Control of cell protein catabolism in rat liver. Effects of starvation and administration of cycloheximide.

1. The loss of liver protein occurring in rats starved for 24 h was largely prevented by the administration of repeated doses of cycloheximide, an inhibitor of protein synthesis. Similar effects were produced on tubulin, a 'fixed' liver protein. 2. Starvation accelerated, whereas cycloheximide markedly lowered, the rate of protein radioactivity decay after labelling with [3H]valine or [14C]bicarbonate, indicating that changes in catabolic rates played an important role in the above regulations of liver protein mass. 3. The total activity of several lysosomal hydrolases showed little change in livers of starved rats, but a marked progressive decline developed after the administration of cycloheximide, particularly in the activities of cathepsins B, D and L as well as acid ribonuclease. There was no evidence that these changes might be due to endogenous inhibitors (at least for cathepsin B activity, which fell to less than 30% of the control values) or enzyme leakage into the bloodstream; rather, plasma beta-galactosidase and beta-N-acetylglucosaminidase activities fell progressively during the cycloheximide treatment. 4. Endogenous proteolytic rates, measured in vitro by incubating subcellular preparations from livers prelabelled in vivo with [3H]valine, were markedly decreased in cycloheximide-treated animals. 5. The osmotic fragility of hepatic lysosomes, appreciably enhanced in starved animals, after cycloheximide treatment was found to be even lower than in fed controls. 6. The present data are consistent with the view that in starved animals the loss of liver protein is mostly accounted for by increased breakdown, due, in part at least, to enhanced autophagocytosis. 7. Cycloheximide largely counteracted these effects of starvation, altering the liver from being 'poised' in a proteolytic direction to a protein-sparing condition. The present data suggest that, besides suppression of the autophagic processes, a decrease in the lysosomal proteolytic enzyme system may also play a role in this regulation, and they seem to provide further circumstantial evidence for the existence of co-ordinating mechanisms between protein synthesis and degradation.     

Interrelationship between protein synthesis and mRNA metabolism in rat liver cells]

It is demonstrated that RNA isolated from polyribosomes and postmitochondrial fraction of rat liver cells and bound to nitrocellulose filters (Milliport) represent mRNA. RNA taken from the nitrocellulose filters sedimented in sucrose concentration gradient with a wide peak within the range of 18--6S, attaining a maximum at 12S. The (A+U)/(G+C) ratio of this RNA was equal to 1.04. On the other hand, the same ratio for rRNA was 0.64. Specific radioactivity of polysomal mRNA containing poly-A sequences, was significantly lower at 14-hour labelling with 14C-orotate than at 4-hour labelling (control). Inhibitors (cycloheximide, puromycin, ethionine, actinomycin D) stabilized polysomal mRNA. Specific radioactivity of postmitochondrial fraction mRNA was higher at 14-hour labelling than at 4-hour labelling. Specific radioactivity of postmitochondrial fraction mRNA during protein synthesis blocking by different inhibitors was comparable to those of control animals. It is hypothesized that active translation is necessary for the initiation of rat liver mRNA degradation.     

Regulation of mammalian protein synthesis in vivo. Protein synthesis in rat liver and kidney after the administration of sublethal doses of cyclohyximide.

Protein synthesis in vivo was studied at various times after the administration of sublethal doses of cycloheximide to rats. Cycloheximide caused an inhibition, followed by a dose-and time-dependent stimulation, of incorportation of labelled precursor into proteins of the liver and kidney. The stimulation of protein synthesis at 24h was not due to a change of precursor pool or the specific radioactivity of the precursor used. During the stimulatory period, leucine incorporation into various cellular protein fractions varied; incorporation into total nuclear protein was the most affected.     

Seasonal changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris)

