The effects of cycloheximide and chloroquine on insulin receptor metabolism. Differential effects on receptor recycling and inactivation and insulin degradation

The effects of protein synthesis inhibitors and the lysosomotropic agent chloroquine on the metabolism of the insulin receptor were examined. Through the use of the heavy-isotope density shift technique, cycloheximide was found to inhibit both the synthesis of new insulin receptor and the inactivation of old cellular insulin receptor. Upon investigation of the locus of this effect of protein synthesis inhibition, it was found that cycloheximide did not inhibit 1) the translocation of receptor from the cell surface to an intracellular site, 2) the recycling of receptor from the internal site back to the plasma membrane, nor 3) the degradation of insulin. Cycloheximide did, however, rapidly and completely inhibit the inactivation of the insulin receptor. In the presence of extracellular insulin, this effect of cycloheximide resulted in the long-term (6 h) accumulation of receptor in a trypsin-resistant intracellular compartment. Puromycin and pactamycin, protein synthesis inhibitors with mechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that this effect on receptor metabolism is due to the inhibition of protein synthesis and not a secondary effect of cycloheximide. Actinomycin D also inhibited the inactivation of receptor. Chloroquine inhibited the receptor-mediated degradation of insulin, but had no effect on either the internalization or inactivation of the insulin receptor. The insulin-induced recycling of the internalized receptor was inhibited by chloroquine, possibly through the inhibition of the discharge of insulin from the insulin-receptor complex. From these observations, we suggest that 1) a protein factor is required to inactivate the insulin receptor, 2) this protein and the messenger RNA coding for the protein have short cellular half-lives, and 3) insulin degradation and insulin receptor inactivation are distinct, separable processes which not only occur at different rates, but possibly occur in distinct subcellular locations.     

Mode of Action of Novobiocin in Escherichia coli

The mechanism of action of novobiocin was studied in various strains of Escherichia coli. In all strains tested except mutants of strain ML, the drug immediately and reversibly inhibited cell division, and later slowed cell growth. The previously described impairment of membrane integrity, degradation of ribonucleic acid (RNA), and associated bactericidal effect were found to be peculiar to ML strains. The earliest and greatest effect in all strains was an inhibition of deoxyribonucleic acid (DNA) synthesis; RNA synthesis was inhibited to a lesser extent, and cell wall and protein synthesis were affected later. The inhibition of nucleic acid synthesis was accompanied by an approximately threefold accumulation of all eight nucleoside triphosphates. Since novobiocin does not inhibit nucleoside triphosphate synthesis, degrade DNA, or immediately affect energy metabolism, it must inhibit the synthesis of DNA and RNA by direct action on template-polymerase complexes.     

RNA synthesis and control of cell division in the yeast S. cerevisiae.

Cells of the yeast Saccharomyces cerevisiae rapidly accumulated in the G1 phase of the cell cycle when exposed to the chelating agents o-phenanthroline (OP) or 8-hydroxyquinoline (HQ). Zinc salts fully reversed the growth-inhibitory effect of both OP and HQ. Cells treated with these chelating agents showed limited RNA accumulation and little RNA degradation. Rates of RNA synthesis were drastically reduced by low concentrations of these compounds. Whereas rates of protein synthesis were essentially unaffected. Rates of synthesis of mRNA and tRNA were less affected than were rates of synthesis of high molecular weight RNA. Processing of ribosomal precursor RNA was altered. these results suggest that the primary effect of OP and HQ is on rRNA synthesis. RNA metabolism must therefore have a key role in the regulation of the cell cycle.     

