Theo J.C. Van Berkel

In vivo uptake of human and rat low density and high density lipoprotein by parenchymal and nonparenchymal cells from rat liver.

Abstract The relative contribution of the parenchymal and nonparenchymal rat liver cells to the hepatic uptake of human and rat high density lipoprotein (HDL) and low density lipoprotein (LDL) was determined in vivo. Non-parenchymal cells, isolated 6 h after intravenous injection of iodinated human HDL and LDL, contained respectively 4.2 and 6.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For rat iodinated HDL and LDL these factors were 3.4 and 4.1, respectively. These results indicate that nonparenchymal liver cells play a substantial role in the hepatic uptake of human and rat HDL and LDL in vivo.

Saturable high affinity binding, uptake and degradation of rat plasma lipoproteins by isolated parenchymal and non-parenchymal cells from rat liver

Abstract 1. Freshly isolated rat parenchymal and non-parenchymal liver cells bind iodinated rat very low density lipoprotein (VLDL) remnants, low density lipoproteins (LDL) and high density lipoproteins (HDL) in a saturable way. The apparent K m values for the binding of VLDL remnants are 6–20-fold lower than for LDL or HDL. The binding per mg cell protein to non-parenchymal cells is 5–8-fold higher than to parenchymal cells. Competition experiments indicate that rat VLDL-remnants, LDL and HDL, but not human LDL compete for the same surface receptor. It is concluded that the source of common recognition could be apolipoprotein E and that the interaction with the receptor is also influenced by the apolipoproteins A and C. The high apparent affinity of the receptor for VLDL remnants might be the result of multiple receptor occupancy of this lipoprotein. The presence of a 5–8-fold higher concentration of the described lipoprotein receptor in non-parenchymal cells as compared to parenchymal cells explains the relatively high uptake of VLDL remnants (as compared to LDL and HDL) as well as the relative contribution of parenchymal and non-parenchymal cells to the total hepatic uptake of lipoproteins in vivo. 2. The greater part (70–80%) of the parenchymal and non-parenchymal cell-associated apolipoproteins, LDL or HDL, remains bound to the external surface of the cells, during in vitro incubation at 37°C. High-affinity degradation of apolipoprotein(s) by isolated liver cells is dependent on the specific lipoprotein. During a 3 h incubation at 37°C, 37–49% of the total cell-associated 125 I-abeled HDL is degraded. These percentages are 11–13% for 125 I-labeled LDL and 4–8% for 125 I-labeled VLDL remnants. Degradation of the different lipoproteins by non-parenchymal liver cells occurs at a 3–6-times higher rate per mg cell protein than by parenchymal cells. It is suggested that the rate-limiting step in the degradation of apolipoprotein by isolated liver cells is their transport to intracellular degradation sites.

Saturable high affinity binding of low density and high density lipoprotein by parenchymal and non-parenchymal cells from rat liver

Abstract Freshly isolated parenchymal liver cells bind both low density lipoprotein (LDL) and high density lipoprotein (HDL). With increasing concentrations of LDL and HDL the amount of cell-associated radioactivity approaches saturation and a linear double-reciprocal plot for the binding is obtained. The binding of LDL and HDL to isolated non-parenchymal liver cells is also saturable and the maximal binding of LDL and HDL per mg cell protein is 4–5 times higher than with parenchymal cells. It is suggested that the presence of a 4–5 fold higher concentration of lipoprotein receptor (for LDL and HDL) on non-parenchymal cells as compared to parenchymal cells explains the 4–5 times higher uptake of lipoproteins by the non-parenchymal liver cells, observed in vivo .

Cyclic nucleotide-, pyruvate- and hormone-induced changes in pyruvate kinase activity in isolated rat hepatocytes

Summary Incubation of isolated rat hepatocytes with glucagon (10−6 M), db-cAMP (0.1 mM) and db-cGMP (0.1 mM) causes a decrease in pyruvate kinase activity of 46, 49 and 34% respectively, when measured at 1 mM Mg2+free and suboptimal substrate (P-enolpyruvate) concentrations, while the Vmax is uninfluenced. An increase in activity (25%) is noticed when the cells are incubated with 1 mM pyruvate. The glucagon inactivated enzyme (Lb) shows a decreased affinity for the substrate P-enolpyruvate and for the allosteric activator Fru-1,6-P2 as compared to the activated form (La). The nature of the hormone and cyclic nucleotide-induced changes in pyruvate kinase is discussed. It is concluded that the P-enolpyruvate cycle is under comparable acute hormonal control as the FDPase-PFK cycle. Both cycles are linked by the common effector Fru-1,6-P2 making not only direct but also indirect hormonal control of pyruvate kinase flux possible.

The role of non-parenchymal cells in liver metabolism

Abstract The biochemical characterization o f the different cell types found in the liver is now possible with the advent of methods for isolating and purifying parenchymal and non parenchymal cells. Biochemical and morphometric studies allow us to calculate the contributions that each type of cell makes to the functions of the liver. Nonparenchymal cells appear to specialize in the (specific) binding, uptake and degradation of materials from the blood circulation. It is also suggested that metabolic co-operation exists between the parenchymal and non-parenchymal cells.

Iodine labeled human and rat low-density and high-density lipoprotein degradation by human liver and parenchymal and non-parenchymal cells from rat liver.

