Indomethacin inhibits the insulin-induced increases in RNA and protein synthesis in L6 skeletal muscle myoblasts

Rates of accretion of RNA and protein and rates of protein synthesis were measured in sub-confluent cultures of L6 myoblasts. Insulin (100 microU/ml) stimulated protein synthesis by 15% within 30 min and by 40% at two and six hours. By six hours insulin also increased the accretion of RNA (+15%). The cyclo-oxygenase inhibitor indomethacin did not reduce the basal rate of RNA or protein accretion in L6 cells but reduced the rate of protein synthesis by 16%. When added together with insulin, indomethacin inhibited the hormonally-stimulated rate of protein synthesis and also significantly reduced the accretion of RNA. Indomethacin still reduced the effects of insulin on protein synthesis when added to the cells two hours after the hormone. Synthesis of RNA measured by the incorporation of [3H]-uridine was also stimulated by insulin but was inhibited by indomethacin only when the drug was present throughout the incubation. Inhibition of protein synthesis by cyclo-oxygenase inhibitors may be the result of both a direct action on translational efficiency and an effect on RNA synthesis.     

The cyclo-oxygenase inhibitors indomethacin and ibuprofen inhibit the insulin-induced stimulation of ribosomal RNA synthesis in L6 myoblasts.

Insulin stimulated total RNA accretion and the incorporation of [3H]uridine into RNA in L6 skeletal-muscle myoblasts. Incorporation of uridine into the rRNA was measured after either separation of 18 S and 28 S rRNA species by agarose-gel electrophoresis or separation of dissociated 40 S and 60 S ribosomal subunits on sucrose density gradients. Both methods showed a stimulation by insulin of uridine incorporation into the RNA of the two subunits. Two non-steroidal anti-inflammatory drugs, indomethacin and ibuprofen, which inhibit the metabolism of arachidonic acid by the cyclo-oxygenase pathway, inhibited the insulin-induced accretion of total cellular RNA and the incorporation of uridine into the RNA of both ribosomal subunits. The effect of insulin was observed both by using a tracer dose of [3H]uridine (5 microM) and in the presence of a high concentration (1 mM) of uridine to minimize possible changes in intracellular precursor pools. Neither insulin nor indomethacin was found to affect the incorporation of uridine into the total intracellular nucleotide pool, or the conversion of uridine into UTP. The ability of inhibitors of arachidonic acid metabolism to prevent insulin-induced increases in RNA metabolism suggests that a prostaglandin or other eicosanoid is involved in the signal mechanism whereby insulin stimulates RNA synthesis.     

Dexamethasone-induced catabolism and insulin resistance in L6 myoblasts are reversed by the removal of serum.

1. One hundred nanomolar dexamethasone reduced protein synthesis by 16% and also decreased the accretion of protein and RNA in L6 myoblasts when foetal calf serum was present; these effects were reversed when serum was omitted from the medium. 2. Insulin (100 microU/ml) increased protein synthesis, protein accretion and RNA accretion both in the presence and the absence of serum. 3. Dexamethasone inhibited the effects of 100 microU insulin/ml in the presence of serum and induced insulin resistance; in the presence of 25 or 100 nM dexamethasone insulin was ineffective at concentrations below 250 microU and 1 mU/ml respectively.     

Arachidonate activation of protein kinase C may be involved in the stimulation of protein synthesis by insulin in L6 myoblasts

Insulin stimulated protein synthesis in L6 myoblasts but did not increase the labelling of DAG or the release of phosphocholine from phosphatidylcholine. The DAG lipase inhibitor, RHC 80267, more than doubled the amount of label appearing in DAG but did not stimulate protein synthesis. Even in the presence of the DAG lipase inhibitor insulin failed to have any effect on DAG labelling, and conversely RHC 80267 did not modify the insulin-induced increase in protein synthesis. These results suggest that endogenous DAG production is not involved in the stimulation of protein synthesis by insulin. However, exogenous diacylglycerols (1-oleoyl-2-acetyl glycerol and 1-stearoyl-2-arachidonoyl glycerol) both stimulated protein synthesis in L6 myoblasts. The efficacy of the former (arachidonatefree) DAG suggested that their action was by activation of protein kinase C rather than by arachidonate release and prostaglandin formation. Ibuprofen, an inhibitor of cyclo-oxygenase failed to block the effects of insulin whereas a second cyclo-oxygenase inhibitor, indomethacin had only a partial inhibitory effect. The protein kinase C (PKC) inhibitor, RO-31-8220, totally blocked the effect of insulin. Since indomethacin is also recognised to inhibit phospholipase A₂, the data suggests that insulin acts on protein synthesis in myoblasts by arachidonate activation of PKC.     

