Molecular identification of GHS-R and GPR38 in Suncus murinus

We previously identified ghrelin and motilin genes in Suncus murinus (suncus), and also revealed that motilin induces phase III-like strong contractions in the suncus stomach in vivo, as observed in humans and dogs. Moreover, repeated migrating motor complexes were found in the gastrointestinal tract of suncus at regular 120-min intervals. We therefore proposed suncus as a small laboratory animal model for the study of gastrointestinal motility. In the present study, we identified growth hormone secretagogue receptor (GHS-R) and motilin receptor (GPR38) genes in the suncus. We also examined their tissue distribution throughout the body. The amino acids of suncus GHS-R and GPR38 showed high homology with those of other mammals and shared 42% amino acid identity. RT-PCR showed that both the receptors were expressed in the hypothalamus, medulla oblongata, pituitary gland and the nodose ganglion in the central nervous system. In addition, GHS-R mRNA expressions were detected throughout the stomach and intestine, whereas GPR38 was expressed in the gastric muscle layer, lower intestine, lungs, heart, and pituitary gland. These results suggest that ghrelin and motilin affect gut motility and energy metabolism via specific receptors expressed in the gastrointestinal tract and/or in the central nervous system of suncus.     

Motilin Stimulates Gastric Acid Secretion in Coordination with Ghrelin in Suncus murinus

Motilin and ghrelin constitute a peptide family, and these hormones are important for the regulation of gastrointestinal motility. In this study, we examined the effect of motilin and ghrelin on gastric acid secretion in anesthetized suncus (house musk shrew, Suncus murinus), a ghrelin- and motilin-producing mammal. We first established a gastric lumen-perfusion system in the suncus and confirmed that intravenous (i.v.) administration of histamine (1 mg/kg body weight) stimulated acid secretion. Motilin (0.1, 1.0, and 10 μg/kg BW) stimulated the acid output in a dose-dependent manner in suncus, whereas ghrelin (0.1, 1.0, and 10 μg/kg BW) alone did not induce acid output. Furthermore, in comparison with the vehicle administration, the co-administration of low-dose (1 μg/kg BW) motilin and ghrelin significantly stimulated gastric acid secretion, whereas either motilin (1 μg/kg BW) or ghrelin (1 μg/kg BW) alone did not significantly induce gastric acid secretion. This indicates an additive role of ghrelin in motilin-induced gastric acid secretion. We then investigated the pathways of motilin/motilin and ghrelin-stimulated acid secretion using receptor antagonists. Treatment with YM 022 (a CCK-B receptor antagonist) and atropine (a muscarinic acetylcholine receptor antagonist) had no effect on motilin or motilin-ghrelin co-administration-induced acid output. In contrast, famotidine (a histamine H₂ receptor antagonist) completely inhibited motilin-stimulated acid secretion and co-administration of motilin and ghrelin induced gastric acid output. This is the first report demonstrating that motilin stimulates gastric secretion in mammals. Our results also suggest that motilin and co-administration of motilin and ghrelin stimulate gastric acid secretion via the histamine-mediated pathway in suncus.     

Induction of fatty liver by fasting in suncus

We found that a fatty liver was easily induced in a novel experimental animal, Suncus murinus (suncus), by withholding food. Hepatic triglyceride content increased linearly for up to 24 h after fasting in these animals. Serum levels of neutral lipids are very low in the fed state compared with those in rats, and decreased significantly after 24 h fasting. On the other hand, serum free fatty acids, which are at the same level in fed animals as in rats, increased threefold in the fasting suncus. In order to learn whether the fatty liver induced by fasting is an unusual physiological state or a pathological on-going state in suncus, they were refed after 24 h fasting. Refeeding resulted in a decrease in hepatic triglyceride content to the level of fed animals. Serum lipid levels, which decreased with fasting, returned to those of fed animals. This evidence indicates that hepatic lipid secretion is impaired even in a physiological state to some extent and that starvation causes increasing influx of free fatty acid to the liver, which might be followed by esterification and result in triglyceride accumulation in the liver. In conclusion, hepatic lipid and lipoprotein metabolism is unique to the suncus, which is a useful animal model for the study of intra-hepatic lipid transport.     

