthesis was reversed by hepatic vagotomy and atropine, an anticholinergic compound, but not by hepatic sympathectomy or 6-hydroxydopamine, which chemically depletes noradrenergic nerve fibers via biosynthetic adrenergic intermediates.22 These findings suggest that parasympathetic and cholinergic pathways participate in the proliferative growth response in the liver to centrally active TRH. These results are consistent with related findings indicating a central action of TRH on gastric functions including secretion, motility, mucosal blood flow, and resistance to mucosal injury.9,31,32 Our present results are also supported by a recent study that indicates that vagal hyperactivity following VMH lesioning stimulates hepatic DNA synthesis through vagal and cholinergic pathways.16,34 Moreover, cell proliferation is regulated by cholin-
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ergic factors in rat cornea,35 and cholinergic stimulation results in an increase in hepatic DNA synthesis after carbon tetrachloride–induced hepatic damage.15 The involvement of insulin in central TRH analog-induced hepatic DNA synthesis was investigated in the present study. Insulin is known to stimulate hepatic proliferation,36 and central administration of TRH elevates plasma insulin levels.37 However, administration of anti-insulin antibody did not affect the hepatic DNA synthesis induced by intracisternal injection of TRH analog, suggesting that the stimulation of hepatic DNA synthesis by intracisternal TRH analog is not secondary to the increase in plasma insulin induced by central TRH. Centrally injected TRH induces parasympathetic nerve activation38 by acting in the medullary nuclei, including the dorsal vagal complex and nucleus ambiguous,39,40 which are important sites for parasympathetic outflow.41 Although microinjections of TRH analog into specific brain medullary nuclei are not possible in conscious rats, the dorsal vagal
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TABLE 2. Effect of Hepatic Sympathectomy, 6-Hydroxydopamine, Hypophysectomy, and Anti-Insulin Antibody on Intracisternal TRH Analog–Induced Stimulation of Hepatic DNA Synthesis in Rats 3
Treatment
FIG. 5. Effect of hepatic branch vagotomy on intracisternal injection of TRH analog–induced stimulation of hepatic DNA synthesis. Hepatic branch vagotomy was performed 72 hours before the intracisternal injection of RX 77368 (10 ng). For details, see legends of Figs. 1 and 2. **P õ .01 compared with respective sham-operation group.
complex, including the vagal motor nucleus and the nucleus of the solitary tract, are probable sites of TRH-induced hepatic DNA synthesis. TRH-immunoreactive nerve terminals and receptors are localized in the dorsal vagal complex,42,43 and various central TRH-induced gastric functions are reproduced by microinjection into the dorsal vagal complex.39,40 In addition, vagotomy blocks hepatic [Methyl-3H]thymidine uptake induced by intracisternal injection of TRH analog in this study. These facts support that the dorsal vagal complex may be the specific site for TRH action on hepatic DNA synthesis. Kiba et al. reported that vagal hyperactivity caused by a lesion of the VMH induces cell proliferation not only in the liver, but also in the intestine and the pancreas.16,24,25 It is
FIG. 6. Effect of atropine treatment on intracisternal injection of TRH analog–induced stimulation of hepatic DNA synthesis. Atropine methyl nitrate (0.15 mg/kg) was injected intraperitoneally 60 minutes before the intracisternal injection of RX 77368 (10 ng). For details, see legends of Figs. 1 and 2. **P õ .01 compared with respective sham-operation group.
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No pretreatment / intracisternal saline (n Å 5) Sham hepatic sympathectomy / intracisternal TRH analog (n Å 6) Hepatic sympathectomy / intracisternal TRH analog (n Å 7) Intraperitoneal saline / intracisternal TRH analog (n Å 7) Intraperitoneal 6-hydroxydopamine / intracisternal TRH analog (n Å 6) Sham hypophysectomy / intracisternal TRH analog (n Å 5) Hypophysectomy / intracisternal TRH analog (n Å 6) Intravenous saline / intracisternal TRH analog (n Å 5) Intravenous anti-insulin antibody / intracisternal TRH analog (n Å 5)
H-Thymidine Incorporation Into Hepatic DNA 24 Hours After TRH Analog Injection (dpm/mg DNA)
95 { 6 724 { 68 694 { 82 695 { 125 723 { 105 688 { 95 678 { 101 712 { 98 689 { 112
NOTE. Rats were injected intracisternally with RX 77368 (10 ng), and, 24 hours later, the animals were decapitated, and the liver was removed for determination of thymidine incorporation into hepatic DNA.