Methyl-beta-D-glucopyranoside in higher plants: accumulation and intracellular localization in Geum montanum L. leaves and in model systems studied by 13C nuclear magnetic resonance

Using (13)C-NMR, methyl-beta-D-glucopyranoside (MeG) was characterized as a major compound in the leaves of the alpine herb Geum montanum L. MeG continuously accumulated during the life span of G. montanum leaves, and accounted for up to 20% of the soluble carbohydrates in aged overwintering leaves, without being reallocated during senescence. Incubating intact plant tissues, culture cells, and purified organelles with (13)C-labelled substrates showed that MeG was synthesized in the cytosol of cells, directly from glucose and methanol molecules. There was no contribution of the C-1 pathway. MeG was subsequently stored in the vacuole without being re-exported to the cytoplasm. All the dicots tested contained the enzymatic machinery permitting MeG synthesis from methanol and glucose, but the plants accumulating this compound at concentrations higher than 1 micromol g(-1) wet wt were mainly members of the Rosaceae family belonging to the Rosoideae subfamily. It is suggested that the synthesis of MeG may contribute to reduce the accumulation in the cytoplasm of methanol and its derived compounds.     

Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse.

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.     

The effects of elevated summer temperature and sublethal pollutants (ammonia, low pH) on protein turnover in the gill and liver of rainbow trout (Oncorhynchus mykiss) on a limited food ration.

Protein synthesis, degradation and growth of the liver and gills were determined in juvenile rainbow trout (Oncorhynchus mykiss) fed a limited ration and exposed for 90 days to normal or elevated summer temperatures (+2 degrees above ambient) and either low pH (5.2) in softwater or 70 microM total ammonia in hardwater. The limited ration resulted in low rates of growth (< 0.80% per day) and protein synthesis in all fish. In softwater, whole-body growth was significantly inhibited by elevated temperature but stimulated by low pH, although tissue protein metabolism was generally unaffected by these treatments. There was no significant difference in final size between the groups of fish in hardwater, but liver protein synthesis and degradation were significantly lower at +2 degrees C, the reduction in synthesis being due to an inhibition of both the capacity for protein synthesis, Cs and the RNA translational efficiency, kRNA. Gill protein metabolism was unaffected by the experimental treatments in trout in hardwater. The authors conclude that a global warming scenario would be detrimental to protein synthesis and growth in freshwater fish under conditions of food limitation in summer, and when late summer temperatures approached the upper thermal limit of the species, regardless of food availability.     

Methylated nucleotides block 5' terminus of HeLa cell messenger RNA.

Polyadenylylated [poly(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poly(A)+ RNA was calculated from the 3H/14C ratios and known degrees of methylation of 18S and 28S ribosomal RNAs. All four 2'-O-methylribonucleosides, but only two base-methylated derivatives, 7-methylguanosine (7MeG) and 6-methyladenosine (6MeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyl-labeled ribonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5' terminus of the poly(A)+ RNA and could be removed by periodate oxidation and beta elimination. Evidence for a 5' to 5' linkage of 7MeG to adjacent 2'-O-methylribonucleosides through at least two and probably three phosphates to give structures of the type 7MeG5'ppp5pNMep- and 7MeG5'ppp5'NMepNmep- was presented. The previous finding of similar sequences of methylated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5' termini may be a characteristic feature of mRNAs that function in eucaryotic cells.     

Inhibition of glucose transport by phenylglyoxal: Both dissipation of ΔpH and essential arginyl residues are involved

The effects of the arginine modifying reagent phenylglyoxal (PGO) on solute transport was studied in two cellular systems: protoplasts isolated from the mesophyll of Vicia faba L. and XD cell suspension culture of Nicotiana tabacum L. cv. Xanthi. The solutes in the case of the protoplasts were the non‐metabolizable glucose analog 3‐O‐methyl‐D‐glucose (MeG), and a non‐metabolizable amino acid analog α‐aminoisobutyric acid (AIB), whereas the solutes for the cell suspension were AIB and nitrate. Solute transport in both systems was rapidly inhibited by PGO. Exposure of the protoplasts to light enhanced the initial rate of MeG uptake. PGO rapidly inhibited MeG uptake in both the light and the dark, the half‐time for inactivation being less than 3 min. Flux analysis of double‐labeled MeG showed that initial MeG uptake was mediated mainly by the plasma membrane transport system and that it was inhibited by PGO. Maximal inhibition of initial MeG uptake rate was observed at PGO concentrations of 1 m M and above. PGO treatment altered rapidly the equilibrium distribution of the ΔpH probe dimethyloxazolidine (DMO) in both cellular systems, indicating dissipation of ΔpH between cell and medium. In the protoplasts, PGO inhibited both DMO and MeG uptake at pH 5.5; however, at pH 7.0, where ΔpH is minimal, only MeG uptake was inhibited. Our results suggest that PGO has two effects on glucose uptake: an indirect effect through ΔpH dissipation and a direct effect through interaction with essential arginyl residues in the glucose transporter.     