Cloning of putative uncoupling protein 1 cDNA in a cold-intolerant mammal, the house musk shrew (Suncus murinus)

The house musk shrew (Suncus murinus), or suncus, is a unique experimental animal. We recently showed that this mammal is cold intolerant and hypothesized that its sensitivity to cold is caused by low thermogenic activity in brown adipose tissue (BAT). Thermogenesis in BAT is performed by a unique mitochondrial protein, uncoupling protein 1 (UCP1). Interestingly, only eutherians possess UCP1, and some traits in the suncus resemble those in the Ucp1-ablated mouse, including cold intolerance, histology of BAT, and obesity resistance. In a previous study, we hypothesized that UCP1 may not be present in BAT of the suncus or may be dysfunctional. Therefore, we performed cDNA cloning of suncus Ucp1 and compared it to homologs from other species. The deduced amino acid sequence showed high similarity to other mammalian UCP1. Northern blot analysis revealed mRNA in BAT, as in other mammals. However, a difference in an amino acid residue was observed in an important residue for thermogenesis. Genomic sequence analysis showed that this difference existed in our two genetically distant laboratory colonies. These results suggest that cold intolerance in the suncus is derived from low thermogenic activity of UCP1 and may exist in wild house musk shrews.     

Scanning, transmission and immunoelectron microscopical studies of the tonsil-like lymphoid organ of normal and horseradish-peroxidase-injected laboratory suncuses

Fine structures of normal and horseradish peroxidase (HRP)-stimulated tonsil-like lymphoid organs of the laboratory suncus (Suncus murinus) were examined by scanning, transmission and immunoelectron microscopies. The normal organs appear as a pair of small oval protrusions at the upper lateral sites of the fauces, and consist of a single lymph nodule with a germinal center and a crypt-like epithelium with prominent lymphoid cell infiltration. Postcapillary venules (PCV) and efferent lymphatics are associated within the marginal region of the nodule and separated from the neighboring pharyngeal tissues. Numerous lympho-plasma cells, interdigitating cells and reticulum cells occurred within the lymphoid parenchyma, as well as in the intraepithelial infiltrating cell populations. In the HRP-stimulated animals, the anti-HRP antibodies producing lympho-plasma cells were often seen in the parenchyma and epithelia; however, similar HRP-antibody-positive lymphocytes were rarely detected in the PCV lumina. In addition, some HRP antibody bearing interdigitating cells were also identified in the same parenchyma. These data indicate that the suncus' tonsil-like organs have a positive immune function to oral antigens, together with the suncus' systemic immune system and it is hypothetically presumed that the organ may correspond to a homologous organ of the human palatine tonsil in comparative anatomy.     

Comparative aspects of microsomal cytochrome P-450-dependent monooxygenase activities in musk shrew (Suncus murinus), Mongolian gerbil (Meriones unguiculatus), harvest mouse (Micromys minutus) and rat.

1. The cytochrome P-450 content (0.75 +/- 0.13 nmol/mg microsomal protein) in musk shrew (suncus, Suncus murinus) liver microsomes was lower than that (1.30 +/- 0.26) in rat liver microsomes, but it is approximately the same level as in the Mongolian gerbil (Meriones unguiculatus, 1.18 +/- 0.14), harvest mouse (Micromys minutus, 1.11 +/- 0.02) and rat. 2. The hydroxylation activity (based on cytochrome P-450) of medium-chain fatty acids (otanoic, decanoic, lauric and tridecanoic acids) is much higher in suncus, Mongolian gerbil and harvest mouse than in rat, with the exception of the activity of decanoic and tridecanoic acids in Mongolian gerbil. 3. This suggests that cytochrome P-450 species catalyzing the hydroxylation of medium-chain fatty acids are present in these laboratory animals in higher concentrations. 4. The aminopyrine N-demethylation activity based on microsomal protein or cytochrome P-450 in suncus is significantly lower than that in rat, but the N-demethylation activity in Mongolian gerbil and harvest mouse is approximately 1.7-2.0-fold greater than that in rat.     