interesting to know whether central TRH administration also increases cell proliferation in these organs besides the liver. In the study of Kiba et al., hepatic DNA synthesis reached a maximum 3 days after the VMH lesion, and hepatic DNA content and hepatic weight kept increasing for 7 days. However, hepatic DNA synthesis peaked on the second day after intracisternal TRH analog injection, and the increase in hepatic DNA content and hepatic weight occurred 2 days after central TRH administration. These discrepancies may be explained by the difference in the duration of vagal stimulation by these two procedures. Although central TRH injection stimulates vagal activity for only a few hours,38 the VMH lesion may induce an enduring stimulation of the vagus nerve. The liver is known to be richly innervated,11 and there is abundant evidence indicating important roles for the central and autonomic nervous systems in hepatic function, including hepatic proliferation.12-14 Very little is revealed about central neuropeptides as neurotransmitters inducing modulation of hepatic function. To date, only an effect of administration of central neuropeptide Y with bile secretion has been reported.44-46 After partial hepatectomy, the liver remnant spontaneously regenerates to restore hepatic volume to normal.36,47 In this phenomenon, the autonomic nervous system plays an important role.12-14,48 However, the identity of the centrally acting neurotransmitters and neuromediators are unknown. In our study, the centrally administered TRH analog, RX 77368, stimulates hepatic DNA synthesis mediated through a vagal-dependent cholinergic pathway. Because the dorsal vagal complex is richly innervated by TRH-immunoreactive fibers and receptors,42,43 and because central TRH activates vagal efferent fibers,38 we suggest that TRH is a likely candidate for a centrally acting chemical messenger involved in CNS regulation of hepatic proliferation. It is also of interest
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to study the significance of endogenous brain TRH on hepatic proliferation after partial hepatectomy and experimental acute liver injury by using an immunoneutralizing TRH monoclonal antibody.
23. 24.
Acknowledgment: The authors thank Dr. Kent C. Lloyd, D.V.M., Ph.D. (University of California, Davis), for editorial assistance, and Dr. Yvette Tache´, Ph.D. (UCLA), for valuable discussion.
25. 26.
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27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.
46. 47. 48.
tion of adrenergic nerve endings with 6-hydroxydopamine. Arch Exp Pathol Pharmacol 1968;261:271-288. Ashrif S, Gillespie JS, Pollock D. The effect of drugs and denervation on thymidine uptake into rat regenerating liver. Eur J Pharmacol 1974; 29:324-327. Kiba T, Tanaka K, Endo O, Inoue S. Ventromedial hypothalamic lesions increase gastrointestinal DNA synthesis through vagus nerve in rats. Gastroenterology 1993;104:475-484. Kiba T, Tanaka K, Numata K, Hoshino M, Misugi K, Inoue S. Ventromedial hypothalamic lesion-induced vagal hyperactivity stimulates rat pancreatic cell proliferation. Gastroenterology 1996;110:885-893. Porter JC, Vale W, Burgus R, Mical RS, Guillemin R. Release of TSH by TRF infused directly into a pituitary stalk portal vessel. Endocrinology 1971;89:1054-1056. Wei E, Sigel S, Loh H, Way EL. Thyrotrophin-releasing hormone and shaking behaviour in rat. Nature 1975;253:739-740. Brown M, Rivier J, Vale W. Actions of bombesin, thyrotropin releasing factor, prostaglandin E2 and naloxone on thermoregulation in the rat. Life Sci 1977;20:1681-1687. Vijayan E, McCann SM. Suppression of feeding and drinking activity in rats following intraventricular injection of thyrotropin releasing hormone (TRH). Endocrinology 1977;100:1727-1730. McCown TJ, Hedner JA, Towle AC, Breese GR, Mueller RA. Brainstem localization of a thyrotropin-releasing hormone-induced change in respiratory function. Brain Res 1986;373:189-196. Tache Y, Vale W, Brown M. Thyrotropin-releasing hormone-CNS action to stimulate gastric acid secretion. Nature 1980;287:149-151. Yoneda M, Tache´ Y. Vagal regulation of gastric prostaglandin E2 release by central TRH in rats. Am J Physiol 1993;264:G231-G236. Mattila J, Bunag R. Sympathomimetic pressor responses to thyrotropinreleasing hormone in rats. Am J Physiol 1986;251:H86-H92. Kiba T, Tanaka K, Inoue S, Endo O, Takamura Y. Comparison of DNA contents of visceral organs in rats with ventromedial hypothalamic lesions and fed a high fat diet. Neurosci