Inhibition of Replication of Ribonucleic Acid Bacteriophage f2 by Superinfection with Bacteriophage T4

Superinfection by phage T4 of cells infected by the ribonucleic acid (RNA) phage f2 results in inhibition of further f2 production. Experiments using rifampin show that the exclusion of f2 requires T4 gene function soon after T4 infection. By using a sensitive new peptide-mapping procedure to identify f2 coat protein in infected cells, we show that synthesis of the f2 coat occurs at a reduced level until 4 min after T4 superinfection and then ceases abruptly. Within 4 min after T4 superinfection, there are also several changes in f2 RNA metabolism, all of which require T4 gene function: preexisting f2 replicative intermediate RNA and f2 single-stranded RNA are degraded to small but still acid-precipitable fragments, and most f2-specific RNA is released from polyribosomes. We favor the hypothesis that T4 induces the synthesis of a specific endoribonuclease which degrades f2 RNA and that the inhibition of f2 protein synthesis may be a consequence of this degradation, rather than a direct effect of T4 upon translation.     

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.     

Temperature sensitive mutations affecting ribosome synthesis in Saccharomyces cerevisiae

Studies have been carried out on the effects of mutations in nine distinct genes which regulate ribosome biosynthesis in yeast. Although some mutants have more extensive effects than others, in each case there is an inhibition by 50 to 80% of the synthesis of ribosomal precursor RNA. In each case there is an inhibition of the processing of that RNA which is made, ranging from 80 to 95%. In each case there is degradation of 50 to 70% of that RNA which is processed. Much of the RNA which survives to become the mature 25 s and 18 s species is found in functioning polyribosomes.     

The breakdown of Tetrahymena ribosomes in vivo. The effects of inhibitors.

1. When Tetrahymena were deprived of nutrients 50% of the polysomes disaggregated within 20 min and 20% of the total RNA broke down in 2 h. Ribosomal RNA accounted for 75% of the RNA breakdown. 2. RNA labelled by a long incubation with [14C]uridine was stable in growing cells and in the presence of actinomycin D, but broke down at the same rate as bulk RNA in starved cells. 3. The following substances inhibited the loss of RNA during starvation: cycloheximide (which inhibited both polysome disaggregation and protein synthesis), inhibitors of energy metabolism and puromycin (all of which caused polysome disaggregation and inhibited protein synthesis), and chloroquine and 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK') (neither of which affected polysomes or protein synthesis). 4. Starvation appears to activate a ribosome degradation mechanism that may involve lysosomal and non-lysosomal enzymes.     

Thyroid hormones and muscle protein turnover. The effect of thyroid-hormone deficiency and replacement in thryoidectomized and hypophysectomized rats.

We have investigated the effects of thyroidectomy, hypophysectomy and 3,3',5-tri-iodothyronine replacement on protein synthesis and degradation in skeletal muscle in vivo. Thyroidectomy resulted in a decrease in the rate of protein synthesis as a result of a loss of RNA. However, RNA activity, the rate of protein synthesis per unit of RNA, was not decreased. This was the case in both young growing rats and mature nongrowing rats. Tri-iodothyronine treatment of thyroidectomized rats increased protein synthesis by increasing RNA concentration without changes in RNA activity, and this occurred even when food intake was restricted to prevent any increase in growth. The rate of protein degradation was decreased by thyroidectomy and increased by tri-iodo-thyronine replacement in both animals fed ad libitum and food-restricted animals. Hypophysectomy decreased protein synthesis by decreasing both RNA concentration and activity. these changes were reversed by tri-iodothyronine treatment even in the presence of persistent marked hypoinsulinaemia. This indicates that tri-iodothyronine can activate athe translational phase of protein synthesis in muscle in the absence of significant quantities of insulin. However, tri-iodothyronine does not seem to be obligatory for the maintenance of normal RNA activity in muscle, since in the thyroidectomized rat, in which plasma insulin concentrations are normal, RNA activity is maintained. From a consideration of the magnitude of changes in RNA activity observed in these experiments, it would appear that alterations in rates of elongation as well as initiation are involved in the changes in RNA activity.     