Abstract 1. 1. The abilities of homogenates of human liver, rat liver parenchymal cells, rat liver non-parenchymal cells and total rat liver to catabolize human and rat iodinated high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were determined by measuring the amount of trichloroacetic acid-soluble (noniodide) radioactivity liberated upon incubation at the optimum pH of 4.2. 2. 2. A comparison of the capacities of human liver, rat liver and parenchymal and non-parenchymal cells from rat liver indicated that these different preparations are all able to degrade rat iodine-labeled LDL and HDL, with a 5–6-fold higher capacity for HDL as compared to LDL. 3. 3. Iodine-labeled human HDL can be degraded by rat liver, rat parenchymal and rat non-parenchymal cells with a 5–7-fold higher rate than human iodinelabeled LDL. Human liver homogenate was more active in the degradation of both human and rat iodine-labeled LDL and rat HDL as compared to rat liver. 4. 4. A comparison of the capacities of parenchymal and non-parenchymal cells for the degradation of iodine-labeled human and rat LDL and HDL indicates that non-parenchymal cells possess a considerable higher capacity to degrade these lipoproteins per mg of cell protein. 5. 5. The results indicate that a high proportion of the total rat liver capacity for lipoprotein degradation is localized in the non-parenchymal liver cells and this, together with the active endocytic activity, suggests an important role of these liver cells in hepatic lipoprotein catabolism.

Identity and activities of superoxide dismutase in parenchymal and nonparenchymal cells from rat liver.

Abstract Intact and pure parenchymal and nonparenchymal cells were isolated from rat liver. The activities of Superoxide dismutase in these cell types were determined by two different methods. With both methods the specific activity of this enzyme is 1.5 times higher in parenchymal than in nonparenchymal liver cells. It can be calculated that about 7% of the total rat liver Superoxide dismutase activity is localized in the nonparenchymal liver cells. Electrophoresis on polyacrylamide gels indicates that the isolated parenchymal cells contain both cytosolic and mitochondrial isoenzymes, whereas with nonparenchymal cells only the cytosolic enzyme could be detected. The mitochondrial band observed in isolated parenchymal cells is absent in the original total liver homogenate. This isoenzyme seems to be activated during the parenchymal cell isolation procedure. Isoelectrofocusing indicates that the cytosolic Superoxide dismutase consists in four different isoelectric forms in both parenchymal and nonparenchymal cells. With the mitochondrial isoenzyme two bands are obtained. The possibility that O 2 − is an important intermediate in H 2 O 2 formation in nonparenchymal liver cells is discussed. In this respect, Superoxide dismutase might not only protect the cell against a toxic reagent as O 2 t - , but might also help to regulate the level of the important antimicrobial agent, H 2 O 2 .

Cellular localization of the receptor-dependent and receptor-independent uptake of human LDL in the liver of normal and 17α-ethinyl estradiol-treated rats

The cellular localization in the liver of the receptor‐dependent and ‐independent uptake of human low density lipoprotein (LDL) in normal and 17α‐ethinyl estradiol‐treated rats was investigated by the simultaneous in vivo injection of human 131I‐LDL and human reductive methylated 125I‐LDL. The cells were subsequently isolated by a low temperature method. In untreated rats, after 30 min of in vivo circulation of human LDL, 57% of the receptor‐dependent liver‐association of human LDL occurs in non‐parenchmal cells and 43% in parenchymal cells. Estradiol treatment of rats for 3 days selectively increases the receptor‐dependent cell‐association of human LDL with hepatocytes (17‐fold), while the receptor‐dependent cell‐association with non‐parenchymal cells is not affected.

High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.

The effect of Ca2+ and trifluoperazine on the processing of human acetylated low density lipoprotein by non-parenchymal liver cells

Studies with human fibroblasts have defined a receptor-mediated pathway by which cells take up and degrade low density lipoprotein (LDL), the major cholesterol(ester)-transport protein in human plasma [ 1 ]. Internalization of the particle is followed after recognition by a specific receptor. These receptors are clustered in coated regions of the cell membrane, so-called coated pits. Upon LDL binding the coated pits invaginate and form coated endocytotic vesicles which fuse subsequently with lysosomes. The protein moiety and the cholesterol esters are then degraded by the action of cathepsins and acid cholesterol esterase, respectively. Although the different steps involved in receptor-mediated endocytosis have been described, the molecular mechanisms involved in the process are largely unknown. Peritoneal macrophages possess a binding site for acetylated LDL which is distinct from the native LDL receptor [2]. On macrophages this binding site is coupled to a very active internalization and degradation process. This property facilitates the study of the molecular mechanism of the uptake and degradation pathway. This is also illustrated by the finding that after injection of lzSI-labeled acetyl-LDL into rats, these particles are rapidly cleared from the circulation (<.3 rain). The radioactivity is subsequently merely recovered in the non-parenchymal liver cells (unpublished). This study describes the in vitro binding of both native and acetylated LDL to freshly isolated non-parenchymal liver cells. Binding of acetylated LDL to these cells is only twice as high as the binding of native LDL but the degradation is increased 30-50-fold after acetylation of the LDL. The degradation of acetylated LDL is inhibited by chloroquine and NH4C1, indicating a lysosomal process. Mg-EDTA at 2 mM inhibits the degradation of acetyl-LDL by 50% and trifluoperazine (50/IM), an inhibitor of calmodulin [3,4], blocks the degradation completely. The rate of association of acetyl-LDL with non-parenchymal cells is only slightly inhibited by trifluoperazine. It is concluded that the main action of trifluoperazine is exerted on the route of acetyl-LDL to the lysosomes after the initial binding process. The data are consistent with a role of calmodulin in the receptor-mediated endocytotic process although it cannot be excluded that trifluoperazine interacts with another still unknown target.