Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

After stimulation of the protein secretion by pilocarpine or feeding the rate of incorporation of [3H]-leucine increases in the acinar cells of the parotid gland of the rat while the secretory cells of the submandibular gland show a moderate decrease (Kuijper et al., 1975b). Since the rate of labelled amino acid incorporation depends on the specific radioactivity of the amino acid used, which is not easy to determine in vivo, experiments in vitro were performed to get an idea of the influence of this factor on the measured changes in [3H]-leucine incorporation. In vitro both cell types showed a more pronounced but essentially identical reaction as in vivo. Since in these experiments the specific radioactivity of the extracellular leucine is the same whether fragments of stimulated or unstimulated glands incorporate the radioactive amino acid, the increase of incorporation in the parotid and the decrease in the submandibular cells cannot be ascribed to differences in specific radioactivity of leucine, unless the intracellular leucine pool should show great differences between secreting and non-secreting cells. However, in vitro the submandibular gland cells under both conditions appear to use the extracellular leucine for their protein synthesis (or a small compartmentalized pool in rapid exchange with the extracellular pool). In the parotid cells the whole intracellular pool showed such a rapid exchange with the extracellular one that for practical reasons one may say that these cells, too, rely on the extracellular specific radioactivity of leucine in their protein synthesis. We conclude that the rat parotid gland cells show a rapid and substantial increase of protein synthesis after stimulation of their enzyme secretion, while the submandibular gland cells do not.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Vitellogenesis by the American cockroach: Haemolymph and follicle protein patterns during vitellogenin synthesis

Radioactive ¹⁴C-leucine is removed from the blood within 4 hr of injection during the first 2 days of the vitellogenic cycle. Injections during the 3rd to 6th day result in leucine retention and a rise in labelled protein.Label appears in the follicle by day 3 with most of the protein being incorporated during day 5. Comparison of haemolymph and follicle proteins suggests that fat body synthesis, subsequent haemolymph transport and follicle uptake all occur primarily on days 4 and 5 of the cycle.In vivo follicle incubations reveal ¹⁴C-leucine uptake during the last 4 days of the cycle. During days 4, 5, and 6, leucine is incorporated into protein by the follicle. Injections of ¹⁴C-haemolymph proteins into 6 day females result in the incorporation of label into the terminal oöcytes.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Effect of temperature on protein synthesis and leucine transport by yeast mitochondria

Protein synthesis in yeast mitochondria shows biphasic Arrhenius plots both in vivo and in vitro, with a twofold increase in the activation energy below the transition temperature suggesting a functional association between mitochondrial protein synthesis and the inner membrane. Analysis by gel electrophoresis of mitochondrial translation products labeled in vivo showed that the same proteins are synthesized and then inserted into the membrane above and below the transition temperature of the membrane. The rate of leucine uptake into mitochondria was decreased at least fivefold in the presence of chloramphenicol, suggesting that leucine is used mainly for protein synthesis. In the absence of chloramphenicol, the rate of leucine uptake was always slightly higher but comparable to the incorporation rate of leucine into protein at all temperatures studied, suggesting that the transport of leucine into mitochondria is not rate-limiting for protein synthesis. The ionophore valinomycin or the uncoupler carbonyl phenylhydrazone (CCCP) inhibited 75-80% of the leucine uptake in the presence of chloramphenicol. In addition, the omission of respiratory chain substrates and the ATP-regenerating system led to a 93% inhibition of uptake, suggesting that leucine uptake may occur by an active transport mechanism.     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice.

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

PROTEIN SYNTHESIS IN THE BRAIN OF OCTOPUS VULGARIS, LAM

Abstract— The process of protein synthesis in the brain of Octopus vulgaris Lam has been examined after systemic administration of [³H]leucine and upon incubation of the tissue in sea water containing the radioactive precursor. After injection of [³H]leucine in the branchial heart, the radioactivity of the TCA-soluble fractions of the three main brain divisions reached a maximum in about 30 min and decreased thereafter, while incorporation into the protein fractions was complete in approx. 2 h. Per unit wet weight the radioactivity of brain proteins was higher than that of most other organs. In vitro the rate of incorporation of [³H]leucine in the protein fraction of the optic lobe remained low for more than 1 h, but increased several fold thereafter. Preincubation of the tissue in sea water abolished the lag period. Similar effects were observed in the vertical lobe as well as in the optic lobe of young and adult octopuses but not in the white body, a non-nervous organ. The process of protein synthesis in the optic lobe is markedly inhibited by puromycin, cycloheximide and chloramphenicol. Electrophoretic analysis on polyacrylamide gels indicated that the soluble proteins labelled in vitro and in vivo are similar.     

Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.     

Control by insulin and insulin-related growth factor 1 of protein synthesis in a cell-free translational system from chick-embryo fibroblasts.

Insulin and insulin-related growth factor 1 (IGF-1) increase by 1.5-1.6-fold the rate of [3H]leucine incorporation into protein in primary monolayer cultures of chick-embryo fibroblasts (CEF); half-maximal hormone concentrations are 10 and 0.25 nM respectively. To investigate the mechanism of this effect, a rapid method is used to prepare a lysate from CEF which is active in protein synthesis. Lysate derived from cells treated for 30-150 min with insulin synthesized protein at 1.8-3.0-fold greater rate than did controls; the increased rate persisted for 20 min in vitro. Pactamycin (0.5 microM), an inhibitor of peptide-chain initiation, inhibited protein synthesis by 50% in lysates derived from insulin-treated and control cells. Thus insulin and IGF-1 cause an increase in the protein-synthesis rate in vivo, which persists in cell-free protein-synthesizing lysates of CEF.     

Kinetics of insulin action on protein synthesis in isolated adipocytes. Ability of glucose to selectively desensitize the glucose transport system without altering insulin stimulation of protein synthesis

When adipocytes were exposed to [3H]leucine for times ranging from 5 to 180 s, leucine was found to enter cells rapidly and equilibrate with the cell interior within 5 s. After an additional 15-30 s [3H]leucine was incorporated into nascent protein, and the rate of incorporation was linear for up to 6 h in both control and insulin-treated cells. Since treatment of adipocytes with 10 ng/ml insulin enhanced the rate of leucine incorporation 2-3-fold with minimal or no effect on the rate of protein degradation or leucine uptake, we conclude that the predominant effect of insulin is on enhancement of protein synthesis. To assess the time required for insulin to stimulate protein synthesis, we preincubated cells with 10 ng/ml of insulin for various times from 2 to 30 min and then measured [3H]leucine incorporation into protein during a 4-min assay. These results revealed that the insulin stimulation of protein synthesis is rapid (t 1/2 of 4.4 min), but 9-fold slower than insulin activation of glucose transport (t 1/2 less than 0.5 min under identical conditions). In contrast to the rapidity of insulin activation, we found that deactivation proceeded at much slower rates (t 1/2 of 32 and 21 min for protein synthesis and glucose transport, respectively). Desensitization of the glucose transport system has previously been shown to occur after adipocytes are exposed to high glucose and insulin. To examine the specificity of desensitization, we treated cells for 6 h with 20 mM glucose and 25 ng/ml insulin and then examined insulin sensitivity and maximal insulin responsiveness of both the glucose transport and protein synthesis systems. After treatment, the glucose transport was markedly insulin-resistant (60% loss in maximal insulin responsiveness and a marked loss in insulin sensitivity), whereas the protein synthesis system exhibited neither diminished insulin responsiveness nor loss of insulin sensitivity. In fact, insulin sensitivity actually increased, as indicated by the finding that less insulin was required to stimulate protein synthesis (insulin ED50 values of 0.25 and 18 ng/ml at 0 and 6 h of treatment). From these studies we conclude that desensitization of the glucose transport system by glucose and insulin treatment appears to be specific for this particular effector system and does not reflect a state of generalized cellular insulin resistance.     

