Taku Hebiguchi

Onset of Apoptosis in the Cystic Duct During Metamorphosis of a Japanese Lamprey, Lethenteron reissneri

A nonparasitic lamprey in Japan, Lethenteron reissneri, stops feeding prior to the commencement of metamorphosis. Resumption of feeding cannot take place due to major alterations in the digestive system, including loss of the gall bladder (GB) and biliary tree in the liver. This degeneration of bile ducts is considered to depend on programmed cell death or apoptosis, but molecular evidence of apoptosis remains lacking. Using terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining and immunohistochemistry with an antibody against active caspase‐3, we showed that epithelial cells of the cystic duct (CD) and GB became TUNEL‐positive by the early metamorphosing stage. Immunohistochemical staining of active caspase‐3, a key mediator in the apoptotic cascade, showed that the apoptotic signal was initiated in the region around the CD in the late larval phase. In later stages, active caspase‐3‐positive epithelial cells were also observed in the large intrahepatic bile duct (IHBD) and peripheral small IHBDs. At the early metamorphosing stage, bile canaliculi between hepatocytes were dilated and displayed features resembling canaliculi in cholestasis. Onset of apoptosis around the CD, which is the pathway for the storage of bile juice, and progression of apoptosis towards the large IHBD, which is the pathway for the secretion of bile juice, may lead to temporary intrahepatic cholestasis. The present study represents the first precise spatial and temporal analysis of apoptosis in epithelial cells of the biliary tract system during metamorphosis of any lamprey species. Anat Rec 293:1155–1166, 2010.

Uptake and storage of vitamin A as lipid droplets in the cytoplasm of cells in the lamina propria mucosae of the rat intestine.

Vitamin A (retinyl palmitate) was injected subcutaneously or administered to rats by tube feeding. After subcutaneous injection, vitamin A was taken up and stored in cells of the lamina propria mucosae of the rat intestine. After oral administration, vitamin A was absorbed by the intestinal absorptive epithelial cells and transferred to cells of the lamina propria mucosae, where cells took up and stored the transferred vitamin A. The morphology of these cells was similar to that of hepatic stellate cells (also called vitamin A‐storing cells, lipocytes, interstitial cells, fat‐storing cells or Ito cells). Thus, these cells in the intestine could take up vitamin A from the systemic circulation and as well as by intestinal absorption, and store the vitamin in the lipid droplets in their cytoplasm. The data suggest that these cells are extrahepatic stellate cells of the digestive tract that may play roles in both the absorption and homeostasis of vitamin A.

Differential Increases in the Expression of Intermediate Filament Proteins and Concomitant Morphological Changes of Transdifferentiating Rat Hepatic Stellate Cells Observed In Vitro

The primary function of hepatic stellate cells (HSCs) is the storage of vitamin A. However, they are also responsible for liver fibrosis and are therapeutic targets for treatment of liver cirrhosis. Among the many molecular markers that define quiescent or activated states of HSCs, the characteristics of type III intermediate filaments are of particular interest. Whereas vimentin and desmin are upregulated in activated HSCs, glial fibrillary acidic protein is downregulated in activated HSCs. The functional differences between vimentin and desmin are poorly understood. By time-course quantifications of several molecular markers for HSC activation, we observed that the expression of vimentin preceded that of desmin during the transdifferentiation of HSCs. The immunoreactivity of vimentin in transdifferentiated HSCs was more intense in perinuclear regions compared to that of desmin. We propose that the delayed expression of desmin following the expression of vimentin and the peripheral localization of desmin compared to vimentin are both related to the more extended phenotype of transdifferentiating HSCs observed in vitro.

Massive bowel resection upregulates the intestinal mRNA expression levels of cellular retinol-binding protein II and apolipoprotein A-IV and alters the intestinal vitamin A status in rats

Short bowel (SB) syndrome causes the malabsorption of various nutrients. Among these, vitamin A is important for a number of physiological activities. Vitamin A is absorbed by epithelial cells of the small intestine and is discharged into the lymphatic vessels as a component of chylomicrons and is delivered to the liver. In the present study, we used a rat model of SB syndrome in order to assess its effects on the expression of genes associated with the absorption, transport and metabolism of vitamin A. In the rats with SB, the intestinal mRNA expression levels of cellular retinol-binding protein II (CRBP II, gene symbol Rbp2) and apolipoprotein A-IV (gene symbol Apoa4) were higher than those in the sham-operated rats, as shown by RT-qPCR. Immunohistochemical analysis revealed that absorptive epithelial cells stained positive for both CRBP II and lecithin retinol acyltransferase, which are both required for the effective esterification of vitamin A. In the rats with SB, the retinol content in the ileum and the retinyl ester content in the jejunum were lower than those in the sham-operated rats, as shown by quantitative analysis of retinol and retinyl esters by high performance liquid chromatography. These results suggest that the elevated mRNA expression levels of Rbp2 and Apoa4 in the rats with SB contribute to the effective esterification and transport of vitamin A.

The role of retinoic acid receptors in activated hepatic stellate cells

Hepatic stellate cells (HSCs), also known as Ito cells, fat-storing cells, vitamin A-storing cells or lipocytes, reside in the spaces between hepatocytes and liver sinusoids. Vitamin A storage within the HSCs is achieved through the cooperative action of two proteins, cellular retinol-binding protein (CRBP) I and lecithin:retinol acyltransferase (LRAT). After the discovery that HSCs are responsible not only for the storage of vitamin A, but also for the development of liver fibrosis and subsequent liver cirrhosis, HSCs have been considered a therapeutic target for prevention or reversal of liver fibrogenesis. We have reported that HSCs acquire retinoid responsiveness after in vitro activation by post-transcriptional upregulation of retinoic acid receptor α gene expression. Here we extend this observation in relation to the functions of CRBP I and LRAT, and propose a hypothesis that increased retinoid signaling in activated HSCs forms a feedback loop toward vitamin A restoration in the liver.

How Do Lampreys Avoid Cholestasis After Bile Duct Degeneration

Biliary atresia (BA) is the most common cause of cholestasis during infancy for which an etiology remains undetermined. Patients require hepatic portoenterostomy within the first 2-3 months of life in order to restore bile flow from the liver into the intestinal tract. Even with successful surgery, in most patients the disease advances to end-stage cirrhosis due to the progressive destruction of bile ducts, and requires liver transplantation in order for long term survival to be viable (Mack & Sokol, 2005). Without a better understanding of the etiology and pathogenesis of the progressive cholangitic mechanisms in BA, improvement in the prognosis of non-transplantation patients should not be expected. Despite the importance of understanding these underlying mechanisms, research of BA has been hindered by the lack of a suitable animal model.