Lett 1991;126:127-130. Cavanagh HD, Colley AM. The molecular basis of neutrophic keratitis. Acta Ophthalmol 1989;67(Suppl 192):115-134. Leffert HL, Koch KS, Moran T, Rubalcava B. Hormonal control of rat liver regeneration. Gastroenterology 1979;76:1470-1482. Brown M. Thyrotropin releasing factor: a putative CNS regulator of the autonomic nervous system. Life Sci 1981;28:1789-1795. Somiya H, Tonoue T. Neuropeptides as central integrators of autonomic nerve activity: effect of TRH, SRIF, VIP and bombesin on gastric and adrenal nerves. Regul Peptides 1984;9:47-52. Ishikawa T, Yang H, Tache Y. Medullary sites of action of the TRH analogue, RX 77368, for stimulation of gastric acid secretion in the rat. Gastroenterology 1988;95:1470-1476. Stephens RL, Ishikawa T, Weiner H, Novin D, Tache´ Y. TRH analogue, RX 77368, injected into dorsal vagal complex stimulates gastric secretion in rats. Am J Physiol 1988;254:G639-G643. Leslie RA, Gwyn DG, Hopkins DA. The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Brain Res Bull 1982;8:37-43. Manaker S, Rizio G. Autoradiographic localization of thyrotropin-releasing hormone and substance P receptors in the rat dorsal vagal complex. J Comp Neurol 1989;290:516-526. Rinaman L, Miselis RR, Kreider MS. Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat. Neurosci Lett 1989;104:7-12. Farouk M, Geoghegan JG, Pruthi RS, Thomson HJ, Pappas TN, Meyers WC. Intracerebroventricular neuropeptide Y stimulates bile secretion via a vagal mechanism. Gut 1992;33:1562-1565. Yoneda M, Tamasawa N, Takebe K, Tamori K, Yokohama S, Sato Y, Nakamura K, et al. Central neuropeptide Y enhances bile secretion through vagal and muscarinic but not nitric oxide pathways in rats. Peptides 1995;16:727-732. Yoneda M, Yokohama S, Tamori K, Sato Y, Nakamura K, Makino I. Neuropeptide Y in the dorsal vagal complex stimulates bicarbonatedependent bile secretion in rats. Gastroenterology 1997;112:1673-1680. Michalopoulos GK. Control mechanisms of liver regeneration. J Gastroenterol 1994;29:23-29. Kato H, Shimazu T. Effect of autonomic denervation on DNA synthesis during liver regeneration after partial hepatectomy. Eur J Biochem 1983; 190:473-478.
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Central neuropeptides play a role as physiological regulators in the autonomic nervous system. One of these neuropeptides, thyrotropin-releasing hormone (TRH), is distributed throughout the central nervous system (CNS) and acts as a neurotransmitter to regulate gastric functions through the vagus nerve. However, the autonomic nervous system is also involved in hepatic regeneration, but the effect of TRH is unknown. Therefore, the CNS’s effect of TRH on hepatic DNA synthesis was studied in rats. Hepatic DNA synthesis was assessed by [Methyl-3H]thymidine incorporation 6, 12, 24, 48, and 72 hours after intracisternal injection of the TRH analog, RX 77368 (1, 5, 10, and 100 ng), and by 5-bromo-2*deoxyuridine (BrdU) labeling of the liver section. Hepatic DNA synthesis was stimulated by intracisternal TRH analog (10 ng), with a peak response at 24 hours after peptide injection, and returned to baseline by 72 hours. This stimulatory effect by central TRH analog on hepatic DNA synthesis was dose-related, ranging from 1 ng to 10 ng (dpm/mg DNA at 24 hours [mean { SE]: saline, 95 { 6; 1 ng, 114 { 14; 5 ng, 318 { 57; 10 ng, 693 { 78; 100 ng, 710 { 135). Hepatocytes were randomly labeled by BrdU 24 hours after intracisternal TRH analog (10 ng). Intravenous TRH analog (10 ng) did not influence hepatic DNA synthesis. The stimulatory effect of TRH analog was blocked by hepatic branch vagotomy and atropine, but not by hepatic sympathectomy, 6-hydroxydopamine, insulin antibody, or hypophysectomy. These results indicate that TRH acts in the CNS to stimulate hepatic DNA synthesis through vagal and cholinergic mechanisms, and that TRH may be the chemical messenger involved in brain regulation of hepatic proliferation. (HEPATOLOGY 1997;26:12031208.) Although abundant anatomical, physiological, and pharmacological evidence suggests that the autonomic nervous system plays an important role in regulating hepatic functions,1,2 the transmitters in the central nervous system (CNS) mediating these effects are not well characterized. In addition to the classical neurotransmitters, such as acetylcholine and