Regulation of RNA metabolism in relation to insulin production and oxidative metabolism in mouse pancreatic islets in vitro

This study was undertaken to investigate the long-term effects of different substrates, in particular glucose, on the regulation of islet RNA metabolism and the relationship of this regulation to the metabolism and insulin production of the islet B-cell. For this purpose collagenase-isolated mouse islets were used either in the fresh state or after culture for 2 or 5 days in RPMI 1640 plus 10% calf serum supplemented with various test compounds. Islets cultured with 16.7 mM glucose contained more RNA than those cultured with 3.3 mM glucose. Culture of islets in glucose at low concentrations inhibited glucose-stimulated RNA synthesis and this inhibitory effect was reversed by prolonged exposure to high glucose concentrations. Culture with 10 mM leucine and 3.3 mM glucose or with 10 mM 2-ketoisocaproate and 3.3 mM glucose increased the total RNA content of islets as compared to that of islets cultured with 3.3 mM glucose alone. Islets cultured with 5 mM theophylline maintained a high RNA content in the presence of 3.3 mM glucose. Theophylline also increased the islet RNA content when added together with 16.7 mM glucose, as compared to 16.7 mM glucose alone. Theophylline probably exerted this effect by decreasing the rate of RNA degradation. Changes in islet RNA metabolism showed a close correlation to changes in islet total protein biosynthesis, whereas islet (pro)insulin biosynthesis and insulin release exhibited different glucose-dependency patterns. The response of islet oxygen uptake to glucose was similar to that of islet RNA and protein biosynthesis. It is concluded that the RNA content of the pancreatic islets is controlled at the levels of both synthesis and degradation. Glucose stimulates the RNA synthesis and inhibits its degradation. Moreover, the results suggest that regulation of RNA synthesis may be mediated through islet metabolic fluxes and the cAMP system.     

Regulation of protein metabolism by a physiological concentration of insulin in mouse soleus and extensor digitorum longus muscles. Effects of starvation and scald injury.

1. Although high concentrations of insulin affect both synthesis and degradation of skeletal-muscle protein, it is not known to what extent these effects occur with physiological concentrations. The effects of a physiological concentration of insulin (100 mu units/ml) on muscle protein synthesis, measured with [3H]tyrosine, and on muscle protein degradation, measured by tyrosine release in the presence of cycloheximide, were studied in mouse soleus and extensor digitorum longus muscles in vitro. 2. Insulin significantly stimualated protein synthesis in both muscles, but an inhibition of degradation was seen only in the extensor digitorum longus. 3. Starvation for 24 h decreased the rate of protein synthesis and increased the rate of breakdown in the extensor digitorum longus. Sensitivity to insulin-stimulation of proteins synthesis in the soleus was increased by starvation. 4. ;a 20%-surface-area full-skin-thickness dorsal scald injury produced a fall in total protein content in soleus and extensor digitorum muscles, maximal on the third day after injury. Soleus muscles 2 days after injury showed an impairment of protein synthesis; degradation was unaffected and neither synthesis nor degradation in vitro was significantly affected in the extensor digitorum longus. 5. The advantages and limitations of studies of protein metabolism in vitro are discussed.     