Arachidonate metabolism in the mouse thyroid implication of the lipoxygenase pathway in thyrotropin action

The present study of compares the effects of various inhibitors of arachidonate metabolism on mouse thyroid cyclo-oxygenase and lipoxygenase activities and thyrotropin-augmented cyclic-AMP accumulation. Mouse thyroid homogenate converts [1-14C]- arachidonate to several products of the cyclo-oxygenase pathway as well as one major product of the lipoxygenase pathway, 12-L-hydroxyeicosatetraenoic acid (12-Hete). Prostaglandin (PG) formation in thyroid homogenates is inhibited by 1-10 microM indomethacin and etya. 12-HETE accumulation is reduced by 91%, 83% and 20% by 5 microM ETYA, 15-HETE, and indomethacin, respectively. Thyrotropin-stimulated cyclic-AMP accumulation, measured in whole thyroid lobes by radioimmunoassay, is reduced by 45% and 73% by 50 microM and 100 microM ETYA, respectively; indomethacin is without effect at these concentrations. 15-HETE reduces thyrotropin-augmented cyclic-AMP accumulation by 57% and 100 microM. In product inhibition studies, 10 microM 12-HETE reduced the formation of radiolabeled 12-HETE by 20%. 10 microM PGE2, PGF2 alpha or PGD2 had no effect on [1-14C]-PG formation. 12-HETE, however, reduced PG synthesis by 76% at 10 microM. This is the first report implicating the arachidonate lipoxygenase pathway in thyrotropin action at the level of cyclic-AMP regulation. Additionally, our finding that 12-HETE inhibits prostaglandin synthesis suggests that the cyclo-oxygenase and lipoxygenase pathways in the mouse thyroid may be highly integrated.     

Time dependent effect of indomethacin on the stimulation of protein synthesis in isolated rabbit muscle by insulin

Insulin (100 U/ml) stimulated protein synthesis and PGF₂ release in isolated rabbit muscle, but had little effect on the rate of protein degradation. The effect of insulin persisted for at least 5 h after removal of the hormone. Indomethacin, added at the start of the incubation, inhibited the stimulatory effect of insulin on protein synthesis and PGF₂ release, but did not block the binding of iodinated insulin. When added 2 h after insulin, indomethacin did not inhibit the stimulation of protein synthesis but completely inhibited the increase in PGF₂ release. The results suggest that the stimulation of protein synthesis by insulin is mediated by metabolites of membrane phospholipids but that these changes are involved during the phase of response that immediately follows the binding of insulin to its receptor.     

Interleukin-1 beta stimulates decidual stromal cell cyclo-oxygenase enzyme and prostaglandin production.

The cytokine interleukin-1 (IL-1 beta) increased prostaglandin production by decidual stromal cells in culture in a time and dose dependent manner. Optimum conditions for stimulation were found to be for 24 hours at a concentration of 100 pg IL-1 beta/ml. An apparent increase in cyclo-oxygenase enzyme synthesis accompanied the increase in prostaglandin production, and both changes were inhibited by the protein synthesis inhibitor cycloheximide. This implicates protein synthesis in the stimulatory effects of IL-1 beta, which may be mediated through the increase in cyclo-oxygenase enzyme. A pre-incubation period of 72 hours was found to be necessary to observe the stimulatory effect of IL-1 beta on prostaglandin production, but this did not seem to be due to any change in the sensitivity of the cells to IL-1 beta; the increase in the number of cyclo-oxygenase positive cells was the same if IL-1 beta was added on day 1, day 2 or day 3 of culture, even though prostaglandin production was not stimulated on day 1 or day 2. Cycloheximide increased prostaglandin production on the first two days of culture and had no effect on the third day of culture. This was interpreted as indicating that a factor inhibiting cyclo-oxygenase activity was synthesised during the initial period of culture, which prevented any increase in prostaglandin production following the increase in enzyme synthesis.     