Abbreviations: CNS, central nervous system; TRH, thyrotropin-releasing hormone; VMH, ventromedial hypothalamus; BrdU, 5-bromo-2*-deoxyuridine. From the Second Department of Medicine and Second Department of Surgery, Asahikawa Medical College, Asahikawa, Japan. Received February 20, 1997; accepted July 11, 1997. Supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan (0767554 and 09670503). Address reprint requests to: Masashi Yoneda, M.D., Second Department of Medicine, Asahikawa Medical College, Nishikagura 4-5-3, Asahikawa 078, Japan. Fax: 81166-65-1182. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2605-0019$3.00/0
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noradrenaline, peptides have also been identified as possible neurotransmitters in the central and peripheral nervous system.3 These neuropeptides have been shown to act centrally to regulate many physiological functions, including behavior, glucose metabolism, body temperature, and cardiovascular and gastrointestinal functions.4-7 In particular, thyrotropinreleasing hormone (TRH) in the medulla plays a physiological role in the vagal regulation of the digestive system.6,8-10 The liver is richly innervated,11 and the autonomic nervous system has an important role in the process of hepatic proliferation after partial hepatectomy and experimental liver necrosis.12-14 Recent experiments have shown that vagal activation by lesioning of the ventromedial hypothalamus (VMH) stimulates hepatic proliferation in rats, and that cholinergic stimulation results in an increase of hepatic DNA synthesis after carbon tetrachloride–induced hepatic damage.15,16 These studies have prompted us to examine a possible role for TRH as a centrally acting chemical messenger involved in the CNS regulation of hepatic proliferation. We report here that TRH acts in the CNS to elicit a vagal-dependent stimulation of hepatic DNA synthesis as a prerequisite to hepatic proliferation. MATERIALS AND METHODS Animals. Male Wistar rats weighing 200 to 220 g (Charles River Japan Inc., Yokohama, Japan) were used. Rats were housed in group cages under controlled conditions of temperature and illumination, and fed ad libitum for at least 7 days before experiments. Protocols describing the use of rats were approved by the Animal Care Committee of Asahikawa Medical College, and in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. All experiments were performed according to the same time schedule, starting at 8 AM in 24-hour food-deprived rats with free access to water up to the beginning of the study. Chemicals. The following substances were used: the stable TRH analog, RX 77368; p-Glu-His-(3,3*-dimethyl)-Pro-NH 2 (Reckitt and Colman, Kingdom-upon-Hill, England); atropine methyl nitrate (Sigma Chemical, St. Louis, MO); 5-bromo-2*-deoxyuridine (BrdU) (Sigma); 6-hydroxydopamine (Sigma); and anti-insulin antibody (IgG class, Linco Research, Inc., St. Louis, MO). RX 77368 has similar binding characteristics as natural TRH on rat brain membranes but is more stable compared with natural TRH.17,18 RX 77368 was aliquoted in 0.5% bovine serum albumin and 0.9% saline (pH 7.0) at a concentration of 1.5 nmol/mL and kept frozen at 0207C. The stock solution was diluted in 0.9% saline before the experiment and injected intracisternally in 10-mL volume. Other chemicals were freshly dissolved in 0.9% saline before use and injected intravenously in 0.2-mL or intraperitoneally in 0.4-mL volume. Effect of Intracisternal Injection of TRH Analog on Hepatic DNA Content and Synthesis. Under light ether anesthesia, rats were mounted on
ear bars of a stereotaxic apparatus (Kopf model 900, David Kopf Instruments, Tujunga, CA), and RX 77368 (1, 5, 10, and 100 ng) or saline vehicle was injected intracisternally. The doses of TRH
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analog were selected based on previous studies that indicated the central effects of RX 77368 on gastric functions.7,10,19 Rats regained light reflex within 7 minutes and returned to their home cages. The accuracy of the intracisternal injection was ascertained by the aspiration of cerebrospinal fluid before and after injection of the peptide. Hepatic DNA synthesis was assessed by [methyl-3H]thymidine incorporation. Rats were intraperitoneally injected with [methyl-3H]thymidine (20 mCi/100 g) (Amersham, Tokyo, Japan) 6, 12, 24, 48, or 72 hours after the peptide injection, and then were killed 2 hours later. The right lateral lobe of the liver was removed and then homogenized in 10% ice-cold trichloroacetic acid with a Polytron homogenizer. DNA was extracted with hot 5% trichloroacetic acid according to Schneider’s method.20 DNA content was determined