Imidazole ring-opened DNA purines and their biological significance

Fragmentation of purine imidazole ring and production of formamidopyrimidines in deoxynucleosides (Fapy lesions) occurs upon DNA oxidation as well as upon spontaneous or alkali-triggered rearrangement of certain alkylated bases. Many chemotherapeutic agents such as cyclophosphamide or thiotepa produce such lesions in DNA. Unsubstituted FapyA and FapyG, formed upon DNA oxidation cause moderate inhibition of DNA synthesis, which is DNA polymerase and sequence dependent. Fapy-7MeG, a methylated counterpart of FapyG-, a efficiently inhibits DNA replication in vitro and in E.coli, however its mutagenic potency is low. This is probably due to preferential incorporation of cytosine opposite Fapy-7MeG and preferential extension of Fapy-7MeG:C pair. In contrast, FapyA and Fapy-7MeA possess miscoding potential. Both lesions in SOS induced E.coli preferentially mispair with cytosine giving rise to A-->G transitions. Fapy lesions substituted with longer chain alkyl groups also show simult aneous lethal and mutagenic properties. Fapy lesions are actively eliminated from DNA by repair glycosylases specific for oxidized purines and pyrimidines both in bacteria and eukaryotic cells. Bacterial enzymes include E.coli formamidopyrimidine-DNA-glycosylase (Fpg protein), endonuclease III (Nth protein) and endonuclease VIII (Nei protein).     

Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores

LéJohn, Herbert B. (Purdue University, Lafayette, Ind.), and James S. Lovett. Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores. J. Bacteriol. 91:709-717. 1966.-The uniflagellate zoospores of Rhizophlyctis rosea display active motility and a high endogenous respiratory metabolism, but neither growth nor net ribonucleic acid (RNA) or protein synthesis can be measured by ordinary procedures. Nevertheless, synthesis can be detected with isotopic precursors. Uracil-C(14) is incorporated slowly into both the soluble and ribosomal RNA. Analysis of zoospore extracts (on diethylaminoethyl cellulose columns or sucrose gradients) after various periods of labeling suggested that most of the uracil incorporation represents slow synthesis of ribosomal precursor RNA and, ultimately, ribosomes. Actinomycin D caused an 80% inhibition of uracil incorporation. The most rapidly labeled RNA was susceptible to extensive degradation in cells treated with actinomycin, but the percentage of stable RNA increased with the time of incorporation before addition of the antibiotic. Neither the effects of actinomycin nor the results of chase experiments have established unequivocally the existence of turnover or the presence of a short-lived "messenger" fraction in motile spores. Both leucine and methionine were slowly incorporated into a spectrum of cellular proteins. The methyl group of C(14)-methylmethionine also served as a methyl donor for the methylation of soluble RNA but not of ribosomal RNA. The observations that some of the newly synthesized RNA and protein occur in the intact 82S ribosomes and that actinomycin inhibits the low level of protein synthesis provide some indirect evidence for a very low rate of "messenger" synthesis and turnover in zoospores.     

The temperature sensitive mutant 72c. II. Accumulation at high temperature of ppGpp and pppGpp in the presence of protein synthesis.

A heat sensitive mutant of E. coli has been analyzed. A shift to restrictive temperature leads to an accumulation of ppGpp and pppGpp in both the parental and the mutant strains (both are relA+). The pool of these compounds is shown to decrease with time after the temperature shift in the case of the parental strain, but remains at the same elevated level in the case of the mutant. The temperature shift of the mutant leads to an apparent reduction of stable RNA synthesis; this inhibition can be released by chloroamphenicol or tetracycline. Gross protein synthesis is more or less unaffected at restrictive temperature. In the parental strain little effect is seen on RNA and protein synthesis after the temperature shift. A relA derivative of the mutant does not show the same inhibition of RNA synthesis at high temperature. Sedimentation analysis suggests that mutant 70S ribosome are more stable, when exposed to a lowered Mg2+ concentration, than are 70S ribosomes from the parental strain. In addition, the relative amounts of the two forms of ribosomal protein S6, which can be obtained on DEAE chromatography (Held et al., 1973), are significantly changed in the mutant.     

The lysine analog L-oxalysine is an inhibitor of RNA synthesis.

1. The lysine analog L-4-oxalysine was found to be a potent inhibitor of RNA synthesis in Candida albicans. 2. The compound was a weak inhibitor of protein synthesis and DNA synthesis was not affected. 3. The inhibition of RNA synthesis was reversed by L-lysine but not D-lysine. 4. The decrease in the level of newly synthesized RNA in cells treated with L-oxalysine was due to inhibition of de novo synthesis rather than to degradation of RNA.