Fatty acid metabolism and triacylglycerol synthesis are critical processes for the survival of hibernating mammals that undergo a prolonged fasting period. Fatty acid synthase, fatty-acid-CoA ligase, diacylglycerol acyltransferase, and monoacylglycerol acyltransferase activities were measured in liver and in white and brown adipose tissue, in order to determine whether enzymes of lipogenesis and triacylglycerol synthesis vary seasonally during hibernation in the yellow-bellied marmot (Marmota flaviventris). Compared with mid-winter hibernation, fatty acid synthase activity was higher in all three tissues during early spring when marmots emerged from hibernation and in mid-summer when they were feeding, consistent with the synthesis of fatty acids from the carbohydrate-rich summer diet. Fatty-acid-CoA ligase and diacylglycerol acyltransferase activities were highest in summer in white adipose tissue when triacylglycerol synthesis would be expected to be high; diacylglycerol acyltransferase activity was also high in brown adipose tissue during spring and summer. In liver, however, diacylglycerol acyltransferase specific activity was highest during hibernation, suggesting that triacylglycerol synthesis may be prominent in liver in winter. Monoacylglycerol acyltransferase activity, which may aid in the retention of essential fatty-acids, was 80-fold higher in liver than in white or brown adipose tissue, but did not vary seasonally. Its dependence on palmitoyl-CoA suggests that a divalent cation might play a role in enzyme activation. The high hepatic diacylglycerol acyltransferase activity during hibernation suggests that the metabolism of very low density lipoprotein may be important in the movement of adipose fatty acids to brown adipose tissue and muscle during the rewarming that occurs periodically during hibernation. These studies suggest that enzymes of lipid metabolism vary seasonally in the marmot, consistent with requirements of this hibernator for triacylglycerol synthesis and metabolism.Abbreviations BAT brown adipose tissue - DGAT diacylglycerol acyltransferase - FAS fatty acid synthase - K _m Michaelis constant - MGAT monoacylglycerol acyltransferase - RQ respiratory quotiant - VLDL very low density lipoprotein - WAT white adipose tissue     

Role of changes in protein degradation in the growth of regenerating livers.

The significance of changes in rates of synthesis, export, and degradation of proteins during liver regeneration was assessed. (a) Proteins were pulse labeled by the intravenous injection of radioactive leucine and, 5 min later, pactamycin (an inhibitor of the initiation of protein synthesis). One-half of the protein radioactivity was lost from the normal liver within 3 hours. From the radioactivity of the plasma proteins at that time and a study of the disappearance of these proteins from the circulation, it was calculated that 28% of the newly synthesized proteins were exported. Serum albumin accounted for a third of the exported proteins. Thirty-six hours after partial hepatectomy the proportion of albumin to total protein synthesis remained constant, while that of the other plasma proteins increased by 50%. The fraction of the newly synthesized proteins retained by the liver after 3 hours decreased by 20%. (b) During the first 36 hours of liver regeneration the average rates of protein degradation slowed down to one-half the normal values. This was determined either by the loss of radioactivity from total protein (or the guanidino-C of protein-bound arginine) in livers labeled with [14C]bicarbonate, or calculated as the balance between protein synthesis and net protein gain. (c) From these results, and those of our previous study of the protein synthetic machinery of normal and regenerating livers (Scornik, O.A. (1974)J. Biol. Chem. 249, 3876-3883), we conclude that changes in the rate of protein degradation are the single most important factor determining the increase in protein content during liver compensatory growth.     

Studies on the assembly and secretion of a multicomponent protein. The biosynthesis of vitellogenin, an oestrogen-induced glycolipophosphoprotein

1. Incorporation of [(32)P]P(i) and [(3)H]leucine into vitellogenin secreted in vitro by liver slices from oestrogen-treated Xenopus laevis is accompanied by a 2h lag; no lag is apparent for the incorporation into total tissue protein. 2. The addition of cycloheximide was found immediately to inhibit further incorporation of radioactive leucine into total tissue protein. The incorporation into secreted vitellogenin, however, continued for 2h after the addition of cycloheximide. 3. Pulse-labelling of liver slices with [(3)H]leucine for 30min, followed by a chase with a large excess of unlabelled leucine, resulted in the appearance of radioactivity in secreted vitellogenin from 90min after the end of the pulse period. 4. Evidence is presented which suggests that of the radioactivity from [(3)H]leucine incorporated into proteins by the liver of oestrogen-treated Xenopus some 70% is present in the single protein vitellogenin. 5. The incorporation of [(32)P]P(i) into vitellogenin followed a pattern identical with that found for [(3)H]leucine in the pulse-labelling experiments and this indicates that synthesis of the polypeptide chain and incorporation of P(i) are closely linked processes. 6. The cumulative evidence suggests that the 2h lag phase represents the time required for the assembly and secretion of this multicomponent protein.     

