Pathways of glycogen synthesis in Novikoff ascites-hepatoma cells.
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ABSTRACT:
SUBMITTER: Nigam VN
PROVIDER: S-EPMC1185104 | biostudies-other | 1969 Nov
REPOSITORIES: biostudies-other
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Project description:A study of the enzymes of the glycogen pathway in Novikoff ascites hepatoma shows that glycogen synthetase has the lowest activity and that the tumour contains no high-K(m) soluble glucokinase. However, incubation of tumour cells with metabolizable sugars in vitro, or intraperitoneal administration of glucose into the tumour-bearing rat, results in glycogen accumulation by the tumour cells. Glycogen synthesis in the tumour is supported by aerobically produced ATP but is decreased anaerobically and by uncouplers of oxidative phosphorylation. Absence of P(i) from the incubation medium increases glycogen synthesis and decreases glycolysis. The optimum temperature for glycogen synthesis is 37 degrees . The capacity of the intact tumour cell to degrade deposited glycogen is low, but is accelerated by 2,4-dinitrophenol. Tumour homogenates prepared after osmotic shock do not incorporate [(14)C]glucose into glycogen. The glucose moiety of glucose 1-phosphate and of UDP-glucose is incorporated into glycogen by the homogenates and the incorporation of glucose 1-phosphate is greatly enhanced by AMP. Glucose 6-phosphate is a poor precursor of glycogen in the homogenate system, probably because it inhibits activation of phosphorylase b by AMP.
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Project description:Novikoff ascites-hepatoma cells show no Crabtree effect on the addition of glucose to tumour-cell suspensions, and convert a significant part of the added glucose into glycogen. Treatment of the cells with 2-deoxyglucose or glucose in the presence of iodoacetate inhibits respiration and decreases glycogen synthesis from glucose. Short-term experiments indicate a slight inhibition of glucose uptake for a brief period, due either to ATP accumulation in the mitochondria or to glucose 6-phosphate-mediated inhibition of hexokinase. Utilization of glucose metabolites and ATP for glycogen synthesis appears to remove inhibition of glucose uptake, and perhaps accounts for the absence of respiratory inhibition, by relieving a deficiency of ADP for the mitochondria. Decreased respiration in the presence of 2-deoxyglucose or glucose in the presence of iodoacetate could be due to the change in mitochondrial structure or permeability, caused by the significant loss of adenine nucleotides.
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Project description:Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.
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Project description:Glycogen storage, conversion and utilization in astrocytes play important roles in brain energy metabolism. The conversion of glycogen to lactate through glycolysis occurs through the coordinated activities of various enzymes, and inhibition of this process can impair different brain processes including formation of long-lasting memories. To replenish depleted glycogen stores, astrocytes undergo glycogen synthesis, a cellular process that has been shown to require transcription and translation during specific stimulation paradigms. However, the detailed nuclear signaling mechanisms and transcriptional regulation during glycogen synthesis in astrocytes remain to be explored. In this report, we study the molecular details of vasoactive intestinal peptide (VIP)-induced glycogen synthesis in astrocytes. VIP is a potent neuropeptide that triggers glycogenolysis followed by glycogen synthesis in astrocytes. We show evidence that VIP-induced glycogen synthesis requires CREB-mediated transcription that is Protein Kinase C and calcium-dependent but is independent of Protein Kinase A. In parallel to CREB activation, we demonstrate that VIP also triggers nuclear accumulation of the CREB coactivator CRTC2 only in astrocytic nuclei that also requires Protein Kinase C activity. Transcriptome profiles of VIP-induced astrocytes identified robust CREB-dependent transcription of glycogenic genes including the upregulation of Ppp1r3c along with robust repression of Phkg1, the catalytic subunit of phosphorylase kinase. Overall, our data demonstrates the importance of CREB-mediated transcription in astrocytes during stimulus-driven glycogenesis.