by Burton’s method,21 and radioactivity of the solubilized DNA was measured in a liquid scintillation counter. In another experiment, RX 77368 (10 ng) or saline vehicle was injected through the jugular vein under identical conditions. To exclude the possibility that food intake was affected by either intracisternal or intravenous peptide injection, rats were pair-fed with respective vehicle-treated rats after the peptide injection. Effect of Intracisternal TRH Analog on Hepatic BrdU Labeling. Twentyfour hours after intracisternal injection of TRH analog (10 ng), BrdU (50 mg/kg) was injected intraperitoneally, and rats were killed 1 hour later. The liver was removed, fixed in cold 70% ethanol for 12 hours, embedded in paraffin, and then cut in 6-mm sections. After deparaffinization of liver sections, endogenous peroxidase was inactivated in 0.3% H2O2 in absolute methanol. The sections were washed in 0.5% Tween 20 in phosphate-buffered saline, and DNA was denatured in 2 N HCl for 1 hour. After neutralization in 0.1 mol/L borate buffer (pH 8.5), the liver sections were incubated with mouse monoclonal antibody to BrdU (Becton Dickinson, San Jose, CA) overnight at 47C. After treating with normal horse serum and washing with phosphate-buffered saline, the sections were stained by the avidin-biotin-peroxidase method using a commercially available kit (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA). The labeling index of hepatocytes was determined by counting more than 2,000 nuclei of three randomly selected fields under a light microscope. Effect of Atropine, 6-Hydroxydopamine, Hepatic Vagotomy, Hepatic Sympathectomy, Hypophysectomy, and Anti-Insulin Antibody on Intracisternal TRH Analog-Induced Modulation of Hepatic DNA Synthesis. Atropine
methyl nitrate (0.15 mg/kg) dissolved in saline was injected intraperitoneally 60 minutes before the peptide injection. 6-Hydroxydopamine dissolved in saline was intraperitoneally injected twice (100 mg/kg on the first day, 80 mg/kg on the fourth day), and intracisternal injection of TRH analog was performed on the seventh day.22 Either hepatic branch vagotomy or hepatic sympathectomy, or the respective sham operation, was performed 72 hours before the peptide injection. Hepatic branch vagotomy was achieved under a dissection microscope by selective section of the hepatic branch of the vagus nerve branching off from the left main vagal trunk a few millimeters proximal to the cardia.14 Hepatic sympathectomy was performed with the use of a dissection microscope by resecting the hepatic branch of the splanchnic nerves around the proper hepatic artery.23 Hypophysectomy was performed transaurally by Japan SLC, Inc. (Hamamatsu, Japan) 7 days before the intracisternal injection of TRH analog. The hypophysectomized rats were fed with standard laboratory chow supplemented with 0.5% glucose solution. Only animals showing no weight gain during a 1-week stabilization period were used. The completeness of the removal of the gland was checked by testis size and visual inspection at autopsy. Anti-insulin antibody was injected 30 minutes before intracisternal TRH analog injection and once a day at 8 AM through the tail vein. The amount of anti-insulin antibody used in this study had the ability to block the effect of insulin action with a plasma insulin level up to at least 120 ng/mL.16,24,25 To exclude the effect of atropine, 6hydroxydopamine, hepatic vagotomy, hepatic sympathectomy, hypophysectomy, and insulin antibody on food intake, rats were pairfed with respective vehicle-treated or sham-operated rats.
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FIG. 1. Effect of intracisternal injection of TRH analog, RX 77368 (10 ng), on hepatic DNA synthesis. Hepatic DNA was assessed by [methyl3 H]thymidine uptake. Rats fasted for 24 hours were injected intracisternally with saline or TRH analog (10 ng). [Methyl-3H]thymidine (20 mCi/100 g) was injected intraperitoneally 6, 12, 24, 48, or 72 hours after peptide injection; rats were killed 2 hours later, and the liver was removed for determination of [methyl-3H]thymidine incorporation into hepatic DNA. Each point represents the mean { SE. **P õ .01 as compared with the saline-treated group decapitated at the same time postinjection.
Statistical Analysis. Data were expressed as means { SE. Comparison between two groups was performed by the Student’s t test, and multiple comparison was calculated by ANOVA, followed by Duncan’s contrast. P õ .05 was considered statistically significant.
