Insulin-responsive cultured foetal-rat hepatocytes. Their preparation and characterization.
Ontology highlight
ABSTRACT:
SUBMITTER: DeSante DC
PROVIDER: S-EPMC1144261 | biostudies-other | 1984 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Insulin-stimulated glycogenesis and insulin degradation were studied simultaneously at 37 degrees C in cultured foetal hepatocytes grown for 2-3 days in the presence of cortisol. Degradation of cell-associated insulin, as measured by trichloroacetic acid precipitation, was significant after 4 min in the presence of 1-3 nM-125I-labelled insulin. This process became maximal (30% of insulin degraded) after 20 min, a time when binding-state conditions were achieved. No insulin-degradative activity was detected in a medium that had been exposed to cells. At steady-state, the appearance of insulin degradation products in the medium was linearly dependent on time (1.5 fmol/min per 10(6) cells at 1nM-125I-labelled insulin). Chloroquine (3-50 microM), bacitracin (0.1-10 mM) and NH4Cl (1-10 mM) inhibited insulin degradation as soon as this became detectable and caused an increase in the association of insulin to hepatocytes after 20 min. Lidocaine and dansylcadaverine had similar effects, whereas N-ethylmaleimide, aprotinin, phenylmethanesulphonyl fluoride and leupeptin were found to be ineffective. Chloroquine, and also bacitracin, at concentrations that inhibited insulin degradation, decreased the insulin-stimulated incorporation of [14C]glucose into glycogen over 2 h. This effect of chloroquine was specific, since it did not modify the basal glycogenesis, or the glycogenic effect of a glucose load in the absence of insulin. It therefore appears that the receptor-mediated insulin degradation (or some associated pathway) is functionally related to the glycogenic effect of insulin in foetal hepatocytes.
| S-EPMC1158116 | biostudies-other
Project description:Analysis of proteins in biological samples opens up the possibility of discovering new markers of toxicity. The liver is one of the primary targets of drug-induced toxicity, and it also secretes many plasma proteins, which can be measured clinically. Most of the plasma proteins produced by the liver are secreted by hepatocytes, but there is little information regarding the protein profile secreted by these cells. The purpose of this study was to analyze the secreted proteome of primary rat hepatocytes in a collagen gel sandwich configuration by a gel-LC-MS/MS procedure. We identified over 600 peptides corresponding to more than 200 proteins. The protein profile included over 50 plasma proteins, suggesting that the cultured hepatocytes secrete many of the proteins that they produce in vivo. Our data also suggests that the hepatocytes are actively remodeling their environment, since we identified several structural extracellular matrix proteins as well as some proteins known to be secreted specifically during liver regeneration. We also identified two proteins, alpha1-antitrypsin and alpha2-macroglobulin, whose secretions appear to be down-regulated in cells exposed to aflatoxin B1. It was noted that a 15 nM dose of aflatoxin B1 led to substantially diminished levels of these proteins and that day 6 of incubation was the ideal time point for medium collection. These data suggest that proteins in the conditioned medium of hepatocyte sandwich culture might lead to the discovery of biomarkers for drug-induced chemical toxicity.
| S-EPMC4113969 | biostudies-literature
Project description:Binding and degradation of 125I-labelled insulin were studied in cultured foetal hepatocytes after exposure to the protein-synthesis inhibitors tunicamycin and cycloheximide. Tunicamycin (1 microgram/ml) induced a steady decrease of insulin binding, which was decreased by 50% after 13 h. As the total number of binding sites per hepatocyte was 20000, the rate of the receptor degradation could not exceed 13 sites/min per hepatocyte. Cycloheximide (2.8 micrograms/ml) increased insulin binding by 30% within 6 h, an effect that persisted for up to 25 h. This drug had a specific inhibitory effect on the degradation of proteins prelabelled for 10 h with [14C]glucosamine, without affecting the degradation of total proteins. Chronic exposure to 10 nM-insulin neither decreased insulin binding nor modified the effect of the drugs. The absence of down-regulation of insulin receptors cannot be attributed to rapid receptor biosynthesis in foetal hepatocytes. Cellular insulin degradation, which is exclusively receptor-mediated, was determined by two different parameters. First, the rate of release of degraded insulin into the medium was 600 molecules/min per hepatocyte with 1 nM labelled hormone, and increased (preincubation with cycloheximide) or decreased (tunicamycin) as a function of the amount of cell-bound insulin. Secondly, the percentage of cell-bound insulin degraded was not changed by the presence of protein-synthesis inhibitors (25-30%). The stability of insulin degradation suggested that this process was dependent on long-life proteinase systems. Such differences in degradation rates and cycloheximide sensitivity imply that hormone- and receptor-degradation processes utilize distinct pathways.
