Effects of assay and acclimation temperatures on incorporation of amino acids into protein of isolated hepatocytes from the european eel, Anguilla anguilla L.

Hepatocytes of adult eels acclimated to 5° C, 10° C and 20° C, respectively were isolated by perfusion of the liver with collagenase. The liver-somatic index and the protein content of liver cells showed significantly higher values in fish kept at the lower temperatures. However, in the adenine nucleotide content and energy charge no significant differences were observed between the 5° C and the 20° C acclimation groups. The incorporation of radioactivity from a ¹⁴C-labelled amino acid mixture into perchloric acid precipitates was used as an estimate of over-all protein synthesis. When eel hepatocytes were incubated in Hanks' solution containing tracer amounts of amino acids, labelling of perchloric acid precipitates showed linear time courses over at least 60 min at 10° C and 20° C assay temperatures. The total cellular radioactivity, however, exhibited non-linear time courses. In the measurement range from 5° C to 25° C Arrhenius plots of protein labelling exhibited a discontinuity in both groups of fish. Hepatocytes from 10° C-acclimated eel showed almost twice the incorporation rates of amino acids as those from the 20° C-acclimated fish. It is concluded that high temperature dependencies in the low temperature range require an increase in the capacity of the apparatus for protein synthesis during cold acclimation.     

Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes.

Protein synthesis in isolated rat hepatocytes, as measured by the incorporation of [14C]-valine at constant specific radioactivity, proceeded at a rate of 0.3-0.5%/h in an unsupplemented medium, i.e. only about one-tenth the rate of protein degradation (4%/h). Leupeptin, which inhibits lysosomal protein degradation (previously found to be 75% of the total degradation in hepatocytes), had no effect on protein synthesis, showing that endogenous protein degradation supplied amino acids in excess of the substrate requirements for protein synthesis. The inhibition of protein synthesis by NH4Cl (another inhibitor of lysosomal protein degradation) as well as the stimulation by a physiological amino acid mixture must therefore represent indirect effects, either on general energy metabolism, or on unknown regulatory processes.     

胰岛素对鹅原代肝细胞增殖及蛋白合成的影响

通过用不同浓度胰岛素培养天府肉鹅（Ansercygnoides）原代肝细胞探讨胰岛素对鹅原代肝细胞增殖及蛋白合成的影响。结果表明：与对照组相比,0—200nmol／L胰岛素对肝细胞上清液中谷草转氨酶（AST）、谷丙转氨酶（ALT）浓度没有显著影响,表明细胞功能正常;100、150和200nmol／L胰岛素显著增加细胞培养上清液中总蛋白（什）和白蛋白（ALB）浓度;Brdu．ELISA法检测DNA合成实验结果表明经胰岛素处理后显著增加鹅原代肝细胞增殖率。因此,胰岛素能促进鹅原代肝细胞的细胞增殖及蛋白合成。     

Effects of antibiotics on protein synthesis and degradation in primary cultures of rat hepatocytes

Summary In primary hepatocyte cultures, maintained in a protein-free medium, streptomycin, penicillin, and Garamycin (gentamicin) all inhibited protein synthesis at concentrations above 0.1 mM. Some inhibition was also observed with the fungicide Mycostatin at 100 U/ml. Hepatocytic protein degradation was markedly inhibited by penicillin at concentrations above 0.1 mM, whereas streptomycin and Garamycin only showed slight inhibition at concentrations in excess of 1 mM. None of the antibiotics had any detectable effect on the structural integrity (viability) of the cells. The work was supported by grants from The Norwegian Cancer Society and The Norwegian Council for Science and the Humanities     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

Protein turnover,synthesis and secretion of albumin in hepatocytes isolated from rats bearing walker 256 carcinoma

Summary Hepatocytes isolated from rats bearing line A of Walker 256 carcinoma (WA) were used to study the turnover of total liver protein and the synthesis of albumin in comparison with ad libitum (AL) and pair-fed (PF) healthy controls. The rates of total protein synthesis by hepatocytes of WA animals were 40 and 90% higher than in AL and PF controls, respectively. The degradation of fast-turnover proteins was not affected by nutrition or by the tumor, whereas the degradation of slow-turnover proteins was slightly but significantly increased—about 24% higher in hepatocytes from WA rats than in PF controls. The combination of the two processes, synthesis and degradation, was in favor of an increased synthesis which explains the increase in liver protein content observed in vivo in WA rats. Dietary restriction did not affect the synthesis and secretion of albumin, whereas the tumor significantly reduced its synthesis by about 30%. The plasma concentration of albumin in WA rats dropped by about the same percentage compared with AL and PF animals.     