Suncus murinus: a new experimental model in emesis research

Effects of various emetic and antiemetic drugs were studied using Suncus murinus for its potential use as an experimental model in emetic research. Subcutaneous injection of nicotine bitartrate (10-15 mg/kg), veratrine sulfate (0.5-1.0 mg/kg), emetine dihydrochloride (40-80 mg/kg) and oral administration of copper sulfate (20-100 mg/kg) caused dose-dependent emesis in suncus. The ED50 of nicotine, veratrine, emetine and copper sulfate were 7.9, 0.4, 47.6 and 21.4 mg/kg, respectively. However, subcutaneously injected apomorphine hydrochloride (0.1-100 mg/kg), digitoxin (0.5-1.0 mg/kg) and orally administered emetine dihydrochloride (10-80 mg/kg) did not induce the vomiting. Chlorpromazine and promethazine decreased the emetic effect of nicotine, veratrine and copper sulfate, but scopolamine hydrobromide was not effective. These results indicate that the Suncus murinus is sensitive to various emetic and antiemetic drugs and can be used as a new experimental animal model for the emesis. Emetic behavior of suncus was discussed in comparison with other animals.     

The morphological and histological characters of the male external genitalia of the house musk shrew, Suncus murinus

External genitalia are the reproductive organs necessary for efficient copulation and internal fertilization in various mammalian species. Their morphogeneses display significant morphological and developmental differences among species. The house musk shrew, Suncus murinus (hereafter described as suncus) is a species of the order Insectivora, which has been considered as primitive and one of the earliest eutheria phylogenetically. Comparative anatomical analyses of phylogenetically different mammals will contribute to the better understanding of morphological diversity of external genitalia. This study performed various anatomical and histological analyses concerning the organization of the external genitalia of male suncus. It was shown that the external genitalia of suncus possessed a muscular structure, which we proposed as musculus ischiocavernosus dorsalis of suncus. The musculus ischiocavernosus dorsalis is originated from the inner surface of the tuber ischiadicum and was allocated adjacent to the corpus cavernosum penis. In addition, a pair of alpha-smooth muscle actin positive muscles was located bilaterally to the urethra. This unique morphology of the external genitalia of suncus males may provide a unique model system to investigate genital morphogenesis.     

Length and weight of gastrointestinal tracts of pikas, suncus, millardias, mice and rats

The length and weight of gastrointestinal tracts including contents of ten week old male pikas (Ochotona rufescens rufescens) and suncus (Suncus murinus) were measured and they were investigated and compared with those of millardias, ICR-mice and Wistar-rats. The length from the duodenum to the anus of pikas was much longer and the weight from the stomach to the anus was about 16g per 100g of body weight. The weight of cecum was about 7g per body weight and they were heavier than those of other species. The length from the duodenum to the anus of the suncus was short but the weight of the small intestine plus colon and rectum per body weight did not differ from that of other species. The suncus has no cecum but the weight from the stomach to the anus was almost the same as that of rats.     

A high-level expression of CYP2A in the lung of the suncus (Suncus murinus) and its role in the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Northern blot analysis of mRNA prepared from the lung of Suncus murinus (suncus), which was classified as an ancestor of primates, revealed that the expression level of cytochrome P450 2A (CYP2A) mRNA was about 100-fold higher than in the lung from rats and mice. To confirm that the pulmonary CYP2A of the suncus had a catalytic activity, the metabolism of a specific substrate for CYP2A6, (+)-cis-3,5-dimethyl-2-(3-pyridyl) thiazolidin-4-one hydrochloride (SM-12502), was determined. The intrinsic clearance for SM-12502 S-oxidation by the suncus lung microsomes was calculated to be 99-fold higher than that by rat liver microsomes. The mutagen-producing activity of a 9,000 g supernatant fraction prepared from suncus lung was examined using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as a promutagen. The results showed that the suncus lung possessed 82-fold higher mutagen-producing activity than the rat lung, indicating that NNK was efficiently activated by the CYP2A isoform expressed in the suncus lung and that the suncus was a sensitive animal species to the genotoxicity of NNK contained in tobacco smoke.     