| S-EPMC1153126 | biostudies-other
Project description:The activity changes of the urea-cycle enzymes were monitored in cultured foetal hepatocytes after dexamethasone and insulin treatments. Addition of dexamethasone induced the development of carbamoyl-phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase activities as soon as day 16.5 of gestation. When insulin was added together with dexamethasone, it markedly inhibited the steroid-induced increase in carbamoyl-phosphate synthetase, argininosuccinate synthetase and argininosuccinase activities.
| S-EPMC1144581 | biostudies-other
Project description:The insulin-receptor cycle was investigated in cultured foetal rat hepatocytes by determining the variations in insulin-binding sites at the cell surface after short exposure to the hormone. Binding of 125I-insulin was measured at 4 degrees C after dissociation of prebound native insulin. Two protocols were used: exchange binding assay and binding after acid treatment; both gave the same results. Cell-surface 125I-insulin-receptor binding decreased sharply (by 40%) during the first 5 min of 10 nM-insulin exposure (t1/2 = 2 min) and remained practically constant thereafter; subsequent removal of the hormone restored the initial binding within 10 min. This fall-rise sequence corresponded to variations in the number of insulin receptors at the cell surface, with no detectable change in receptor affinity. The reversible translocation of insulin receptors from the cell surface to a compartment not accessible to insulin at 4 degrees C was hormone-concentration- and temperature-dependent. SDS/polyacrylamide-gel electrophoresis after cross-linking of bound 125I-insulin to cell-surface proteins with disuccinimidyl suberate showed that these variations were not associated with changes in Mr of binding components, in particular for the major labelled band of Mr 130,000. The insulin-receptor cycle could be repeated after intermittent exposure to insulin. Continuous or intermittent exposure to the hormone gave a similar glycogenic response, contrary to the partial effect of a unique short (5-20 min) exposure. A relationship could be established between the repetitive character of the rapid insulin-receptor cycle and the maximal expression of the biological effect in cultured foetal hepatocytes.
| S-EPMC1147330 | biostudies-other
Project description:The antagonistic effects of insulin and glucagon on glycogen formation and mobilization were studied in cultured 18-day foetal rat hepatocytes with regard to different modes of exposure. Hormone combinations were achieved with a constant dose of 10 nM-insulin (maximal for the glycogenic effect of this hormone) and increasing doses of glucagon [from 0.03 to 10 nM: concn. causing half-maximal response (ED50) = 0.3 nM)]. When insulin and glucagon were added simultaneously, increasing glucagon concentrations progressively depressed the glycogenic effect of insulin and 0.3 nM-glucagon antagonized the insulin effect completely. The maximal glycogenolytic effect of glucagon was observed at concentrations greater than 1 nM. When the two hormones were introduced successively, with an interval of 4 h between additions, the effect of the second hormone was always fully expressed between 4 and 8 h. at which time the effect of the first hormone had ceased; the dominance of glucagon over insulin was also lost, due to cell desensitization to glucagon. Both continuous or intermittent (10 min on/10 min off periods) exposure to insulin and/or glucagon gave similar antagonistic effects, while in cells exposed to insulin plus glucagon alternating with exposure to insulin or glucagon alone, the glycogenic effect of insulin was less or more antagonized respectively by glucagon. Whatever the situation, the results obtained could not be related to antagonism by a glucagon-induced rise in either cyclic AMP levels (ED50 = 3 nM) or cell-surface hormone binding. Thus, depending on the hormonal state and the mode of hormone administration, regulation of glycogenesis in cultured foetal hepatocytes appears to be different from that predicted by the insulin/glucagon molar ratio, which is strikingly altered in the perinatal period.
| S-EPMC1151198 | biostudies-other
Project description:The pathways of glycogen synthesis from glucose were studied using double-isotope procedures in 18-day cultured foetal-rat hepatocytes in which glycogenesis is strongly stimulated by insulin. When the medium containing 4 mM-glucose was supplemented with [2-3H,U-14C]glucose or [3-3H,U-14C]glucose, the ratios of 3H/14C in glycogen relative to that in glucose were 0.23 +/- 0.04 (n = 6) and 0.63 +/- 0.09 (n = 8) respectively after 2 h. This indicates that more than 75% of glucose was first metabolized to fructose 6-phosphate, whereas 40% reached the step of the triose phosphates prior to incorporation into glycogen. The stimulatory effect of 10 nM-insulin on glycogenesis (4-fold) was accompanied by a significant increase in the (3H/14C in glycogen)/(3H/14C in glucose) ratio with 3H in the C-2 position (0.29 +/- 0.05, n = 6, P less than 0.001) or in the C-3 position (0.68 +/- 0.09, n = 8, P less than 0.01) of glucose, whereas the effect of a 12 mM-glucose load (3.5-fold) did not alter these ratios. Fructose (4 mM) displaced [U-14C]glucose during labelling of glycogen in the presence and absence of insulin by 50 and 20% respectively, and produced under both conditions a similar increase (45%) in the (3H/14C in glycogen)/(3H/14C in glucose) ratio when 3H was in the C-2 position. 3-Mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis from lactate/pyruvate, further decreased the already poor labelling of glycogen from [U-14C]alanine, whereas it increased both glycogen content and incorporation of label from [U-14C]serine and [U-14C]glucose with no effect on the relative 3H/14C ratios in glycogen and glucose with 3H in the C-3 position of glucose. These results indicate that an alternative pathway in addition to direct glucose incorporation is involved in glycogen synthesis in cultured foetal hepatocytes, but that insulin preferentially favours the classical direct route. The alternative foetal pathway does not require gluconeogenesis from pyruvate-derived metabolites, contrary to the situation in the adult liver.