RESULTS Effect of Intracisternal TRH Analog on Hepatic DNA Synthesis and Content, and Liver Weight. Intracisternal injection of saline
vehicle did not influence hepatic DNA synthesis as assessed by [Methyl-3H]thymidine incorporation into hepatic DNA in rats (Fig. 1). Intracisternal injection of RX 77368 at 10 ng caused an increase in [Methyl-3H]thymidine incorporation into hepatic DNA (Fig. 1). The response reached a maximum within 24 hours of peptide injection, and thereafter returned to the basal level by 72 hours (dpm/mg DNA: 0 hours, 92 { 6; 6 hours, 131 { 11; 12 hours, 195 { 42; 24 hours, 693 { 78; 48 hours, 378 { 81; 72 hours, 120 { 27). A significant dose-related stimulatory effect was observed following intracisternal injection of RX 77368 in doses ranging from 1 to 10 ng, and reached a plateau at 10 ng as measured 24 hours after peptide injection (dpm/mg DNA at 24 hours: saline, 95 { 6; 1 ng, 114 { 14; 5 ng, 318 { 57; 10 ng, 693 { 78; 100 ng, 710 { 135) (Fig. 2). Intracisternal injection of RX 77368 (10 ng) increased hepatic DNA content at 24 hours after the peptide injection, and this stimulatory effect was maintained over the 72-hour observation period after the peptide (Fig. 3). In contrast, although intracisternal RX 77368 (10 ng) slightly increased the liver weight at 48 and 72 hours after the peptide, this change did not reach a statistically significant level (Fig. 3). RX 77368 injected intravenously at 10 ng did not alter hepatic DNA synthesis (Table 1). Effect of Intracisternal TRH Analog on Hepatic BrdU Labeling. Although the labeled hepatocytes undergoing DNA syn-
thesis in intracisternal saline-injected rats was 0.25 { 0.32% (n Å 5), that in intracisternal RX 77368 (10 ng)-injected rats was significantly elevated 24 hours after the peptide (8.9 { 2.6%; n Å 5; P õ .01) (Fig. 4A and 4B). The labeled hepato-
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FIG. 2. Dose-dependent stimulatory effect of intracisternal injection of RX 77368 on hepatic DNA synthesis. Hepatic DNA synthesis was assessed by [Methyl-3H]thymidine uptake. Rats fasted for 24 hours were injected intracisternally with saline or TRH analog (1-100 ng). [Methyl-3H]thymidine (20 mCi/100 g) was injected intraperitoneally 24 hours after peptide injection; rats were decapitated 2 hours later, and the liver was removed for determination of [Methyl-3H]thymidine incorporation into hepatic DNA. Each column represents the mean { SE. *P õ .05; **P õ .01 as compared with the saline-trated group.
cytes were located randomly in the lobules in intracisternal RX 77368–injected rats (Fig. 4B). Effect of Atropine, 6-Hydroxydopamine, Hepatic Vagotomy, Hepatic Sympathectomy, Hypophysectomy, and Anti-Insulin Antibody on the Intracisternal TRH Analog-Induced Stimulation of Hepatic DNA Synthesis. Hepatic branch vagotomy performed 72 hours
before, or atropine methyl nitrate (0.15 mg/kg) injected intraperitoneally 60 minutes before, the peptide injection completely abolished intracisternal TRH analog (10 ng)-induced stimulation of hepatic DNA synthesis (Figs. 5 and 6). Hepatic sympathectomy 72 hours before and 6-hydroxydopamine treatment 7 days before the peptide injection did not modify the stimulation of hepatic DNA synthesis induced by intracisternal injection of TRH analog (10 ng) (Table 2). Intracisternal TRH analog (10 ng) stimulated hepatic DNA synthesis even in hypophysectomized rats and anti-insulin–treated rats (Table 2). DISCUSSION
In the present study, we demonstrate that the TRH analog, RX 77368 (1-100 ng), injected intracisternally stimulates hepatic DNA synthesis in conscious rats as assessed by [Methyl3 H]thymidine incorporation. Hepatic BrdU labeling reveals that DNA synthesis in hepatocytes is stimulated by central TRH analog. The stimulatory effect of intracisternal injection of TRH analog is dose-related in doses ranging from 1 to 10 ng. Intracisternal TRH analog (RX 77368, 10 ng) injection significantly increases hepatic DNA synthesis, with a peak response at 24 hours, and thereafter the enhanced hepatic DNA synthesis gradually returns to the baseline by 72 hours. The dose of 10 ng induces 693 { 78 dpm/mg DNA of hepatic DNA synthesis, which represents the maximal response because 100 ng does not further stimulate hepatic DNA synthesis (710 { 135 dpm/mg DNA). In contrast, TRH analog in-
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FIG. 3. Effect of intracisternal injection of RX 77368 (10 ng) on hepatic DNA content and hepatic weight. Hepatic DNA was extracted by Schneider’s method, and the DNA content was determined by Burton’s method. Rats were killed 6, 12, 24, 36, 48, and 72 hours after intracisternal injection of TRH analog. Each point represents the mean { SE. **P õ .01 as compared with the saline-treated group decapitated at the same time postinjection.
