Henk F. J. Hendriks

Perisinusoidal fat-storing cells are the main vitamin A storage sites in rat liver

Highly purified sinusoidal (fat-storing, Kupffer and endothelial cells) and parenchymal cells were isolated to assess the cellular distribution of vitamin A in liver of adult vitamin A-sufficient rats. A modified simple procedure was developed for the purification of fat-storing cells from rat liver. This was achieved by a single centrifugation step in a two-layer density Nycodenz gradient. Endothelial and Kupffer cells were obtained from the same gradient and further purified by centrifugal elutriation. Reverse-phase HPLC analysis showed that fat-storing cells contained about 300-fold the amount of retinyl esters present in parenchymal cells on a mg cell protein basis. In fat-storing cells, the same retinyl esters, viz. retinyl palmitate, retinyl stearate and retinyl oleate, were present as in whole liver. It was also observed that, within 12 h after intravenous injection of chylomicron [3H]retinyl ester, most of the radioactivity had accumulated in the fat-storing cells. It is concluded that fat-storing cells are the main storage sites for vitamin A in rat liver.

Liver parenchymal cells differ from the fat-storing cells in their lipid composition

The neutral lipid and phospholipid compositions of purified sinusoidal (fat-storing, endothelial and Kupffer) cells, parenchymal cells and liver homogenates were determined by thin layer chromatography. In addition, the retinoid content of the same purified cell populations was determined by high performance liquid chromatography. From each cell type, both a lipid droplet fraction and a pellet fraction (containing the majority of the remaining cell organelles) were prepared by differential centrifugation. Electron microscopic analysis showed that lipid droplets isolated from fat-storing cells were larger (up to 8 μm) than those isolated from parenchymal cells (up to 2.5 μm). Moreover, the parenchymal lipid droplets seemed to be surrounded by a membranous structure, while the fat-storing lipid droplets seemed not to be. Both fat-storing and parenchymal cells contained high concentrations of neutral lipids, 57.9 μg and 71.0 μg/106 cells, respectively, while endothelial and Kupffer cells contained only 8.6 μg and 13.8 μg/106 cells of neutral lipids, respectively. Sixty-five percent of fat-storing cell lipid droplet fractions comprised esters of retinol and cholesterol. This combined ester fraction contained mainly retinyl esters. In addition, considerable quantities (20%) of triglycerides were present. Parenchymal cell lipid droplet fractions comprised triglycerides (62%) and cholesteryl esters (up to 30%). The pellet fractions prepared from all four cell types consisted mainly of cholesterol (41–67%) and free fatt acids (20–28%). The phospholipid content was much higher in parenchymal cells than in the sinusoidal liver cell types. The relative proportions of the four major phospholipid classes were comparable in all liver cell types analyzed. It is concluded that parenchymal cell lipid droplets comprised mainly triglycerides and cholesteryl esters, which is in agreement with the function of parenchymal cells in lipid metabolism. Fat-storing cell lipid droplets consisted of retinyl esters and triglycerides, which correlates well with their function in retionid storage and metabolism.

Isolation, purification, and characterization of liver cell types

Publisher Summary The liver plays a central role in the uptake, storage, and mobilization of retinol (vitamin A) in the body. The metabolism of retinoids in the liver, where over 95% of the retinoids in the body is found, is both complex and highly regulated. Specific functions in retinoid metabolism have been described for parenchymal and fat-storing cells. Possibly, Kupffer cells may have a function in retinoid metabolism as well. Liver cell isolation procedures have been widely applied to study cell-specific functions in liver retinoid metabolism. This chapter describes methods available for the isolation, purification, and characterization of parenchymal, fat-storing, Kupffer, and endothelial cells. Isolated liver cells have been widely used to study the cellular distribution of retinoids, retinoid-binding proteins, and enzyme activities important in retinoid metabolism. The results obtained are generally consistent with in vivo data. Cell isolation procedures are currently being used to define further the respective roles of the different liver cells types in retinoid metabolism.

