Adriaan Brouwer

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.

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.

Alcohol in combination with malnutrition causes increased liver fibrosis in rats

Rats were malnourished for 12 months with a highly inadequate fat-rich, calorie-sufficient but otherwise poly-deficient liquid diet composed of mashed potatoes with mayonnaise, comparable with the nutritional intake of many chronic alcoholics. When alcohol was incorporated into this diet, administered as whisky in drinking water available ad libitum, the livers of all eight rats showed increased fibrosis and cirrhosis as compared to the livers of the eight non-alcohol-treated, isocalorically fed, paired control rats. Alcohol-treated rats developed fibrosis and cirrhosis on a dietary fat content of 38% of total caloric intake and low blood alcohol levels, ranging from 50 to 126 mg/dl, due to gradual intake over the day and to low absolute intake (mean 11.9 +/- 0.6 g/kg per day). None of the rats died spontaneously. Malnutrition is likely to be an important factor in the development of the fibrosis of alcoholic liver disease, and this rat model may be used to study aspects of the pathogenesis.

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.

Changes in endotoxin senstivity in ageing. Absorbtion, elimination and mortality

In this paper we describe the influence of ageing on responses to intravenously-injected endotoxin in two rat strains. Old age had no apparent effect on the absorption of 51Cr-labelled endotoxin from either jejunum or colon. Notwithstanding, aged animals appeared much more sensitive than their young counterparts to the lethal effects of intravenously injected endotoxin. Old animals exhibited virtually 100% mortality over the dose range 1-4 mg/100 g body weight while only sporadic deaths were seen in young animals. One consistent feature of dying animals was a profound and progressive hypothermia. At post mortem examination, the major findings were in the liver (leukocyte infiltrates and hepatocellular necrosis) and kidneys (acute tubular necrosis). Ageing was associated with slower removal of endotoxin from the circulation but not to an extent that could reasonably account for the enhanced sensitivity to endotoxin toxicity.

Effects of acute graft-vs-host disease on the liver of the brown Norway rat.

In this study we examined the effects of acute graft‐vs‐host disease (aGVHD) on the Brown Norway (BN) rat liver. When clinical signs of the disease appeared, rats were inoculated with fluorescent latex beads and 30 min later nonparenchymal cells were isolated from the liver. The cells were then analyzed via flow cytometry, histochemistry, and electron microscopy. Flow cytometry demonstrated that 58% of the cells from the 80 ml/min elutriation fraction (normally rich in Kupffer cells) of the non‐GVHD liver had high fluorescence intensity compared to 8% in rats with aGVHD. Determination of the cellular composition of the various fractions with electron microscopy confirmed flow cytometry observations in that only 9% of the 80 ml/min elutriation fraction of GVHD livers had peroxidase‐positive rough ER and the morphological appearance of macrophages as compared to 60% in the non‐GVHD liver. The low percentage of fluorescent‐positive Kupffer cells in the 80 ml/min elutriation fraction of the GVHD liver is attributed to a massive lymphocytic invasion of the liver and not necessarily to a defect in the mononuclear phagocyte system.