PHA stimulation of human lymphocytes during amino acid deprivation. Protein,RNA and DNA synthesis

Resting lymphocytes are in the G₀ phase of the cell cycle. Upon activation by PHA, they progress into G₁ with accompanying increased protein and RNA synthesis, initiate DNA synthesis and divide. We have studied the kinetics of inhibition of macromolecular synthesis during activation in the absence of single amino acids. Three types of kinetics are observed. In the absence of tryptophan or isoleucine, stimulated lymphocytes show a normal increase in protein and RNA synthesis during the first 30 hours of stimulation, initiate DNA synthesis but are subsequently inhibited. In phenylalanine-deficient medium, no DNA synthesis occurs in spite of a slight increase in protein synthesis. No increase in macromolecular synthesis is observed in medium lacking any one of the other essential amino acids (eg: lysine). Our results indicate that the kinetics of macromolecular synthesis in tryptophan-deficient medium is the result of a limited reserve of protein-bound tryptophan which becomes exhausted after 30 hours. On the other hand, delayed inhibition of synthesis in isoleucine-deficient medium probably reflects an initially low requirement for this amino acid followed by inhibition of the synthesis of isoleucine-rich proteins involved in some late event of stimulation. Partial deprivation of lysine results in kinetics of protein synthesis similar to that in tryptophan- or isoleucine-deficient media. The results indicate that the kinetics of macromolecular synthesis during activation of lymphocytes in the absence of an essential amino acid is a function of the quantitative requirement for that amino acid, at a given time during stimulation. Upon replacement of lysine, lymphocytes inhibited by lysine deficiency begin RNA and protein synthesis immediately and at a rate faster than that of unstimulated cultures to which PHA is added. They also initiate DNA synthesis earlier and therefore, are closer to the S phase than resting lymphocytes. It is concluded that lymphocytes stimulated in the absence of lysine are activated even though no overall increase in macromolecular synthesis is observed. Furthermore, the kinetics of DNA synthesis following reversal of inhibition by phenylalanine suggests that lymphocytes stimulated during phenylalanine deprivation become arrested at most six hours before S. These results indicate that amino acid deficiencies lead to arrest of activated lymphocytes at various stages of stimulation, depending on how stringent these deficiencies are.     

Rates of protein turnover in vivo and in vitro in ventricular muscle of hearts from fed and starved rats.

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.     

The influence of changes in tension on protein synthesis and prostaglandin release in isolated rabbit muscles.

Intermittent stretching of isolated rabbit muscles increased the rate of protein synthesis by 70% and prostaglandin-F2 alpha release by 105%. Both effects of intermittent stretching were blocked by indomethacin and meclofenamate; the absence of Ca2+ together with added EGTA also inhibited both processes. The residual influence of intermittent stretching on protein synthesis was closely correlated with prostaglandin-F2 alpha release, which may be the link between mechanical activity and protein synthesis in skeletal muscle.     

Amiloride inhibits protein synthesis in isolated rat hepatocytes

The effects of amiloride on Na⁺ ion influx, amino acid transport, protein synthesis and RNA synthesis have been studied in isolated rat hepatocytes. The initial rate of ²²Na⁺ uptake and the amount of ²²Na⁺ taken up at later time points were decreased in hepatocytes incubated in the presence of amiloride. Amiloride inhibited by about 25% the influx of α-methylamino[1?¹⁴C]isobutyric acid, a specific substrate for the A (Alanine preferring) system of neutral amino acid transport. By contrast, the activity of system L (Leucine preferring) was not affected by amiloride. Rates of protein synthesis were determined by using high extracellular concentrations of [¹⁴C]valine in order to maintain a constant amino acid precursor pool. Amiloride inhibited protein synthesis by 85% and had no effect on RNA synthesis. Half-maximal inhibition of protein synthesis occurred with amiloride at about 150 μM. In the absence of Na⁺ in the incubation medium, the rate of protein synthesis was reduced by about 35% and no further inhibition was observed with amiloride. These results suggest that in isolated rat hepatocytes protein synthesis is partially dependent on Na⁺, and that amiloride is an efficient inhibitor of protein synthesis.     

Effect of leucine and metabolites of branched chain amino acids on protein turnover in heart.

Leucine, but not isoleucine or valine, inhibited protein degradation and accelerated protein synthesis in hearts perfused with buffer that contained glucose (15 mM) and normal plasma levels of other amino acids, except for the branched chain compounds. Products of leucine, isoleucine, and valine metabolism also inhibited protein degradation and stimulated protein synthesis. These compounds included the transamination and decarboxylation products, as well as acetate, acetoacetate, and propionate. In some, but not all instances, inhibition of degradation and acceleration of synthesis were accompanied by an increase in intracellular leucine. When insulin was added to the perfusate, the rate of degradation was reduced by 40%, but addition of leucine was ineffective in the presence of the hormone. Insulin, leucine (2 mM) and a mixture of branched chain amino acids at normal plasma levels increased latency of cathepsin D in hearts that were perfused with buffer containing glucose. A combination of leucine and insulin increased latency more than either substance alone. These studies indicate that leucine as well as a variety of substrates that are oxidized in the citric acid cycle are involved in regulation of protein turnover in heart muscle.     