Effect of acclimatization temperatures on incorporation of amino acids into proteins of abdominal muscle from the shrimp Palaemon serratus

The effect of thermal acclimatization on the incorporation of [³H]-l-leucine into muscular acid-soluble amino acids, soluble proteins and into the lactate dehydrogenase was studied in the shrimp Palaemon serratus As far as protein synthesis is concerned, a period of time of 10 to 15 days is required for the achievement of the process. During acclimatization to 10° or 20° three periods can be distinguished. During the first 3–4 days the temperature increment exert a direct decreasing or increasing effect on the protein synthesis. The system then tends to reach a new equilibrium respectively below or above the initial level: the synthesis mechanisms were able to partially compensate the effect of thermal variations. The synthesis of LDH follows the general pattern of incorporation of the labelled precursor into the macromolecules     

Effect of experimental diabetes on the incorporation of amino acids into protein in rat aorta.

The incorporation of 14C-labelled leucine or phenylalanine into alkali-soluble protein was determined under in vitro conditions in aortic intima-media of normal and streptozotocin-diabetic rats. Two weeks after the induction of diabetes the incorporation of the amino acids into aortic protein was reduced. When determined after diabetes of one week's duration the leucine-14C incorporation was not significantly reduced, while after 5 weeks of diabetes it was severely impaired. After administration of insulin to diabetic rats in vivo for 2 weeks there was no difference in leucine-14C incorporation between normal and diabetic rats. Addition of insulin (0.1 U/ml) in vitro had no effect on the leucine-14C incorporation in either normal or diabetic aorta during incubation times of 3 or 6 h. Elevation of the glucose concentration in vitro from 5.6 to 22.2 mmol/l did not influence the leucine incorporation in diabetic aorta. Both the aortic wet weight and the aortic content of alkali-soluble protein were decreased after 5 weeks of diabetes. The decrease in the protein content of aorta of diabetic animals suggest that the protein synthesis is impaired in vivo.     

Studies on the differential response to insulin on the stimulation of amino acid incorporation into protein in isolated hepatocytes containing different levels of glycogen.

Effect of insulin on amino acid incorporation into protein by isolated rat liver hepatocytes was studied. A two to three-fold increase in the incorporation of U-¹⁴C-Leucine and U-¹⁴C-Phenylalanine into protein by insulin (100 μUnits) was observed in isolated hepatocytes containing high glycogen. This effect was abolished by the addition of glucagon (3 × 10^?6M). No stimulation in amino acid incorporation by insulin was observed when isolated hepatocytes contained low or no glycogen. Electron micrographs of incubated cells show that in the presence of insulin more normal parallel strands of polyribosomes are maintained as compared to control cell preparation.     

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

Summary High yields of human hepatocytes (up to 23×10⁶ viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10⁻⁸ M insulin, and 10⁻⁸ M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg⁻¹·min⁻¹) similar to that reported for human liver. Insulin at 10⁻⁸ M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10⁻⁹ M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α₁-antitrypsin, α₁-antichymotrypsin, α₁-acid glycoprotein, haptoglobin, α₂-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10⁻⁹ M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg⁻¹ cell protein·min⁻¹, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg⁻¹). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.     

Synchronising the availability of amino acids and glucose increases protein retention in pigs

Effects of synchronising the availability of amino acids and glucose within a day on protein and energy metabolism were studied in growing pigs. Ten pigs of on average 54 (s.e. 1.0) kg live weight were assigned to each of two dietary treatments (synchronous v. asynchronous nutrient supply) in a change-over design. On the synchronous treatment (SYN), pigs received two balanced meals: one at 0800 h and one at 1600 h. On the asynchronous treatment (ASYN), pigs received virtually all protein at 0800 h and all carbohydrates at 1600 h. The dietary supply of ingredients and nutrients to pigs was similar for both treatments. Pigs were housed individually in respiration chambers. Faecal apparent nutrient digestibility was determined and nitrogen and energy balances were measured. Faecal apparent digestibility of energy, organic matter and non-starch polysaccharides was higher ( P < 0.05) for SYN than for ASYN. The efficiency of utilisation of digestible protein with protein gain was higher ( P = 0.001) for SYN (56.7%) than for ASYN (47.1%). The substantial decrease ( P < 0.05) in respiratory quotient and ¹³C enrichment of the expired CO₂ after the morning meal indicated higher amino acid oxidation for ASYN than for SYN. Heat production and energy retention as fat were not affected by nutrient synchrony. In conclusion, an asynchronous availability of glucose and amino acids within a day increases amino acid oxidation, resulting in a substantial reduction in protein utilisation but with virtually no effect on fat retention.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.