Effect of dietary restriction on the degradation of proteins in senescent mouse liver parenchymal cells in culture

We previously reported that the half-life of protein degradation in cells from old mice is about 50% longer than that in cells from young or middle-aged ones. In the present study we investigated the degradation rate of microinjected proteins (horseradish peroxidase (HRP) and ovalbumin (OVA] and pulse-labeled proteins in hepatocytes from dietary-restricted old mice. Dietary restriction was initiated when mice were 23 months of age and performed in two steps (first 80% and then 60% of the ad libitum intake), the total period being 70 days. Hepatocytes were isolated from mice fed a restricted diet and fed ad libitum. The half-lives of HRP, OVA, and pulse-labelled proteins in the hepatocytes from mice fed a restricted diet were about 40% shorter than those in the cells from mice fed ad libitum. These values were close to those in the cells of young animals. These results are discussed in relation to our previous findings that a similar regimen reduces the percentage of heat-labile enzymes accumulated in tissues of aged animals.     

Fate and structure of DNA microinjected into mouse TK L cells

Co-microinjection of single linearized molecules of plasmids containing the human β-globin gene (pRK1) and the herpes simplex virus (HSV) type I thymidine kinase gene (pX1) into the mouse TK^? L cell nucleus results in covalent linkage between these (or derived) molecules within the nucleus as revealed by Southern blotting, plasmid rescue, and recovery of plasmid-derived DNA from a Charon 4A phage library of cellular DNA. The microinjected DNA is predominantly found as high molecular weight DNA as determined by Hirt fractionation. Southern blotting data and recombinants from the Charon 4A library suggest that the plasmid DNA is in the form of a head-to-tail linear concatamer of up to 80 copies. Passage of these microinjected cells in selective medium (HAT) results in coordinate amplification of both plasmids, which are maintained in an approx. 3:1 molar ratio of pRK1 to pX1-derived molecules. Hybridization in situ shows the DNA to be integrated on a translocation chromosome, t(4;4). Integration does not appear to be site-specific, since plasmid DNA from another microinjected cell line, C2B, appears on a different translocation chromosome, t(8?;14). Plasmid rescue experiments confirm a previous finding that passage of pBR322 DNA through eukaryotic cells may result in deletions of normally stable plasmid DNA upon subsequent transformation of E. coli. These deletions appear to occur in the bacteria, and originate in a 128 bp region between the Sal I and Hae II sites of pBR322.     

Receptor-mediated endocytosis of ovalbumin by two carbohydrate-specific receptors in rat liver cells. The intracellular transport of ovalbumin to lysosomes is faster in liver endothelial cells than in parenchymal cells.

1. The uptake of ovalbumin (OVA) in rat liver parenchymal cells (PC) and non-parenchymal cells was studied in vivo and in vitro in order to compare the cellular expression of glycoprotein receptors and the kinetics of intracellular transport of ligand endocytosed by these receptors. 2. Ovalbumin was labelled with 125I or with 125I-tyramine-cellobiose (125I-TC). By using 125I-TC-OVA the labelled degradation products were trapped in the cells. 3. 125I-TC-OVA was rapidly cleared from blood mainly by receptor-mediated uptake in the liver. At 30 min after injection, 50% of the ligand was recovered in the liver. The endothelial cells (EC) and the PC were the predominant cell types responsible for uptake. 4. The uptake in PC was strongly inhibited by asialo-orosomucoid (AOM), but not by mannan, indicating that the uptake in these cells was mediated by the galactose receptor and not by the mannose receptor. This finding is compatible with the observation that a proportion of the OVA contains terminal galactose residues in the carbohydrate moiety. 5. In vitro uptake of OVA in cultured EC was saturable and inhibited by mannan, mannose, fructose, N-acetylglucosamine, EDTA or monensin, but not by galactose or AOM. The uptake of OVA in these cells was therefore mediated by the mannose receptor. 6. To label the organelles involved in endocytosis in PC and EC, 125I-TC-OVA was injected intravenously together with an excess of either AOM or mannan. In this way the labelled ligand could be directed selectively to EC or PC respectively. Subcellular fractionation of total liver in sucrose and Nycodenz gradients revealed that in EC the intracellular transport of OVA is so fast that endocytosed ligand accumulates and thus increases the density of the lysosomes. Conversely, in PC transfer of ligand is slower, with the result that accumulation of undegraded ligand in the lysosomes does not occur. These findings are interpreted to mean that in EC the rate-limiting step of handling of endocytosed ligand is intralysosomal degradation, whereas in PC the rate-limiting step is transport of ligand to the lysosomes. 7. Altogether, these findings suggest that endocytosis of OVA by the liver EC and PC is mediated by mannose and galactose receptors respectively, and that the kinetics of intracellular transport of OVA differ in the two cell types.     