Degradation of 18S and 28S ribosomal RNA in the cytoplasm of rat liver cells after inhibition of protein synthesis

Inhibition of protein synthesis (up to 95%) in starved rat liver cells after a single injection of a sublethal dose of cycloheximide (0.3 mg per 100 g of body weight) results in degradation of 18S rRNA during the first 3 hours, whereas the 28S rRNA remains unaffected. However, the increase of 28S rRNA degradation products was observed by the 6th and 12th hours. The rapid decay of 18S rRNA is due to the degradation of this RNA in 40S ribosomal subunits. In contrast to 28S rRNA the specific radioactivity of 18S rRNA is increased by the 6th hour. Presumably the synthesis and processing of 18S rRNA impaired during the 1st hour are recovered partially or completely by this time. A molecular mechanism underlying 18S rRNA degradation in 40S ribosomal subunits is proposed.     

Effect of a nutritional shift on the degradation of abnormal proteins in the mouse liver. Decreased degradation during rapid liver growth.

1. The intravenous injection of puromycin to mice 0.5 min after administration of radioactive leucine resulted in the release of labelled ribosome-bound nascent protein chains with the next 0.5 min. 2. During the subsequent 13 min, 40% of the liver protein radioactivity disappeared. The rate of this process was already maximal 0.5 min after the injection of puromycin, with no apparent lag. 3. Evidence is presented that this phenomenon represents the selective degradation of puromycinyl-peptides: (a) the magnitude of this fraction corresponded to the calculated proportion of protein radioactivity in nascent chains at the time of the puromycin effect; (b) the size distribution of the proteins disappearing between 2 and 14 min was smaller than that of those retained at 14 min; and (c) when the injection of puromycin was delayed for 5 min, or when the leucine pulse was interrupted by the injection of cycloheximide (rather than puromycin), the fraction disappearing within 14 min was much smaller. 4. The degradation of puromycinyl-peptides was much slower in the rapidly growing livers of animals recovering from a protein depletion than in the protein-depleted controls. It is concluded that the large decrease in the overall rates of total liver protein degradation previously described during liver growth is a general phenomenon, also affecting the rate of scavenging of abnormal proteins.     

Invertase inhibitor-control of sucrose transport from carnation petals to other flower parts

An investigation was made of the distribution of metabolites in the receptacles and ovaries, and the activity of an invertase inhibitor after feeding the petals with ¹⁴C-sucrose and cycloheximide during the natural aging of cut carnation flower. The cycloheximide treatment blocked the synthesis of the invertase inhibitor in petals and maintained a relatively high level of invertase activity during flower senscence. Treatment with cycloheximide decreased the movement of radioactivity from petals to receptacles and ovaries from 7 to 10 times, compared to non-treated control flowers. IAA applied on the stigma had no influence on synthesis of the inhibitor and distribution of the radioactivity.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.     

Chalone-antichalone system of the liver in different animals and its state depending on physiological rhythms in amphibia

The chalone-antichalone activity in the mollusc (anodonta), fish, frog, bird, mammalians liver preparations is determined. Inspite of the fact that chalone-antichalone content varies greatly in different animals livers, in most of them the chalone-antichalone ratio is approximately equal and doesn't increases 2.0. In the frog livers during the hibernation period the chalone is not determined. Its synthesis begins at the hibernation exit and it becomes active in the spring-time.     

Depression of protein synthesis during diapause in embryos of the annual killifish Austrofundulus limnaeus

Rates of protein synthesis are substantially depressed in diapause II embryos of Austrofundulus limnaeus. Inhibition of oxygen consumption and heat dissipation with cycloheximide indicates that 36% of the adenosine triphosphate (ATP) turnover in prediapausing embryos (8 d postfertilization [dpf]) is caused by protein synthesis; the contribution of protein synthesis to ATP turnover in diapause II embryos is negligible. In agreement with the metabolic data, incorporation of amino acids (radiolabeled via (14)CO(2)) into perchloric acid-precipitable protein decreases by over 93% in diapause II embryos compared with embryos at 8 dpf. This result represents a 36% reduction in energy demand because of depression of protein synthesis during diapause. Adjusting for changes in the specific radioactivity of the free amino acid pool at the whole-embryo level yields rates of protein synthesis that are artifactually high and not supportable by the observed rates of oxygen consumption and heat dissipation during diapause. This result indicates a regionalized distribution of labeled amino acids likely dictated by a pattern of anterior to posterior cell cycle arrest. AMP/ATP ratios are strongly correlated with the decrease in rates of protein synthesis, which suggests a role for adenosine monophosphate (AMP) in the control of anabolic processes. The major depression of protein synthesis during diapause II affords a considerable reduction in energy demand and extends the duration of dormancy attainable in these embryos.     

Effects of cycloheximide on protein degradation and gluconeogenesis in the perfused rat liver.

