Wakako Fujimoto

Cellular expression of murine Ym1 and Ym2, chitinase family proteins, as revealed by in situ hybridization and immunohistochemistry

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.

Isx Participates in the Maintenance of Vitamin A Metabolism by Regulation of β-Carotene 15,15′-Monooxygenase (Bcmo1) Expression

Isx (intestine specific homeobox) is an intestine-specific transcription factor. To elucidate its physiological function, we generated Isx-deficient mice by knocking in the β-galactosidase gene (LacZ) in the Isx locus (IsxLacZ/LacZ mice). LacZ staining of heterozygous (IsxLacZ/+) mice revealed that Isx was expressed abundantly in intestinal epithelial cells from duodenum to proximal colon. Quantitative mRNA expression profiling of duodenum and jejunum showed that β-carotene 15,15′-monooxygenase (EC1.14.99.36 Bcmo1) and the class B type I scavenger receptor, which are involved in vitamin A synthesis and carotenoid uptake, respectively, were drastically increased in IsxLacZ/LacZ mice. Although mild vitamin A deficiency decreased Isx expression in duodenum of wild-type (Isx+/+) mice, severe vitamin A deficiency decreased Isx mRNA expression in both duodenum and jejunum of Isx+/+ mice. On the other hand, vitamin A deficiency increased Bcmo1 expression in both duodenum and jejunum of Isx+/+ mice. However, Bcmo1 expression was not increased in duodenum of IsxLacZ/LacZ mice by mild vitamin A deficiency. These data suggest that Isx participates in the maintenance of vitamin A metabolism by regulating Bcmo1 expression in the intestine.

Cellular Expression of Gut Chitinase mRNA in the Gastrointestinal Tract of Mice and Chickens

Recently, the second mammalian chitinase, designated acidic mammalian chitinase (AMCase), has been identified in human, mouse, and cow. In contrast to the earlier identified macrophage-derived chitinase (chitotriosidase), this chitinase is richly expressed in the gastrointestinal (GI) tract, suggesting its role in digestion of chitin-containing foods as well as defense against chitin-coated microorganisms and parasites. This in situ hybridization study first revealed cellular localization of the gut-type chitinase in the mouse and chicken. In adult mice, the parotid gland, von Ebners gland, and gastric chief cells, all of which are exocrine cells of the serous type, expressed the gut chitinase mRNA. In the chicken, oxyntico-peptic cells in glandular stomach (proventriculus) and hepatocytes expressed the chitinase mRNA. Because cattle produce the gut chitinase (chitin-binding protein b04) only in the liver, the gut chitinases in mammals and birds have three major sources of production, i.e., the salivary gland, stomach, and liver. During ontogenetic development, the expression level in the parotid gland and stomach of mice increased to the adult level before weaning, whereas in the stomach of chickens intense signals were detectable in embryos from incubation day 7.

Immunohistochemical demonstration of acidic mammalian chitinase in the mouse salivary gland and gastric mucosa

Acidic mammalian chitinase (AMCase) is the sole chitinolytic enzyme that has been identified thus far in the gastrointestinal tract of mammals. AMCase mRNA expression has been demonstrated in the salivary gland and stomach of mice and in the stomach of humans, while a bovine homologue of AMCase is produced in the liver and secreted into the blood. The present study using antibody raised against bovine AMCase demonstrates the cellular distribution of AMCase in salivary and gastric secretions at the protein level. Immunostaining using mouse tissues detected intense immunoreactivity for AMCase in serous-type secretory cells of the parotid gland and von Ebners gland. Gastric chief cells, localized at the bottom of gastric glands, were also immunoreactive for AMCase. Electron-microscopically, the immunoreactivity was localized in granules in the apical cytoplasm of these secretory cells, and not in other structures. Western blot analysis confirmed the existence of AMCase in the parotid gland and stomach, and in their secretions in mice. However, no immunoreactive band was clearly detectable in immunoblots of the human parotid saliva and gastric juice. At least in the mouse, AMCase is secreted into the saliva and gastric juice, and may function as a digestive enzyme or play a defensive role against chitinous pathogens.

Cephalic phase insulin secretion is KATP channel independent

Glucose-induced insulin secretion from pancreatic β-cells critically depends on the activity of ATP-sensitive K⁺ channels (KATP channel). We previously generated mice lacking Kir6.2, the pore subunit of the β-cell KATP channel (Kir6.2(-/-)), that show almost no insulin secretion in response to glucose in vitro. In this study, we compared insulin secretion by voluntary feeding (self-motivated, oral nutrient ingestion) and by forced feeding (intra-gastric nutrient injection via gavage) in wild-type (Kir6.2(+/+) and Kir6.2(-/-) mice. Under ad libitum feeding or during voluntary feeding of standard chow, blood glucose levels and plasma insulin levels were similar in Kir6.2(+/+) and Kir6.2(-/-) mice. By voluntary feeding of carbohydrate alone, insulin secretion was induced significantly in Kir6.2(-/-) mice but was markedly attenuated compared with that in Kir6.2(+/+) mice. On forced feeding of standard chow or carbohydrate alone, the insulin secretory response was markedly impaired or completely absent in Kir6.2(-/-) mice. Pretreatment with a muscarine receptor antagonist, atropine methyl nitrate, which does not cross the blood-brain barrier, almost completely blocked insulin secretion induced by voluntary feeding of standard chow or carbohydrate in Kir6.2(-/-) mice. Substantial glucose-induced insulin secretion was induced in the pancreas perfusion study of Kir6.2(-/-) mice only in the presence of carbamylcholine. These results suggest that a KATP channel-independent mechanism mediated by the vagal nerve plays a critical role in insulin secretion in response to nutrients in vivo.

Dmbx1 is essential in agouti-related protein action

Dmbx1 is a paired-class homeodomain transcription factor. We show here that mice deficient in Dmbx1 exhibit severe leanness associated with hypophagia and hyperactivity and that isolation of a Dmbx1−/− mouse from its cohabitants induces self-starvation, sometimes leading to death, features similar to those of anorexia nervosa in humans. Interestingly, overexpression of agouti in Dmbx1−/− mice failed to induce aspects of the Ay/a phenotype, including hyperphagia, obesity, and diabetes mellitus. In Dmbx1−/− mice, administration of agouti-related protein increased cumulative food intake for the initial 6 h but significantly decreased it over 24- and 48-h periods. In addition, Dmbx1 was shown to be expressed at embryonic day 15.5 in the lateral parabrachial nucleus, the rostral nucleus of the tractus solitarius, the dorsal motor nucleus of the vagus, and the reticular nucleus in the brainstem, all of which receive melanocortin signaling, indicating involvement of Dmbx1 in the development of the neural network for the signaling. Thus, Dmbx1 is essential for various actions of agouti-related protein and plays a role in normal regulation of energy homeostasis and behavior.