Glycolytic and gluconeogenic enzyme activities in parenchymal and non-parenchymal cells from mouse liver

1. Parenchymal cells have been prepared from mouse liver by enzymic and mechanical means. 2. The dry weights, protein and DNA contents of these cells have been determined. 3. Mouse liver ;M-' and ;L-type' pyruvate kinases have been prepared free of contamination with each other; their kinetic properties have been examined and a method has been developed for their assay in total liver homogenates. 4. Recoveries of phosphoglycerate kinase, lactate dehydrogenase and phosphofructokinase in enzymically prepared cells indicate that little, if any, cytoplasmic protein is lost during preparation. 5. Parenchymal cells exhibit a very substantial increase in the activity ratio of glucokinase to hexokinase over that in total liver homogenate; in three out of eight experiments, hexokinase activity was undetectable. 6. ;L-type' pyruvate kinase alone occurs in the parenchymal cell. Non-parenchymal cells are characterized by the presence of ;M-type' activity only. 7. Parenchymal cells contain both glucose 6-phosphatase and fructose 1,6-diphosphatase. The non-parenchymal fraction appears to contain fructose 1,6-diphosphatase, but is devoid of glucose 6-phosphatase. 8. No aldolase A was detectable in the whole liver. Aldolase B occurs in both parenchymal and non-parenchymal tissue. 9. Parenchymal cells prepared by mechanical disruption of mouse liver with 20% polyvinyl alcohol exhibit a similar enzyme profile to those prepared enzymically. 10. The methodology involved in the preparation of isolated liver cells is discussed. The importance of the measurement of several parameters as criteria for establishing the viability of parenchymal cells is stressed. 11. The metabolic implications of the results in the present study are discussed.     

Uptake and degradation of bovine testes beta-galactosidase by parenchymal and nonparenchymal rat liver cells

The plasma half-life of beta-galactosidase in rat was about 1.5 min. Ten minutes after in vivo injection, 45% of the enzyme was recovered in liver, with hepatocytes and endothelial cells as the predominant cell types responsible for uptake. In vitro uptake of beta-galactosidase in hepatocytes and nonparenchymal liver cells was saturable, Ca2+-dependent and it could be partly inhibited by mannose or alpha-methyl-mannoside.     

Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis

Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mt(spr) strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mt(spr) mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.     

Cytoplasmic proteases of rat liver parenchymal cells

Soluble extracts of isolated rat liver parenchymal cells contained three proteases with alkaline pH optima. One protease was a high molecular weight (Mr = 500,000) enzyme which was stimulated by ATP. The other two proteases were totally dependent on calcium for activity and displayed different calcium concentration requirements. One was half-maximally activated by 150 μM Ca²⁺ while the other required only 10 μM Ca²⁺ for half-maximal activation.     

Subcellular distribution of rhodamine-actin microinjected into living fibroblastic cells

The time course and pattern of incorporation of rhodamine-labeled actin microinjected into cultured fibroblastic cells were examined by fluorescence microscopy. Following microinjection, the fluorescent probe was incorporated rapidly into ruffling membranes, and within 5 min faintly fluorescent stress fibers were observed. Levels of fluorescence in ruffling membranes then tended to remain constant while fluorescence of the stress fibers continued to increase until approximately 20-min postinjection. Small, discrete regions of some microinjected cells displayed high levels of fluorescence that appeared initially approximately 5-10 min postinjection. I observed these small areas of intense fluorescence frequently near the cell periphery, which corresponded to focal contacts when examined with interference reflection optics. The results of this study show that a relationship exists between patterns of fluorescent actin incorporation in these cells and cellular areas or structures presumed to play a role in cell movement. These findings suggest that actin within stress fibers and the microfilament network of ruffling membranes undergoes a rapid turnover that may relate directly to the motility of the cell.     

The turnover of tRNAs microinjected into animal cells.

Red cell-mediated microinjection has been used to study tRNA turnover in SV3T3 mouse cells and TC7 cells, an African green monkey kidney line. The turnover of endogenous tRNA, measured by labeling with 3H-methionine, was first-order with half-lives of approximately one day in SV3T3 and two days in TC7 cells. 32PtRNA isolated from E. coli or TC7 cells turned over at the same rate as endogenous tRNA when injected into either SV3T3 or TC7 cells. This demonstrates that cellular processes, not properties inherent to tRNAs, are responsible for the difference in tRNA turnover observed between SV3T3 and TC7 cells. These results further indicate that the mechanism of tRNA turnover in mammaliam cells does not distinguish prokaryotic from eukaryotic tRNAs. In contrast to unmodified tRNA, glyoxalated tRNA was rapidly degraded upon injection. Thus altered tRNA's, like altered proteins, are turned over more rapidly in animal cells.     

Degradation of proteins microinjected into cultured mammalian cells.