Early changes in the synthesis of acidic nuclear proteins in human diploid fibroblasts stimulated to synthesize DNA by changing the medium

When resting WI-38 cells in a confluent monolayer were stimulated to proliferate by changing the medium, the incorporation of leucine-³H into nuclear acidic proteins was promptly stimulated, although its incorporation into total cellular proteins was unchanged or even decreased. Three fractions, all acidic by aminoacid analysis, were extracted from the nuclei: (1) ribonucleoproteins (RNP); (2) a fraction extractable with 0.15 M NaC1; and (3) a fraction tenaciously bound to the insoluble residue (residual fraction). A first increase occurred between one and three hours after stimulation in all three fractions. The synthesis of NaCl-soluble proteins then returned to control levels, while the synthesis of residual and RNP proteins remained high between 6 and 12 hours and increased even further at 18 hours, the peak of DNA synthesis. Pulse chase experiments indicated that the proteins synthesized in the first hour after stimulation have a turnover time of less than four hours, while the same fractions in non-proliferating cells were stable for at least 12 hours. 2-mercapto-1-(β-4-pyridethyl) benzimidazole, when added at the same time as the fresh medium, produced an inhibition of the increase in nuclear protein synthesis at one hour, but, if added at five hours after stimulation, it did not inhibit the increase in nuclear protein synthesis occurring at six hours. Actinomycin D (0.01 μg/ml) inhibited both the stimulation of DNA synthesis and the increases in nuclear acidic protein synthesis occurring at one and six hours after stimulation. These results seem to indicate that the serum factors responsible for the stimulation of WI-38 cells, after binding to cells, induce an early synthesis of acidic nuclear proteins which is sensitive to very low doses of actinomycin D. In turn, the newly synthesized proteins could in some way activate in the nuclei the genes that control DNA synthesis and cell division.     

Ethylene-forming Systems in Etiolated Pea Seedling and Apple Tissue

Auxin-induced ethylene formation in etiolated pea (Pisum sativum L. var. Alaska) stem segments was inhibited by inhibitors of RNA and protein synthesis. Kinetics of the inhibitions is described for actinomycin D, cordycepin, α-amanitin, and cycloheximide. α-Amanitin was the most potent and fast-acting inhibitor, when added before induction or 6 hours after induction of the ethylene-forming system. The ethylene-forming system of postclimacteric apple (Malus sylvestris L.) tissue, which is already massively induced, was not further stimulated by auxin. Ethylene production in apples was inhibited least by α-amanitin and most by actinomycin D. The relative responses of the ethylene system in apples to RNA inhibitors were different from the ethylene system of pea stems. However, the protein synthesis inhibitor, cycloheximide, appeared to act equally in both tissue systems. The effect of cycloheximide on ethylene production in postclimacteric apple tissue, already producing large quantities of ethylene, suggests a dynamic regulating system for the synthesis and degradation of the ethylene-forming system.     

The role of increased proteolysis in the atrophy and arrest of proliferation in serum-deprived fibroblasts

When cultured fibroblasts are deprived of serum, the degradation of long-lived proteins and RNA increases, the cells stop proliferating, and they decrease in size. To determine the role of the increased protein catabolism in these responses, we studied the effects of inhibitors of intralysosomal proteolysis in Balb/c 3T3 cells. When these cells were placed in serum-deficient medium (0.5% serum), the rate of degradation of long-lived proteins increased about twofold within 30 min. This increase was reduced by 50-70% with inhibitors of lysosomal thiol proteases (Ep475 and leupeptin) or agents that raise intralysosomal pH (chloroquine and NH4Cl). By contrast, these compounds had little or no effect on protein degradation in cells growing in 10% serum. Thus, in accord with prior studies, lysosomes appear to be the site of the increased proteolysis after serum deprivation. When 3T3 cells were deprived of serum for 24-48 hours, the rate of protein synthesis and the content of protein and RNA and cell volume decreased two- to fourfold. The protease inhibitor, Ep475, reduced this decrease in the rate of protein synthesis and the loss of cell protein and RNA. Cells deprived of serum and treated with Ep475 for 24-48 hours had about twice the rate of protein synthesis and two- to fourfold higher levels of protein and RNA than control cells deprived of serum. The Ep475-treated cells were also about 30% larger than the untreated cells. Thus, the protease-inhibitor prevented much of the atrophy induced by serum deprivation. The serum-deprived fibroblasts also stopped proliferating and accumulated in the G1 phase of the cell cycle. The cells treated with Ep475 accumulated in G1 in a manner identical to untreated serum-deprived cells. Other agents which inhibited protein breakdown in serum-deprived cells also did not prevent the arrest of cell proliferation. Thus the enhancement of proteolysis during serum deprivation appears necessary for the decrease in size and protein synthesis, but probably not for the cessation of cell proliferation. When cells deprived of serum in the presence or absence of Ep475 were stimulated to proliferate by the readdition of serum, the larger Ep475-treated cells began DNA synthesis 1-2 hours later than the smaller untreated cells. Thus, after treatment with Ep475, the rate of cell cycle transit following serum stimulation was not proportional to the cell's size, protein, or RNA content, or rate of protein synthesis.     