jected intravenously at the maximal effective dose, given intracisternally, does not influence hepatic DNA synthesis under identical conditions. These results indicate that TRH analog, injected into the cisternal magna, acts in the CNS to stimulate hepatic DNA synthesis and not through leakage into the peripheral circulation. The pathways through which central TRH analog stimulates hepatic DNA synthesis were investigated. The neurohumoral mechanisms through which intracisternal injection of TRH analog stimulates hepatic DNA synthesis are unrelated to its regulatory action on the pituitary-thyroidal axis,26 because intracisternal injection of TRH analog-induced [Methyl-3H]thymidine uptake persists in hypophysectomized rats, and intravenous TRH analog did not stimulate hepatic DNA synthesis. TRH has already been shown to have various CNS-mediated actions unrelated to its neuro-endocrine activity, including modification of behavior27; alteration
TABLE 1. Effect of Intravenous Injection of RX 77368 on Hepatic DNA Synthesis in Rats 3
Treatment
H-Thymidine Incorporation Into Hepatic DNA 24 Hours After TRH Analog Injection (dpm/mg DNA)
Saline (intravenously) (n Å 6) RX 77368 (10 ng intravenously) (n Å 7)
89 { 12 92 { 10
NOTE. Rats were injected intravenously (i.v.) with RX 77368 (10 ng) through the jugular vein under light ether anesthesia; 24 hours later, the animals were decapitated, and the liver was removed for determination of thymidine incorporation into hepatic DNA.
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FIG. 4. Distribution of cells undergoing DNA synthesis in the livers of rats 24 hours after intracisternal injection of RX 77368 (10 ng) or saline vehicle. (A) Rats intracisternally injected with saline. (B) Rats intracisternally injected with TRH analog. DNA synthesis was detected by BrdU uptake and following immunochemical staining using monoclonal antibody to BrdU. (Original magnification 140.)
of thermoregulation28; blood pressure5; food intake29; and cardiac,5 respiratory,30 and gastric7,31,32 functions. These central actions of TRH are mediated through the sympathetic or parasympathetic nervous systems.31-33 Central TRH analoginduced stimulation of hepatic DNA synthesis was reversed by hepatic vagotomy and atropine, an anticholinergic compound, but not by hepatic sympathectomy or 6-hydroxydopamine, which chemically depletes noradrenergic nerve fibers via biosynthetic adrenergic intermediates.22 These findings suggest that parasympathetic and cholinergic pathways participate in the proliferative growth response in the liver to centrally active TRH. These results are consistent with related findings indicating a central action of TRH on gastric functions including secretion, motility, mucosal blood flow, and resistance to mucosal injury.9,31,32 Our present results are also supported by a recent study that indicates that vagal hyperactivity following VMH lesioning stimulates hepatic DNA synthesis through vagal and cholinergic pathways.16,34 Moreover, cell proliferation is regulated by cholin-
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ergic factors in rat cornea,35 and cholinergic stimulation results in an increase in hepatic DNA synthesis after carbon tetrachloride–induced hepatic damage.15 The involvement of insulin in central TRH analog-induced hepatic DNA synthesis was investigated in the present study. Insulin is known to stimulate hepatic proliferation,36 and central administration of TRH elevates plasma insulin levels.37 However, administration of anti-insulin antibody did not affect the hepatic DNA synthesis induced by intracisternal injection of TRH analog, suggesting that the stimulation of hepatic DNA synthesis by intracisternal TRH analog is not secondary to the increase in plasma insulin induced by central TRH. Centrally injected TRH induces parasympathetic nerve activation38 by acting in the medullary nuclei, including the dorsal vagal complex and nucleus ambiguous,39,40 which are important sites for parasympathetic outflow.41 Although microinjections of TRH analog into specific brain medullary nuclei are not possible in conscious rats, the dorsal vagal
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TABLE 2. Effect of Hepatic Sympathectomy, 6-Hydroxydopamine, Hypophysectomy, and Anti-Insulin Antibody on Intracisternal TRH Analog–Induced Stimulation of Hepatic DNA Synthesis in Rats 3
Treatment
FIG. 5. Effect of hepatic branch vagotomy on intracisternal injection of TRH analog–induced stimulation of hepatic DNA synthesis. Hepatic branch vagotomy was performed 72 hours before the intracisternal injection of RX 77368 (10 ng). For details, see legends of Figs. 1 and 2. **P õ .01 compared with respective sham-operation group.
