Dick L. Knook

Fat-storing cells of the rat liver: Their isolation and purification

Abstract Fat-storing cells from the lobular area of the rat liver have been isolated by digesting the liver with pronase E and collagenase, and purified by Metrizamide density centrifugation and centrifugal elutriation. More than 70% of the cells in the final fraction were fat-storing cells. Per gram wet weight of liver, 3.1 ± 0.5 × 106 cells were isolated. The purified cells showed a well preserved ultrastructure and contained lipid droplets with a fluorescence characteristic of vitamin A. A HPLC technique demonstrated the presence of large quantities of retinol and retinyl palmitate in the isolated fat-storing cells.

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.

Primary cultures of endothelial cells of the rat liver

SummaryA new isolation and purification procedure for endothelial cells of the rat liver and the conditions for large scale survival of these cells in maintenance culture are reported. Cells isolated by this new method and cultured with homologous rat serum on a collagen matrix show the restoration of several ultrastructural characteristics typical of rat liver endothelial cells in situ, including the broad cytoplasmic extensions that contain the sieve plates. These fenestrated cytoplasmic projections, which cover the liver sinusoids in vivo, are well preserved and are reformed in a manner reminiscent of the situation in situ. Reformation of specific membrane receptors is indicated by the reappearance of the capacity to take up horseradish peroxidase by adsorptive endocytosis, a characteristic that is lost during the cell isolation procedure. From the results obtained in this study, maintenance culture of rat liver endothelial cells seems to be a promising system for studying the regulation of pore size of the fenestrated sieve plates by alcohol and certain hormones, for studying the interaction of endothelial cells with other liver cells and tumor cells, and for studying the mechanisms of adsorptive endocytosis.

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 and culture of Kupffer cells from human liver: Ultrastructure, endocytosis and prostaglandin synthesis

Kupffer cells and other sinusoidal cells were isolated after perfusion and incubation with pronase and collagenase of pieces of liver tissue obtained from organ donors. The resulting cell preparations contained endothelial cells, Kupffer cells and fat-storing cells as well as considerable numbers of leucocytes. Attempts to purify the different sinusoidal cell types by density centrifugation and centrifugal elutriation were successful only for Kupffer cells. Kupffer cells, in contrast to endothelial cells and fat-storing cells, could be kept in maintenance culture for at least 5 days. Cultured Kupffer cells were active in the endocytosis of foreign substances, such as colloidal carbon, latex beads, horseradish peroxidase and bacterial endotoxin. The cultured Kupffer cells synthesized and secreted considerable amounts of prostaglandins PGE2, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane B2. The production of prostaglandins was influenced by the presence of Escherichia coli endotoxin.

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.

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.