Differences in inhibition by IDF45 (an inhibitory diffusible factor) of early RNA synthesis stimulation induced by pp60 v-src and various mitogens.

Factors inhibiting cell growth have been isolated from different cell types. However, little information is available concerning their mode of action. A novel growth inhibitory factor of 45 kDa (IDF45) was recently purified to homogeneity from medium conditioned by 3T3 cells. This molecule was able to inhibit DNA synthesis and the growth of chick embryo fibroblasts (CEF) in a reversible manner. By contrast, DNA synthesis stimulated by v-src expression in CEF was poorly inhibited by IDF45. In order to gain further insight into the IDF45 mode of action in normal and transformed CEF, we compared the effects of IDF45 on early stimulation of RNA synthesis induced in CEF by different mitogenic factors and by v-src gene expression. Stimulation, by serum, of RNA synthesis was inhibited by IDF45; however, inhibition increased when cells were preincubated with IDF45 before addition of serum and cell labeling for 2 h. IDF45 was also able to inhibit partially the stimulation of RNA synthesis induced by PMA and PDGF but was unable to inhibit stimulation of RNA synthesis induced by insulin and v-src expression. By contrast, stimulation of RNA synthesis induced by IGF-I was rapidly 100% inhibited by IDF45. The effect of IDF45 on DNA synthesis stimulated by the different mitogens was also determined and was correlated with the effect of IDF45 on RNA synthesis. These results suggest that the modes of action of IDF45 on stimulation of RNA synthesis by v-src and by insulin are similar. Our present results agree with others showing the bifunctional activity of IDF45 as an IGF-binding protein and as an inhibitory molecule in DNA stimulation induced by serum.     

Studies on the role of RNA synthesis in auxin induction of cell enlargement

Selective inhibitors were used to study the connection between nucleic acid synthesis and indoleacetic acid (IAA) induction of cell enlargement. Actinomycin D (act D) and azaguanine (azaG) almost completely inhibit IAA-induced growth in aged artichoke tuber disks when they are added simultaneously with IAA. In contrast, when they are added 24 hours after the hormone, these inhibitors have little or no effect on the induced growth which continues for 48 hours or more with little or no inhibition. Inhibitors of protein synthesis still stop growth when applied 24 hours after the IAA, thus protein synthesis and presumably supporting metabolism are still essential.

In corn coleoptile sections auxin-induced growth did not show any pronounced tendency to become less sensitive to act D as the IAA treatment progressed. Act D did not completely inhibit the response to IAA unless the sections were pretreated with act D for 6 hours. In contrast to act D, cordycepin produced almost complete inhibition of IAA-induced growth when added with the IAA.

Although IAA has a very large and very rapid stimulatory effect (within 10 min) on incorporation of ³²P-orthophosphate into RNA in disks, it did not cause a detectable change in the base composition of the RNA synthesized. Furthermore, the promotive effect could be accounted for through increased uptake of the ³²P. That much of the RNA synthesis in these tissues is not necessary for auxin action is indicated by the results with fluorouracil (FU). FU strongly inhibits RNA synthesis, probably acting preferentially on ribosomal RNA synthesis, without inhibiting auxin-induced growth in the disks or coleoptile sections. FU also strongly inhibited respiration in auxin-treated disks indicating that the large promotion of respiration by auxin likewise may not be entirely necessary for growth.

At least in the artichoke disks, RNA synthesis is required for auxin induction of cell enlargement and not for cell enlargement itself.

The possible relationships of auxin induction of cell enlargement and RNA synthesis are discussed.