Effect of cycloheximide on protein biosynthesis in rat liver.
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ABSTRACT:
SUBMITTER: Korner A
PROVIDER: S-EPMC1270163 | biostudies-other | 1966 Dec
REPOSITORIES: biostudies-other
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Project description:1. The mechanism of the stimulation of ferritin synthesis by iron in vivo has been studied in rat liver. Ferritin synthesis and turnover was measured by [(14)C]leucine incorporation. 2. Actinomycin D had no inhibitory effect, after administration of iron, on [(14)C]leucine incorporation into ferritin but appeared to augment the effect of iron on ferritin synthesis. 3. Cycloheximide completely abolished the stimulation by iron of [(14)C]leucine into ferritin and was subsequently utilized to show that iron acts in vivo by translational induction of apoferritin synthesis, rather than by stabilization of apoferritin or its precursors. 4. This conclusion was confirmed by showing that 2 days after acute bleeding, when iron was in the process of being removed from hepatic ferritin stores, ferritin synthesis was decreased whereas breakdown rates were unchanged.
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Project description: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.
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Project description:Starvation (24h) increased the maximum activity of carnitine palmitoyltransferase 1 in rat liver and increased the concentration of malonyl-CoA required to cause 50% inhibition of the enzyme (I50). Re-feeding (24h) with a standard cube diet or a high-carbohydrate diet reversed both of these changes, whereas re-feeding with a high-fat diet did not. Administration of cycloheximide (200 micrograms/100 g body wt.) blocked the increases in carnitine palmitoyltransferase 1 activity and I50 on starvation. It is suggested that increase in carnitine palmitoyltransferase 1 activity in starvation may involve synthesis of new enzyme.
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Project description:1. Administration of a single dose of dimethylnitrosamine to rats temporarily fed on a protein-deficient diet causes a high incidence of kidney tumours. The effect of such a dose of dimethylnitrosamine (40mg/kg body wt.) on metabolism of nucleic acids and protein in rat liver and kidneys was examined during the week immediately after administration. 2. Incorporation of [(14)C]leucine and [(14)C]orotate into hepatic macromolecules was inhibited within 5h of injection of dimethylnitrosamine, and did not recover for at least 5 days. Interpretation of these results is complicated by the concomitant extensive hepatic necrosis. 3. Renal RNA synthesis was assayed by incorporation of [(14)C]orotate in vivo and measurement of DNA-dependent RNA polymerase activity in vitro. Both systems indicate biphasic inhibition; minimal activity was recorded 9h and 3 days after treatment. Changes in incorporation of [(14)C]leucine into renal protein were similar but less marked. 4. Sucrose-density-gradient analysis of renal cytoplasmic RNA indicated increased synthesis of rRNA 24h after injection of the nitrosamine. The rate of loss of radioactivity from kidney ribosomes pre-labelled with [(14)C]orotate was not modified by dimethylnitrosamine. 5. Dimethylnitrosamine increased incorporation of [(3)H]-thymidine into renal DNA. The three distinct periods of stimulated synthesis observed are discussed, with particular reference to recently published morphological studies of the sequential development of kidney tumours induced by dimethylnitrosamine in protein-depleted rats.
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Project description:Heparin biosynthesis has been investigated with mouse mastocytoma in vitro. Minced tumour tissue catalysed the incorporation of [35S]sulphate and [3H]glucosamine into heparin and to a smaller extent into chondroitin sulphate. Addition of cycloheximide caused an inhibition (greater than 80%) of incorporation of each labelled precursor into both polysaccharides. Addition of benzyl beta-D-xyloside relieved the inhibition of incorporation into chondroitin sulphate and restored it to more than threefold that of the control incubation. The effect of beta-D-xyloside on incorporation into heparin was less marked although a consistent small increase of incorporation into this polysaccharide was observed. beta-D-Xyloside did, however, cause a marked incorporation of 35S and 3H labels into material of low molecular weight, which appeared to comprise heparin-like fragments. It is proposed that these fragments arise through a breakdown of the usual process of heparin biosynthesis.
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Project description:1. Male rats were injected intraperitoneally with l-[(35)S]methionine, [(32)P]-phosphate and [2-(14)C]acetate. The animals were killed at various times up to 72hr. after injection, and liver mitochondria were prepared and fractionated into soluble protein, insoluble protein and lipid for assay of the radioactivity of each fraction. 2. The maximal specific radioactivity of total mitochondrial phospholipid with respect to both (32)P and (14)C was attained after approx. 6hr. 3. (32)P was incorporated most rapidly into phosphatidylethanolamine, maximal incorporation being attained after approx. 6hr.; maximal incorporation into lecithin occurred after 6-12hr. The specific radioactivity of cardiolipin was still slowly increasing at the end of the experiment (72hr.). 4. There were no major differences between the rates of incorporation of (14)C into the lecithin, phosphatidylethanolamine and cardiolipin fractions of mitochondrial phospholipid, maximal incorporation in each case occurring after approx. 6hr. 5. Maximal incorporation of (35)S into both soluble and insoluble protein fractions was attained less than 12hr. after injection, the maximal specific radioactivity of soluble protein being higher than that of insoluble protein.
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Project description:We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF) rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ), a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL) rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.
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Project description:1. Phytohaemagglutinin stimulates the transformation into blast cells of human lymphocytes incubated in vitro. This transformation is accompanied by an increase in the incorporation of [(14)C]leucine into protein and [(3)H]uridine into RNA. 2. The incorporation of [(14)C]leucine by cultures grown in the presence or absence of phytohaemagglutinin is inhibited to the same extent by cycloheximide, a known inhibitor of protein synthesis. 3. Lymphocytes grown without phytohaemagglutin synthesize mainly non-ribosomal RNA. [(3)H]Uridine incorporation by these cells was increased by cycloheximide. 4. Lymphocytes incubated with phytohaemagglutinin begin to synthesize substantial quantities of ribosomal RNA. Under these conditions [(3)H]uridine incorporation was partially inhibited by cycloheximide. This inhibition is shown to be largely a result of inhibition of the synthesis of ribosomal RNA.
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Project description:Protein synthesis in rat liver in vivo was measured between 0 and 72 h after administration of a non-lethal dose of cycloheximide. There was a period of inhibition of [3H]leucine incorporation into both intra- and extra-cellular proteins at 2 h after administration of the drug, which was followed by a recovery phase in which amino acid incorporation varied significantly among the various proteins evaluated. At 12 h there was a marked stimulation of incorporation into nascent polypeptides released from polyribosomes and plasma fibrinogen, but incorporation into ribosomal proteins as well as albumin was still inhibited. Between 12 and 48 h, nascent-polypeptide synthesis remained elevated, but ribosomal-protein synthesis recovered slowly from the inhibition to normal rates only, and plasma-albumin synthesis increased slowly to above control values up to 48 h before returning to normal. A differential pattern of incorporation was also observed for incorporation into free and membrane-bound polyribosomes.
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