2024-04-30 | GSE243500 | GEO
Project description:The hepatitis C virus (HCV) causes chronic liver disease leading to fibrosis, cirrhosis, and hepatocellular carcinoma. HCV infection triggers various types of cell death which contribute to hepatitis C pathogenesis. However, much is still unknown about the impact of viral proteins on them. Here we present the results of simultaneous immunocytochemical analysis of markers of apoptosis, autophagy, and necrosis in Huh7.5 cells expressing individual HCV proteins or their combinations, or harboring the virus replicon. Stable replication of the full-length HCV genome or transient expression of its core, Е1/Е2, NS3 and NS5B led to the death of 20-47% cells, 72 h posttransfection, whereas the expression of the NS4A/B, NS5A or NS3-NS5B polyprotein did not affect cell viability. HCV proteins caused different impacts on the activation of caspases-3, -8 and -9 and on DNA fragmentation. The structural core and E1/E2 proteins promoted apoptosis, whereas non-structural NS4A/B, NS5A, NS5B suppressed apoptosis by blocking various members of the caspase cascade. The majority of HCV proteins also enhanced autophagy, while NS5A also induced necrosis. As a result, the death of Huh7.5 cells expressing the HCV core was induced via apoptosis, the cells expressing NS3 and NS5B via autophagy-associated death, and the cells expressing E1/E2 glycoproteins or harboring HCV the replicon via both apoptosis and autophagy.
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Project description:Direct acting antivirals (DAAs) revolutionized the therapy of chronic hepatitis C infection. However, unexpected high recurrence rates of hepatocellular carcinoma (HCC) after DAA treatment became an issue in patients with advanced cirrhosis and fibrosis. In this study, we aimed to investigate an impact of DAA treatment on the molecular changes related to HCC development and progression in hepatoma cell lines and primary human hepatocytes. We found that treatment with sofosbuvir (SOF), a backbone of DAA therapy, caused an increase in EGFR expression and phosphorylation. As a result, enhanced translocation of EGFR into the nucleus and transactivation of factors associated with cell cycle progression, B-MYB and Cyclin D1, was detected. Serine/threonine kinase profiling identified additional pathways, especially the MAPK pathway, also activated during SOF treatment. Importantly, the blocking of EGFR kinase activity by erlotinib during SOF treatment prevented all downstream events. Altogether, our findings suggest that SOF may have an impact on pathological processes in the liver via the induction of EGFR signaling. Notably, zidovudine, another nucleoside analogue, exerted a similar cell phenotype, suggesting that the observed effects may be induced by additional members of this drug class.
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Project description:The distribution of hexokinase between bound and soluble forms was studied by digitonin fractionation of Zajdela hepatoma ascites cells maintained under various metabolic conditions. Addition of glucose to Zajdela cells respiring on endogenous substrates induces an immediate inhibition of respiration by 50-60% ( Crabtree effect), and a production of acid due to glycolysis. Acid production decreases abruptly after 60s to 50% of the initial rate. The ATP/ADP ratio is not altered by the addition of glucose or by different rates of glycolysis. The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone decreases the ATP/ADP ratio by 10-fold in cells respiring on endogenous substrate, but has little effect on cells oxidizing glucose. Rapid fractionation of the cells under these various metabolic conditions revealed no change in the distribution of hexokinase. Approx. 75% of hexokinase is bound in all cases, in contrast with lactate dehydrogenase, 95% of which was in the soluble form. Longer-term incubations (to 20 min) revealed only slight (10-15%) increases in soluble hexokinase in cells incubated with glucose. Various metabolic inhibitors had little additional affect on the subcellular distribution of hexokinase. Thus a rapid release of hexokinase from mitochondrial membrane is not a mechanism by which glycolysis is regulated in rapidly growing Zajdela hepatoma.
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Project description:The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.
| S-EPMC1147793 | biostudies-other
Project description:Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.
| S-EPMC4643957 | biostudies-literature