complex, including the vagal motor nucleus and the nucleus of the solitary tract, are probable sites of TRH-induced hepatic DNA synthesis. TRH-immunoreactive nerve terminals and receptors are localized in the dorsal vagal complex,42,43 and various central TRH-induced gastric functions are reproduced by microinjection into the dorsal vagal complex.39,40 In addition, vagotomy blocks hepatic [Methyl-3H]thymidine uptake induced by intracisternal injection of TRH analog in this study. These facts support that the dorsal vagal complex may be the specific site for TRH action on hepatic DNA synthesis. Kiba et al. reported that vagal hyperactivity caused by a lesion of the VMH induces cell proliferation not only in the liver, but also in the intestine and the pancreas.16,24,25 It is
FIG. 6. Effect of atropine treatment on intracisternal injection of TRH analog–induced stimulation of hepatic DNA synthesis. Atropine methyl nitrate (0.15 mg/kg) was injected intraperitoneally 60 minutes before the intracisternal injection of RX 77368 (10 ng). For details, see legends of Figs. 1 and 2. **P õ .01 compared with respective sham-operation group.
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No pretreatment / intracisternal saline (n Å 5) Sham hepatic sympathectomy / intracisternal TRH analog (n Å 6) Hepatic sympathectomy / intracisternal TRH analog (n Å 7) Intraperitoneal saline / intracisternal TRH analog (n Å 7) Intraperitoneal 6-hydroxydopamine / intracisternal TRH analog (n Å 6) Sham hypophysectomy / intracisternal TRH analog (n Å 5) Hypophysectomy / intracisternal TRH analog (n Å 6) Intravenous saline / intracisternal TRH analog (n Å 5) Intravenous anti-insulin antibody / intracisternal TRH analog (n Å 5)
H-Thymidine Incorporation Into Hepatic DNA 24 Hours After TRH Analog Injection (dpm/mg DNA)
95 { 6 724 { 68 694 { 82 695 { 125 723 { 105 688 { 95 678 { 101 712 { 98 689 { 112
NOTE. Rats were injected intracisternally with RX 77368 (10 ng), and, 24 hours later, the animals were decapitated, and the liver was removed for determination of thymidine incorporation into hepatic DNA.
interesting to know whether central TRH administration also increases cell proliferation in these organs besides the liver. In the study of Kiba et al., hepatic DNA synthesis reached a maximum 3 days after the VMH lesion, and hepatic DNA content and hepatic weight kept increasing for 7 days. However, hepatic DNA synthesis peaked on the second day after intracisternal TRH analog injection, and the increase in hepatic DNA content and hepatic weight occurred 2 days after central TRH administration. These discrepancies may be explained by the difference in the duration of vagal stimulation by these two procedures. Although central TRH injection stimulates vagal activity for only a few hours,38 the VMH lesion may induce an enduring stimulation of the vagus nerve. The liver is known to be richly innervated,11 and there is abundant evidence indicating important roles for the central and autonomic nervous systems in hepatic function, including hepatic proliferation.12-14 Very little is revealed about central neuropeptides as neurotransmitters inducing modulation of hepatic function. To date, only an effect of administration of central neuropeptide Y with bile secretion has been reported.44-46 After partial hepatectomy, the liver remnant spontaneously regenerates to restore hepatic volume to normal.36,47 In this phenomenon, the autonomic nervous system plays an important role.12-14,48 However, the identity of the centrally acting neurotransmitters and neuromediators are unknown. In our study, the centrally administered TRH analog, RX 77368, stimulates hepatic DNA synthesis mediated through a vagal-dependent cholinergic pathway. Because the dorsal vagal complex is richly innervated by TRH-immunoreactive fibers and receptors,42,43 and because central TRH activates vagal efferent fibers,38 we suggest that TRH is a likely candidate for a centrally acting chemical messenger involved in CNS regulation of hepatic proliferation. It is also of interest
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to study the significance of endogenous brain TRH on hepatic proliferation after partial hepatectomy and experimental acute liver injury by using an immunoneutralizing TRH monoclonal antibody.
23. 24.
Acknowledgment: The authors thank Dr. Kent C. Lloyd, D.V.M., Ph.D. (University of California, Davis), for editorial assistance, and Dr. Yvette Tache´, Ph.D. (UCLA), for valuable discussion.
25. 26.
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WBS: Hepatology