Serum constituents of Suncus murinus

Blood samples were collected by cardiac puncture from nine-week-old suncus, mice and rats those had been fasted for 16 hours, and the serum was assayed for the levels of corticosterone, cortisol, and other constituents. The following results were obtained: The serum levels of corticosterone and cortisol and corticosterone/cortisol ratio of suncus were about the same as those of human. The SGPT, gamma-GT, ALP, LDH, Ca and K values in suncus were all within the normal range of the respective values in human. SGOT, amylase, BUN, Na, Cl, Fe and inorganic phosphate values were higher, and total cholesterol, triglyceride, phospholipid, creatinine, urea, total protein, albumin and bilirubin levels were lower in suncus than the respective normal values in human. The values of SGOT, amylase, ZTT, and K were higher in female than in male suncus, while the values of gamma-GT and ALP were higher in the male. Feeding the animals individually in the individual cages for a week increased the values of direct bilirubin, total cholesterol, ZTT, Na, K and Cl and lowered the value of urea. The values of SGPT, ALP and triglyceride of suncus obtained in 1984 were higher, the value of albumin was lower, than the respective values obtained in 1983. The value of cholinesterase in suncus was very small.     

Comparison of various motion stimuli on motion sickness and acquisition of adaptation in Suncus Murinus

Effects of various types of motion stimuli were compared to investigate optimum method to elicit motion sickness and adaptation in Suncus murinus (suncus). Three different direction of shaking in the horizontal plane, back and forth, right and left and revolving, induced emetic response to the similar extent. However, vertical shaking was far less effective in inducing motion sickness. Mild and severe horizontal shaking (15 min per day) was continued for 14 days and emetic response to standard motion stimulus was compared before and after the training. The severe daily acceleration strongly depressed the susceptibility to motion stimulus. The mild acceleration which was not emetic stimulus in itself also remarkably attenuated the vomiting response to standard motion stimulus. These results indicate that 1) the emetic responsiveness of the suncus does not depend on the modes of shaking as long as the direction is in the horizontal plane, 2) the suncus is relatively refractory to the vertical linear acceleration and 3) the adaptation to motion stimulus does not develop on the latest peripheral steps of the vomiting reflex pathways.     

Changes in Ucp1, D2 (Dio2) and Glut4 (Slc2a4) mRNA expression in response to short-term cold exposure in the house musk shrew (Suncus murinus).

The house musk shrew (Suncus murinus), or so-called suncus, is a cold-intolerant mammal, but it is unclear why it is susceptible to low temperatures. Cold-intolerance may be the result of lower thermogenic activity in brown adipose tissue (BAT). The early phase of severe cold exposure is a critical period for suncus. Therefore, we exposed suncus to mildly cold temperatures (10-12 degrees C) for 1 to 48 h to increase non-shivering thermogenesis without causing stress and measured changes in the expression of uncoupling protein 1 (Ucp1), type II iodothyronine 5'-deiodinase (Dio2=D2), and glucose transporter 4 (Slc2a4=Glut4) in BAT. These mRNAs play a major role in non-shivering thermogenesis and are mainly regulated by the sympathetic nervous system via direct beta-noradrenergic innervation of BAT. During cold exposure, Ucp1 expression in BAT increased steadily over time, albeit only slightly. Neither D2 nor Glut4 expression in BAT increased immediately; however, they had increased significantly after 24 h and 48 h of cold exposure. These findings suggest that the responsiveness of mRNA regulation is weak and thus may be involved in cold-intolerance in suncus.     

Motilin receptors in the human antrum

Motilin is an intestinal peptide that stimulates contraction of gut smooth muscle. The motilin receptor has not been cloned yet, but motilin-receptor agonists appear to be potent prokinetic agents for the treatment of dysmotility disorders. The aim of this study was to determine neural or muscular localization of motilin receptors in human upper gastrointestinal tract and to investigate their pharmacological characteristics. The binding of (125)I-labeled motilin to tissue membranes prepared from human stomach and duodenum was studied; rabbit tissues were used for comparison. Solutions enriched in neural synaptosomes or in smooth muscle plasma membranes were obtained. Various motilin analogs were used to displace the motilin radioligand from the various tissue membranes. The highest concentration of human motilin receptors was found in the antrum, predominantly in the neural preparation. Human motilin receptors were sensitive to the NH(2)-terminal portion of the motilin molecule, but comparison with rabbit showed that both species had specific affinities for various motilin analogs [i.e., Mot-(1-9), Mot-(1-12), Mot-(1-12) (CH(2)NH)(10-11), and erythromycin]. Motilin receptors obtained from synaptosomes or muscular plasma membranes of human antrum expressed different affinity for two motilin-receptor agonists, Mot-(1-12) and Mot-(1-12) (CH(2)NH)(10-11), suggesting that they correspond to specific receptor subtypes. We conclude that human motilin receptors are located predominantly in nerves of the antral wall, are functionally (and probably structurally) different from those found in other species such as the rabbit, and express specific functional (and probably structural) characteristics dependent on their localization on antral nerves or muscles, suggesting the existence of specific receptor subtypes, potentially of significant physiological or pharmacological relevance.     