Effect of chronic ethanol ingestion on pancreatic protein synthesis

The effect of chronic ethanol feeding on pancreatic protein synthesis was assessed by studying the rate of incorporation of [3H]leucine into proteins in isolated rat pancreatic acini in vitro. Chronic ethanol feeding increased the rate of protein synthesis (2-3-fold) compared to controls fed an isocaloric diet. The onset of the increase in protein synthesis was detectable 2 days after the beginning of ethanol feeding, reached a maximum after 7 days and remained constant for up to 4 months. The increased incorporation of [3H]leucine was not due to an increased turnover of proteins as measured in pulse-chase experiments. After separation of individual digestive enzymes by SDS-polyacrylamide gel electrophoresis and determination of the distribution of radioactivity in different proteins, a general increase in the rate of incorporation of the label into all of the proteins was observed. In contrast to the observations made with isolated acini, there was no significant difference between the control and ethanol-fed groups when the rate of pancreatic protein synthesis was measured in vivo. However, overnight withdrawal of ethanol led to an increase of approx. 70% in protein synthesis in the ethanol-fed group. These results suggest that chronic ethanol ingestion modifies the control of pancreatic protein synthesis; the enhanced protein synthesis is expressed in isolated acini, i.e., in the absence of physiological factors present during chronic ethanol ingestion and in vivo after ethanol withdrawal.     

Incorporation of fucose and leucine into PNS myelin proteins in nerves undergoing early Wallerian degeneration

The simultaneous incorporation of [³H]fucose and [1-¹⁴C]leucine into normal rat sciatic nerve was examined using an in vitro incubation model. A linear rate of protein precursor uptake was found in purified myelin protein over 1/2–6 hr of incubation utilizing a supplemented medium containing amino acids. This model was then used to examine myelin protein synthesis in nerves undergoing degeneration at 1–4 days following a crush injury. Data showed a statistically significant decrease in the ratio of fucose to leucine at 2, 3, and 4 days of degeneration, which was the consequence of a significant increase in leucine uptake. These results, plus substantial protein recovery in axotomized nerves, are indicative of active synthesis of proteins that purify with myelin during early Wallerian degeneration.     

Amino acid transport in the exocrine pancreas. IV. Do glucagon or insulin mediate the in vivo effect of caerulein on amino acid transport and incorporation?

The direct in vitro effect of caerulein on pancreatic protein synthesis and amino acid transport has been investigated. In contrast to in vivo conditions we were unable to demonstrate any effect on alpha-aminoisobutyric acid and leucine uptake and on leucine incorporation usin rat pancreatic lobules. Insulin and glucagon were therefore examined as possible mediators for the in vivo effect of caerulein. Insulin (1--5 microM) slightly enhanced AIB uptake (16% but did not change uptake and incorporation of leucine. Glucagon (0.01--1 microM) was ineffective. Both islet hormones had no influence on the formation of cyclic GMP induced by secretagogue either in rat (40% increase) or in guinea pig lobules (500% increase). It seems unlikely that the two islet hormones exert any direct effect on the exocrine pancreas and thus could serve as mediators for the in vivo synthetic effect of caerulein.     

A mevalonate requirement for maintenance of fatty acid and protein synthesis during hormonally stimulated development of mammary gland in vitro

The effect of compactin on hormonally induced lipogenesis and protein synthesis was studied in vitro in explants of mammary gland from mid-pregnant rabbits. Compactin blocks mevalonate synthesis by the specific inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, and in this system, culture with 10 microM compactin for 24, 48, and 72 h inhibited incorporation of [1-14C]acetate (but not [2-14C]mevalonate) into sterol by 98, 95, and 86%, respectively. Removal of compactin prior to assay rapidly reversed this effect and was associated with increased tissue 3-hydroxy-3-methylglutaryl-CoA reductase activity. Fatty acid synthesis (measured by incorporation of [1-14C]acetate or [4,5-3H]leucine) and protein synthesis (measured by incorporation of [4,5-3H]leucine) were both inhibited by around 50% after culture with compactin. This inhibition was not rapidly reversed by removal of compactin prior to assay, but it was prevented by inclusion of 1 mM mevalonolactone in the culture medium. After removal of compactin and continued culture in its absence for 24 h with hormones, the normal tissue capacity for fatty acid and protein synthesis was restored, indicating no permanent cell damage. The results suggest a specific requirement for mevalonate (or derived products) for the hormonal maintenance of the increased fatty acid and protein synthesis characteristic of the development of the mammary gland.     

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.     