Iodinated proteins were degraded after injection into HeLa cells at first-order rates with half-lives varying from three hours for the trout monhistone chromosomal protein, HMG-T, -to 60 hours for whale myoglobin. Fluoresceinated-bovine serum albumin (fl-BSA) was degraded almost twice as fast as unmodified BSA. The rate of degradation of 125I-BSA was very similar in eight cell lines of mouse, human, monkey and rat origin. Microinjected proteins were analyzed on SDS-acrylamide gels after injection, and for BSA and immunoglobin G, all remaining intracellular 125I migrated at the molecular weight of the injected proteins. By contrasting, more than 80% of the extracellular 125I chromatographed as iodotyrosine. With the exception of fl-BSA, which exhibited perinuclear accumulation in approximately one-half of the injected cells, autoradiography showed that throughout the period of study the injected proteins remained dispersed in the cytoplasm.     

Propranolol inhibits the in vitro conversion of thyroxine into triiodothyronine by isolated rat liver parenchymal cells.

A model for the in vitro study of the conversion of thyroxine into triiodothyronine using isolated rat liver parenchymal cells is described. Isolated liver cells (mean protein content 18 mg/ml) convert approximately 0.8% of 1.3 microM exogenously added T4 into T3 during thirty minutes incubation. Carbimazole (50 microM) has no effect on the conversion process, whereas propylthiouracil (50 microM) inhibits the conversion. The beta-adrenoceptor blocking agent propranolol lowers the conversion ratio when added in concentrations of 580 and 1160 microM, but has no inhibitory effect when 290 microM is added.     

Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelial liver cells

Conditioned media of isolated Kupffer and endothelial liver cells were added to incubations of parenchymal liver cells, in order to test whether secretory products of Kupffer and endothelial liver cells could influence parenchymal liver cell metabolism. With Kupffer cell medium an average stimulation of glucose production by parenchymal liver cells of 140% was obtained, while endothelial liver cell medium stimulated with an average of 127%. The separation of the secretory products of Kupffer and endothelial liver cells in a low and a high molecular weight fraction indicated that the active factor(s) had a low molecular weight. Media, obtained from aspirin-pretreated Kupffer and endothelial liver cells, had no effect on the glucose production by parenchymal liver cells. Because aspirin blocks prostaglandin synthesis, it was tested if prostaglandins could be responsible for the effect of media on parenchymal liver cells. It was found that prostaglandin (PG) E1, E2, and D2 all stimulated the glucose production by parenchymal liver cells, PGD2 being the most potent. Kupffer and endothelial liver cell media as well as prostaglandins E1, E2, and D2 stimulated the activity of phosphorylase, the regulatory enzyme in glycogenolysis. The data indicate that prostaglandins, present in media from Kupffer and endothelial liver cells, may stimulate glycogenolysis in parenchymal liver cells. This implies that products of Kupffer and endothelial liver cells may play a role in the regulation of glucose homeostasis by the liver.     

Metabolism of lipoprotein acylglycerols by liver parenchymal cells

We investigated the metabolism by hepatocyte suspensions of the acylglycerols in lipoprotein remnants as well as those associated with albumin and low or high density lipoproteins. Remnants, albumin and plasma lipoproteins, rich in monoacylglycerol were prepared by short-term incubations of radio-labeled chylomicra or very low density lipoproteins with extrahepatic lipoprotein lipase in the presence of albumin and low and high density lipoproteins. We demonstrated that liver parenchymal cells contain an active monoacyl-glycerol acyltransferase that is located on the extracellular surface of the cell plasma membrane. Further, the enzyme is capable of degrading the monoacyl-glycerol in all the above forms. Triacylglycerol in intact chylomicra and very low density lipoproteins were not metabolized by the cells to any appreciable degree. The degradation of the remnant triacylglycerol appeared to depend solely on the activity of the lipoprotein lipase bound to the lipoprotein remnants. Little uptake of intact lipoprotein acylglycerols by the hepatocytes was observed; instead, hydrolysis of the substrates in the medium always preceded the uptake of the products. The products were then utilized for the synthesis of triacylglycerol and phospholipid within the cells.     

Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts

We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.     

Internalization of erythrocytes into liver parenchymal cells in naturally hibernating frogs (Rana esculenta L.)

Mature and intact red blood cells were found in hepatocytes of frogs during natural underground hibernation. No signs of erythrophagocytosis, e.g., separating membranes between erythrocyte and hepatocyte cytoplasm, and lysosomes, were observed. Red blood cells probably penetrated into hepatocytes by ameboid-like mechanisms, which can be deduced by cytoplasmic protrusions and invaginations. Most of the hepatocytes had large amounts of stored glycogen and few organelles, often segregated in condensed areas. The narrowed bile canalicular lumens without lysosomes and exocytotic vacuoles around them and the reduction of the Disse spaces indicate a low metabolic activity of liver during natural hibernation. The dramatic accumulation of red cells in the hepatocytes of hibernating frogs could share similarities with the phenomenon of internalization of leucocytes into epithelial cells of some vertebrate's tissues via emperipolesis, the mechanisms of which are not well understood.