Cycloheximide at concentrations above 18 muM produced a 93% inhibition of total protein synthesis measured by valine incorporation in the perfused rat liver. Rates of protein degradation were estimated by perfusing livers prelabeled in vivo with L-[1-14C]valine with medium containing 15 mM L-valine. Thus labeled valine released from liver protein during perfusion was greatly diluted and reincorporation of label was minimized. Cycloheximide at 18 muM inhibited protein degradation by over 60%, after a delay of 15-20 min. Associated with these effects were dose-dependent increases in the rates of glucose and urea production. Glucose production increased 3 fold, from 0.54 +/- 0.07 in control to 1.85 +/- 0.24 mumol/min/100 g rat in cycloheximide-treated livers. Urea production increased from 0.24 +/- 0.02 to 0.62 +/- 0.06 mumol/min/100 g rat. No changes in liver glycogen or cyclic AMP content were seen. The data suggest that inhibition of protein synthesis provides an increased availability of intra-cellular amino acids and that many of these are rapidly degraded, yielding urea and glucose. This is supported by the fact that intracellular alanine levels were significantly increased following cycloheximide treatment. It is possible that the inhibition of protein degradation by cycloheximide is due to altered intra-cellular pools of amino acids or their metabolites.     

Melanoprotein: absence of a direct melanin-protein relationship in chick embryo melanocytes.

Short-term synthesis of radioactivity labeled melanin (using DL-[2-14C]tyrosine or 2-[2-14C]thiouracil) by chick retinal pigment tissues in vitro was not influenced by inhibitors of protein synthesis, puromycin and cycloheximide. Co-ordinate synthesis of protein is, therefore, unnecessary for melanin synthesis, and melanoproteins must represent secondary interactions between melanin and protein. Melanin was isolated from chick embryo feather germs by extracting the proteins with hot dodecyl sulfate/mercaptoethanol. Melanin isolated from tissues incubated previously in L-[U-14C]valine medium had no associated radioactivity compared to the radioactivity of melanin prepared from tissues incubated in DL-[2-14C]tyrosine or 2-[2-14C]thiouracil. If melanoproteins exist at all, they are non-covalently bonded associations of melanin and melanosomal proteins.     

Regulation of tyrosine aminotransferase messenger ribonucleic acid in rat liver. effect of cycloheximide on messenger ribonucleic acid turnover

Tyrosine aminotransferase messenger ribonucleic acid (mRNA) activity in rat liver was rapidly increased 3-6-fold following in vivo administration of hydrocortisone acetate, dibutyryladenosine cyclic 3',5'-phosphate, or the protein synthesis inhibitor cycloheximide. Treatment with the steroid hormone or cyclic nucleotide in combination with cycloheximide resulted in levels of tyrosine aminotransferase mRNA 10-20-fold greater than control values. These changes in mRNA activity were not accompanied by changes in albumin mRNA or total liver template activity. The rapid decline in tyrosine aminotransferase mRNA activity following cordycepin inhibition of de novo RNA synthesis was prevented by cycloheximide treatment. This protection was not observed when pactamycin was substituted for cycloheximide, demonstrating that the inhibition of protein synthesis per se was not responsible for the stabilization of tyrosine aminotransferase mRNA. Based upon the effects of cycloheximide and pactamycin on rat liver polysome structure, it is concluded that the cycloheximide-mediated increase in tyrosine aminotransferase mRNA activity is the result of stabilization of the mRNA molecule which renders the message less susceptible to inactivation and degradation in the cytoplasm. The action of cycloheximide is very specific for tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and probably several other mRNAs that code for minor liver proteins that turn over rapidly in response to hormonal or metabolic stimuli.     

Correlation between the rate of protein and nucleic acid synthesis and the intensity of various glucose metabolic pathways in rat liver cells

A sharp and strong suppression of protein synthesis by cycloheximide in liver cells of starving rats is paralleled with activation of RNA synthesis and glucose-6-phosphate dehydrogenase production. Subsequent reconstitution and stimulation of protein synthesis (6-12 hrs after cycloheximide injection) result in activation of hexokinase. Upon stimulation of DNA synthesis (48-60 hrs after cycloheximide injection) the activity of both enzymes is very low. Since glucose-6-phosphate dehydrogenase appears to be the limiting step of glucose decay via the pentose phosphate pathway, and hexokinase is the limiting step of glycolysis, it was assumed that RNA synthesis predominantly occurs via the pentose phosphate pathway, while that of proteins via glycolysis.     

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.