Inhibition of butylated hydroxytoluene-induced mouse lung cell division by oxygen: time-effect and dose-effect relationships

Mice were injected i.p. with 250 or 400 mg/kg of butylated hydroxytoluene (BHT). In vivo incorporation of thymidine into pulmonary DNA was measured on days 1-7 after BHT. 2, 3 and 4 days after BHT, DNA synthesis was inhibited by a 24-h exposure to 100% oxygen, whereas on days 5, 6 and 7 after BHT, oxygen failed to depress synthesis. A similar pattern was observed when incorporation of leucine into protein was measured: 2 and 4 days after BHT, oxygen decreased leucine incorporation, but had no effect 6 days after BHT or in animals not pretreated with BHT. It is concluded that the cells proliferating early after BHT, the type II alveolar cells, are more susceptible to the cytotoxic effects of oxygen than are interstitial and capillary endothelial cells.     

Determination of rates of protein synthesis, gain and degradation in intact hind-limb muscle of lambs.

A model of leucine metabolism in the hind-limb muscles of the milk-fed lamb was developed which permitted simultaneous estimation of the rates of protein synthesis (Ks, days-1), degradation (Kd) and therefore gain (Kg) of muscle in vivo. The conclusions drawn from the model were: the rate of protein synthesis in muscle was related to uptake of leucine; the rate of degradation of protein was related to leucine output, as leucine, or its corresponding oxo acid, 4-methyl-2-oxopentanoic acid, or CO2. These findings support findings drawn from a wide range of studies in vitro. There was no correlation between rate of protein synthesis and rate of protein degradation, which suggests that the method can allow independent estimates of each. Estimates of protein synthesis obtained from the model (of leucine metabolism in muscle) were compared with those obtained simultaneously by constant infusion of radioisotope and analysis of incorporation into tissue. There were no significant differences between the mean values obtained for synthesis (Ks), gain (Kg) and degradation (Kd) by either method (Ks 0.051 +/- 0.002, 0.046 +/- 0.007; Kg 0.016 +/- 0.002, 0.004 +/- 0.008; Kd 0.035 +/- 0.004, 0.041 +/- 0.008 day-1, respectively, for tissue analysis and the model). However, Ks obtained from the model was significantly and positively correlated with uptake of leucine from plasma, whereas Ks obtained from tissue analysis was not.     

The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat.

The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per mug DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. The absence of a short term effect of insulin on the rate of protein synthesis does not exclude a long term effect as suggested by the higher rate of incorporation in the cells of peri-insular acini.     

The effect of insulin on free amino acid pools and protein synthesis in rat skeletal muscle in vitro

1. The effects of insulin in vitro on tissue pools and incorporation into protein of glycine and leucine in the extensor digitorum longus muscle of the rat are reported. 2. It was found that insulin decreased the lag period before the establishment of a linear rate of incorporation of radioactive glycine into protein. 3. The hormone increased the size of the free intracellular glycine pool. No such effect was found for leucine. The accumulation of radioactive glycine in the intracellular fluid compartment was increased. The content of radioactive leucine in the intracellular compartment was decreased. 4. Insulin decreased the specific radioactivity of both glycine and leucine in the extracellular fluid. 5. The hormone also decreased protein catabolism. 6. The effect on protein synthesis was not caused by an increase in the specific radioactivity of the extracellular pool but was possibly related to increased amino acid concentrations in this pool, which could in turn have affected the aggregation of ribosomes.     

Vitellogenin Incorporation into Oocytes of Rainbow Trout, Oncorhynchus mykiss, in Vitro: Effect of Hormones on Denuded Oocytes

An in vitro culture procedure to measure vitellogenin (VTG) incorporation into oocytes without follicle cell layers was developed using oocytes of the rainbow trout, Oncorhynchus mykiss . Oocytes incorporated VTG specifically and linearly for up to 24 hr. The maximum incorporation observed was 314 μg/24 hr/oocyte, using vitellogenic (3.6 mm diameter) oocytes.
The effect of hormones was examined by adding insulin, growth hormone, prolactin, gonadotropins (GTH-I, GTH-II), thyroid hormones, testosterone, estradiol-17β, or 17α, 20β-dihydroxy-4-pregnen-3-one to the medium. The results indicated that insulin and thyroxine stimulated uptake of VTG by 13% and 12%, respectively. Insulin specifically stimulated VTG incorporation and did not cause any change in background uptake of insulin. The lack of an effect of gonadotropins hormones on denuded oocytes suggests that the previously observed stimulation of VTG incorporation into follicle cell-enclosed oocytes in vivo and in vitro by GTH-I is most likely mediated by the somatic cells of the ovarian follicle.