Role of the duodenum in motilin release

In order to study the regulatory mechanism of motilin release, plasma motilin was measured in healthy dogs during the fasting state and after the ingestion of ordinary nutrient. Fasting plasma motilin levels were found to fluctuate intermittently, but ingestion of a meal completely abolished the intermittent motilin release and resulted in low motilin levels lasting for 6–8 h. To clarify the role of the duodenum in this motilin release, an operation was performed in five dogs by which we excluded from the alimentary tract the upper half of the small intestine not including the duodenum from a point 2 cm below the larger pancreatic duct. After this operation meal ingestion still caused a decrease in plasma motilin levels. However, after a modified version of the operation was performed in 5 other dogs by which the upper half of the small intestine together with the duodenum was transected at the pyloric ring, plasma motilin was not suppressed by meal ingestion. These results suggest that motilin secretion is regulated by nutrient ingestion and that the passage of nutrients through the duodenum plays a important role in its regulation.     

Serum constituents and liver photomicrographs of wild Suncus murinus in the south of Taiwan

Serum constituents and liver of the wild male suncus as well as of those bred and fed in our laboratory were examined serologically and histologically. The following results were obtained: The serum glutamic-oxaloacetic transaminase, gamma-glutamyl transaminase, inorganic phosphate, blood urea nitrogen, creatinine, uric acid, and zinc turbidity test levels of wild suncus were higher than those of 4 and 8 week old fed suncus. The total cholesterol level of wild suncus was lower than those of 4 and 8 week old fed suncus. In wild suncus, the total bilirubin and direct bilirubin levels were higher, and the Ca and albumin levels were lower than those of 4 week old fed suncus. However, no significant differences were observed between the wild and the 8 week old fed suncus. Although the alkaline phosphatese and thymol turbidity test levels of wild suncus were larger than those of 8 week old fed suncus, no significant differences were observed between the wild and the 4 week old fed suncus. The histological study revealed the presence of fatty droplets in only 2 of the 67 wild suncus examined while fatty droplets (grade +) were observed in almost all the fed suncus. In one case of the wild suncus, moderate round cell infiltration in interlobular connective tissue was found.     

Analysis of pancreatic polypeptide cDNA from the house musk shrew,Suncus murinus,suggests a phylogenetically closer relationship with humans than for other small laboratory animal species

Pancreatic polypeptide was isolated and sequenced from endocrine cells of the pancreas from an insectivore, the house musk shrew, Suncus murinus. The primary sequence was APLEPAYPGD(10)NATPEQMAQY(20)AAELRKYINM(30)VTRPRYamide. This is the first polypeptide hormone to be characterised from this species and is typical of the primary sequences of pancreatic polypeptide of other animals, being a C-terminal-amidated peptide with 36 residues. Comparison with several vertebrate sequences shows that it has more in common with the human form than do the forms from common laboratory animals such as rabbits, rats, mice and guinea-pigs.     

Serotonergic ligand binding in aging brain of experimental animals

Although the use of aging expeimental animals for studying serotonergic neuronal changes is limited because of species differences, cholinergic neuronal deterioration does appear to be a feature common to mammalian aging brains. In the present study, a recently introduced experimental animal, Suncus murinus (house musk shrew, an insectivore classified as being at the stem of the mammalian phylogenic tree) which in certain physiological characteristics is more closely related to the primate than is the rat, was used as an experimental animal model for serotonergic neuronal deterioration in aging brain. We examined the changes in binding to the membrane fraction of aging brain cortex of the experimental animals Suncus and Fischer rat of the serotonergic ligands, 5-HT, imipramine, and 8-OH-DPAT. Morphological study of the brain stem including the Nucleus raphae by immunohistochemical staining demonstrated that in Suncus all the serotonergic ligands had decreasing affinity to the membrane of aging brain; binding of 8-OH-DPAT and imipramine decreased to a greater extent than that of 5-HT. In contrast, the aging rat brain showed no appreciable change in the binding of serotonergic ligands.