| S-EPMC1133514 | biostudies-other
Project description:DNA microarray is a powerful tool in biomedical research. However, transcriptomic profiling using DNA microarray is subject to many variations including biological variability. To evaluate the different sources of variation in mRNA gene expression profiles, gene expression profiles were monitored using the Affymetrix RatTox U34 arrays in cultured primary hepatocytes derived from six rats over a 26 hour period at 6 time points (0 h, 2h, 5h, 8h, 14 h and 26 h) with two replicate arrays at each time point for each animal. In addition, the impact of sample size on the variability of differentially expressed gene lists and the consistency of biological responses were also investigated. Excellent intra-animal reproducibility was obtained at all time points with 0 out of 370 present probe sets across all time points showing significant difference between the 2 replicate arrays (3-way ANOVA, p <or= 0.0001). However, large inter-animal biological variation in mRNA expression profiles was observed with 337 out of 370 present probe sets showing significant differences among 6 animals (3-way ANOVA, p <or= 0.05). Principal Component Analysis (PCA) revealed that time effect (PC1) in this data set accounted for 47.4% of total variance indicating the dynamics of transcriptomics. The second and third largest effects came from animal difference, which accounted for 16.9% (PC2 and PC3) of the total variance. The reproducibility of gene lists and their functional classification was declined considerably when the sample size was decreased. Overall, our results strongly support that there is significant inter-animal variability in the time-course gene expression profiles, which is a confounding factor that must be carefully evaluated to correctly interpret microarray gene expression studies. The consistency of the gene lists and their biological functional classification are also sensitive to sample size with the reproducibility decreasing considerably under small sample size.
| S-EPMC2759134 | biostudies-literature
Project description:Arsenic is a proven human carcinogen and is associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high-affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)(2)]. In rats, the related canalicular transporter Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)(3) and MMA(GS)(2). The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic efflux was detected only across the basolateral membrane for 9 out of 14 SCHH preparations, 5 had both basolateral and canalicular efflux. Basolateral transport of arsenic was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2-dependence. Treatment of SCHH with As(III) (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1, MRP6, or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of arsenic. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest arsenic basolateral transport prevails over biliary excretion and is mediated at least in part by MRPs, most likely including MRP4.
| S-EPMC6596447 | biostudies-literature
Project description:The role of serine as a possible intermediate of the alternative pathway from glucose to glycogen was investigated under basal and insulin-stimulated conditions in 18-day cultured foetal-rat hepatocytes because these cells cannot use pyruvate-derived metabolites [Bismut & Plas (1989) Biochem. J. 263, 889-895]. Incubation of cells with [U-14C]glucose for 24 h led to a release of labelled serine in the medium concomitantly with a net serine production (100 nmol/24 h per culture). The rate of [14C]serine formation (close to 3 nmol/h per culture) indicated that a large part of newly formed serine originated from glucose. When short-term experiments were performed at day 2, glycogen labelling from [U-14C]serine or [U-14C]glycine, which was increased 3-fold by insulin after 2 h, evidenced their participation as glycogenic precursors. When a double-isotope procedure with [U-14C,3-3H]glucose was used, the direct and the alternative pathways from glucose were found to contribute to glycogenesis by 75 and 25% respectively. Cycloserine (18 mM), a transaminase inhibitor, strongly inhibited glycogen labelling from [U-14C] serine while producing a 70% increase in glucose incorporation by the alternative pathway, in both the presence and the absence of insulin. The inhibitor had no effect on the direct pathway from glucose to glycogen. Supplementation with 1 mM-hydroxypyruvate, a serine-derived metabolite, did not affect direct glucose incorporation, whereas the alternative pathway was stimulated whether insulin was present or not. These results indicate that the sequence glucose----serine----glycogen is operative in cultured foetal hepatocytes. The alternative pathway interferes with hydroxypyruvate utilization, and is likely mediated by the serine aminotransferase pathway, independently of the acute glycogenic action of insulin.
| S-EPMC1151044 | biostudies-other