Alterations in cytokeratin expression precede histological changes in epithelia of vitamin A-deficient rats

SummaryNormal epithelial cell differentiation is charactezied by the production of distinct cytokeratin proteins. It is well known that epithelia of several organs show squamous metaplasia in a vitamin A-deficient status. It is not yet known whether these histological changes are concomitant with a change in cytokeratin expression. Therefore, 3-week-old female rats (BN/BiRij) were fed a vitamin A-deficient diet for 8 weeks. The cytokcratin expression in epithelia of various organs was monitored immunohistochemically during the induction of vitamin A deficiency. Therefore, monoclonal antibodies specific for human cytokeratin 4, 5, 5+8, 7, 10, 14, 18 and 19 were used. In a normal vitamin A status, the distributional pattern for the different cytokeratins in rats was similar to that reported for human tissue. No change in cytokeratin expression was seen in trachea, skin, liver and colon at any time point studied. Squamous metaplasia in urinary bladder and salivary glands was observed after six weeks on the vitamin A-deficient diet. This was concomitant with a substitution of cytokeratins 4, 5+8, 7, 18 and 19 by cytokeratin 10. The latter cytokeratin is specific for keratinzed squamous epithelium. A change in cytokeratin expression was observed in bladder, ureter, kidney, salivary glands, uterus and conjunctiva before histological alterations appeared. In conclusion, the changes in cytokeratin expression observed under vitamin A deficiency in epithelia in vivo are in agreement with those described in other studies for epithelial cells in vitro. The changes in cytokeratin expression and the subsequent differentiation into squamous cells occurs in basal cells of the bladder but not in transitional cells. Furthermore, histological alterations are preceded by changes in cytokeratin expression indicating that vitamin A status controls cytokeratin expression in vivo.

Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions

It is now well documented that lecithin‐retinol acyltransferase (LRAT) is the physiologically important enzyme activity involved in the esterification of retinol in the liver. However, no information regarding the cellular distribution of this enzyme in the liver is presently available. This study characterizes the distribution of LRAT activity in the different types of rat liver cells. Purified preparations of isolated parenchymal, fat‐storing, and Kupffer + endothelial cells were isolated from rat livers and the LRAT activity present in microsomes prepared from each of these cell fractions was determined. The fat‐storing cells were found to contain the highest level of LRAT specific activity (383 ± 54 pmol retinyl ester formed min−1·mg−1 versus 163 ± 22 pmol retinyl ester formed min−1·mg−1 for whole liver microsomes). The level of LRAT specific activity in parenchymal cell microsomes (158 ± 53 pmol retinyl ester formed min−1‐mg−1) was very similar to LRAT levels in whole liver microsomes. The Kupffer + endothelial cell microsome fractions were found to contain LRAT, at low levels of activity. These results indicate that the fat‐storing cells are very enriched in LRAT but the parenchymal cells also possess significant levels of LRAT activity.

Endotoxin-induced liver injury in aged and subacutely hypervitaminotic a rats

The plasma disappearance of endotoxin and endotoxin-induced hepatic injury were studied in two rat models: the aging rat and the subacutely hypervitaminotic A rat. The choice of these models was based on their respective association with a decreased or increased Kupffer cell endocytic activity. The half-life of endotoxin (E. coli O26: B6, phenol extracted) in plasma was significantly prolonged in aged rats as measured by both the Limulus assay (t1/2 = 2.1 +/- 0.1 h in 3-6-month-old, and 3.3 +/- 0.3 h in 24-36-month-old rats) and 51Cr-labeled endotoxin radioactivity assay (t1/2 = 5.3 +/- 0.3 h in 3-6-month old and 7.7 +/- 0.6 h in 24 36-month-old rats). In subacute hypervitaminosis A, the half-life of endotoxin was significantly decreased in the Limulus assay (t1/2 = 2.1 +/- 0.1 h in 3-6-month old and 1.4 +/- 0.2 h in subacutely hypervitaminotic A rats), but not in the radioactivity assay (t1/2 = 5.3 +/- 0.3 h in 3-6-month-old and 5.0 +/- 0.4 h in subacutely hypervitaminotic A rats). Hundred percent mortality was observed at a dose of 2 mg endotoxin/100 g body wt. in old rats, but not in young rats. Only 1 of 7 young subacutely hypervitaminotic A rats died following injection of this dose of endotoxin. The dose of endotoxin which caused only minimal parenchymal liver cell injury in young rats induced substantial parenchymal cell injury in old rats and subacutely hypervitaminotic A rats as determined by both histological and biochemical parameters. It is concluded that some basic characteristics of experimental animals, such as age and nutritional status, can dramatically influence the sensitivity to endotoxin and this is not necessarily correlated with the rate of endotoxin clearance.

Enhancement of Tissue-type Plasmhogen Activator Levels by Retinoids in Rat Tissues in Vivo

We have previously reported that retinoids (vitamin A and derivatives) stimulate the synthesis of tissue-type plasminogen activator (tPA) but not PA inhibitor 1 (PAI-1) in cultured human endothelial cells. * To assess whether a similar effect could be obtained in h, female rats were fed retinoids for various periods of time, and the plasma and tissue concentrations of tPA and PAI-1 were determined.