leupeptins and Starvation

Although sucrose availability is crucial for commitment to plant cell division during G1 phase, it has remained uncertain how protein levels of core cell cycle genes are regulated. We found that Arabidopsis retinoblastoma-related protein1 (AtRBR1) and three E2F proteins were degraded under limited sucrose conditions, while protein abundance increased in response to treatment with the proteasome inhibitor MG132. We conclude that Arabidopsis key cell cycle proteins are degraded in a proteasome-dependent manner during sucrose starvation in Arabidopsis suspension MM2d cells.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Cycle Proteins; Cells, Cultured; Cysteine Proteinase Inhibitors; E2F Transcription Factors; G1 Phase; Gene Expression Regulation, Plant; Leupeptins; Proteasome Endopeptidase Complex; Starvation; Sucrose

The mechanisms of the increased serum leptin in response to feeding are poorly understood. Therefore, we used metabolic labeling to directly assess leptin biosynthesis, secretion, and turnover in adipose tissue from 14 h-starved compared with fed 12-14 week old rats. Starvation decreased serum leptin (-47 +/- 7%), adipose tissue leptin content (-32 +/- 5%), and leptin secretion during 3 h of incubation (-65 +/- 12%). Starvation did not affect leptin mRNA levels but decreased rates of leptin biosynthesis by tissue fragments, as determined by [(35)S]methionine/cysteine incorporation into immunoprecipitable leptin. Insulin in vitro did not acutely increase leptin biosynthesis or rates of (125)I-leptin degradation. Pulse-chase studies showed that in adipose tissue from fed but not starved rats, insulin accelerated the secretion of [(35)S]leptin by approximately 2-fold after 30 and 60 min of chase. Degradation of newly synthesized leptin was slower in adipose tissue of starved than fed rats (half-lives of 50 and 150 min, respectively). Inhibitor experiments showed that both lysosomes and proteosomes contributed to leptin degradation. In conclusion, feeding compared with starvation influences leptin production at multiple posttranscriptional levels: synthesis, tissue storage, turnover, and secretion. The insulin-stimulated release of leptin from a preformed intracellular leptin pool may contribute to increases in serum leptin levels after meals.

Topics: Adipocytes; Adipose Tissue; Animals; Blotting, Northern; Body Weight; Chloroquine; Cycloheximide; Insulin; Iodine Isotopes; Leptin; Leupeptins; Lysosomes; Male; Rats; Rats, Wistar; RNA, Messenger; Starvation; Sulfur Isotopes

We isolated membranes from leupeptin-induced autophagic vacuoles and compared them with lysosomal membranes purified from dextran-administered rats. In protein composition, autophagic vacuole membranes prepared from long term-starved (36 h) rats bear marked resemblance to lysosomal membranes, whereas vacuole membranes prepared from short term-starved (12 h) animals differ significantly from lysosomal membranes. Immunoblotting analyses showed that only autophagic vacuole membranes from short term-starved rats possess endoplasmic reticulum markers such as cytochrome P450 and NADPH-cytochrome c reductase. None of the membranes contain sialyltransferase, a Golgi membrane marker. In experiments in which rats were starved after feeding to induce autophagy, the appearance of the endoplasmic reticulum markers occurred during 6-12 h of starvation, concomitantly with increases in vacuolar proteins and sequestered cytosolic aldolase. The endoplasmic reticulum membrane markers and sequestered aldolase declined gradually after 20-36 h of starvation, suggesting that prolonged starvation causes no further increase in the formation of autophagic vacuoles but an increase in the population of matured autophagic vacuoles. Thus, the prominent markers of endoplasmic reticulum from which autophagosomes originate are well preserved in autophagic vacuole membranes, and retention of these markers is highly dependent on the formation and subsequent maturation process of autophagic vacuoles.

Topics: Animals; Autophagy; Biomarkers; Cytochrome P-450 Enzyme System; Endoplasmic Reticulum; Fructose-Bisphosphate Aldolase; Immunoblotting; Intracellular Membranes; Leupeptins; Liver; Male; Rats; Rats, Inbred Strains; Starvation; Vacuoles

Increase in the density of liver lysosomes after leupeptin administration was marked in starved rats but only slight in starved-refed rats. The levels of several intracellular enzymes in the liver lysosome fraction purified from leupeptin-treated rats were about 10 to 30 times more in starved rats than in refed rats. However, there was no difference between the intralysosomal levels of endocytosed FITC-labeled asialofetuin in starved and refed rats, indicating that refeeding after starvation markedly suppressed autophagy but not heterophagy in vivo. Immunohistochemical studies with cathepsin B and asialofetuin Fab'-peroxidase conjugates showed that refeeding after starvation markedly altered the cellular distribution of cathepsin B in the liver, resulting in a linear arrangement of the enzyme only on the periphery of hepatocytes. In contrast, endocytosed asialofetuin was found only in the periphery of hepatocytes of both starved and starved-refed rats. These results indicate that autophagy and heterophagy are regulated by different mechanisms in vivo.

Topics: alpha-Fetoproteins; Animals; Asialoglycoproteins; Autophagy; Cathepsins; Centrifugation, Density Gradient; Fetuins; Food; Immunoenzyme Techniques; Kinetics; Leupeptins; Liver; Lysosomes; Male; Phagocytosis; Pinocytosis; Rats; Rats, Inbred Strains; Starvation

Effects of chloroquine, colchicine, leupeptin, taxol and vinblastine on the resialylation and degradation of human [125I]asialotransferrin type 3 were studied in rats. An improved experimental technique was applied that permitted the quantification of resialylated ligand produced by individual animals over 3 h by using deconvolution. All three microtubule inhibitors increased the proportion of the dose undergoing resialylation by 35-39%. In addition, colchicine, and, especially, vinblastine enhanced the overall recovery of the dose as protein-bound 125I. The dose recovery was also augmented by leupeptin without any concomitant change in resialylation. Chloroquine suppressed resialylation and this effect could only be partially lifted by the administration of colchicine. The blood of colchicine-treated rats possessed no resialylating activity toward the ligand even when supplemented with additional alkaloid in vitro. The observations support the view that the respective fractions of the ligand destined for resialylation and degradation can, to a certain extent, be varied independently of each other. The effects of short-term starvation (20 h) and refeeding (4 h) on these processes are also presented.

Topics: Alkaloids; Animals; Asialoglycoproteins; Chloroquine; Colchicine; Female; Humans; Leupeptins; Liver; Lysine; Male; Microtubules; Paclitaxel; Protein Biosynthesis; Rats; Starvation; Transferrin; Vinblastine