Somatomedin production by rat liver in organ culture

1982 ◽  
Vol 99 (3) ◽  
pp. 422-430 ◽  
Author(s):  
Michel Binoux ◽  
Claudine Lassarre ◽  
Nathalie Hardouin
Keyword(s):  
Organ Culture ◽  
Rat Liver ◽  
Culture Media ◽  
Gel Filtration ◽  
Base Line ◽  
Rat Serum ◽  
Competitive Protein Binding Assay ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Abstract. Radioligand assays have been used to study the production of insulin-like growth factor (IGF) and its carrier (IGFCP) by rat liver in organ culture. Bound IGF released into the culture medium was dissociated and separated from its carrier by gel filtration in 1 m acetic acid. IGF was measured by a competitive protein-binding assay using human IGF and the specific IGFCP produced by rat liver. IGF CP concentrations were assessed in terms of IGFCP binding to labelled IGF, compared to a reference IGFCP preparation obtained from rat serum. Without added hormone,the ratios IGF:total protein and IGFCP:total protein in the culture media after 3 days' culture were approximately 7 and 70 times higher, respectively, than those in serum. GH and insulin both stimulated IGF production. The response was dose-dependent, and significant at physiological concentrations of hormone (10 ng/ml GH and 10 μU/ml insulin. With 1 μg/ml GH and 1 mU/ml insulin, the IGF concentrations in the media on average reached 2½ times the base-line level. These hormones had no significant effect on IGF CP production. One ng/ml cortisol stimulated IGF production, but, in response to increasing concentrations, there was a dose-dependent decrease in IGF production. By contrast, IGFCP production was stimulated and there was a positive correlation between the carrier concentration in the culture media and the amount of cortisol added. The results indicate that insulin and cortisol, in addition to GH, have a direct influence on IGF production by the liver in vitro. They also suggest that the biosyntheses of IGF and its carrier are subject to different systems of regulation.

Somatomedin production by rat liver in organ culture

1980 ◽  
Vol 93 (1) ◽  
pp. 73-82 ◽  
Author(s):  
M. Binoux ◽  
P. Hossenlopp ◽  
C. Lassarre ◽  
D. Seurin
Keyword(s):  
Organ Culture ◽  
Rat Liver ◽  
De Novo ◽  
Culture Media ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Leucine Incorporation ◽  
Large Molecules ◽  
The Media ◽  
Dose Dependent

Abstract. Organ cultures with livers from normal rats were used to investigate the production of somatomedins (SM) and the control of this activity by hormones. Orosomucoid was used as an indicator of liver activity and measured by radioimmunoassay in the culture media. This protein was released continuously over a period of 6 days and a positive correlation was found between the concentrations of orosomucoid and total proteins. Cycloheximide and actinomycin suppressed the release of orosomucoid. Experiments using [3H]leucine incorporation indicated that the proteins released correspond to proteins synthesized de novo. Orosomucoid release was stimulated by insulin and cortisol and inhibited by growth hormone. Both effects were dose-dependent. These results demonstrate the validity of the technique used for studying the release of export proteins synthetized by the liver. Liver culture media significantly inhibited cartilage sulphation as estimated by [35S]sulphate incorporation in embryonic chick cartilage. Gel filtration (Sephadex G 25) and ultrafiltration experiments showed that the inhibition was caused by large molecules. Heating of the media (100°C for 20 min) reduced, but did not entirely suppress, the inhibitory effect. The addition of GH (1 mU/ml) or insulin (0.25—1 mU/ml) to the culture dishes significantly increased the SM activity of the medium and a dose-dependent effect was observed with insulin. Cycloheximide suppressed the insulin-stimulated SM release. A combination of GH and insulin resulted in an additive effect. These findings suggest that: 1) SM is synthesized by rat liver in organ culture; 2) insulin plays an important role in SM generation, as it itself is capable of both stimulating the release of SM and amplifying the stimulatory effect of GH.

Somatomedin production by rat liver in organ culture

1980 ◽  
Vol 93 (1) ◽  
pp. 83-90 ◽  
Author(s):  
M. Binoux ◽  
C. Lassarre ◽  
D. Seurin
Keyword(s):  
Organ Culture ◽  
Rat Liver ◽  
Culture Medium ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Antagonistic Action ◽  
Large Molecules ◽  
Sites Of Action ◽  
Dose Dependent

Abstract. Inhibitors of cartilage sulphation have been found to be released by the rat liver in organ culture. They cause a decrease in [35S]sulphate uptake by embryonic chick cartilage and, when added to a constant amount of serum, counteract the somatomedin (SM) activity of the serum. Both of these effects are dose-dependent. Their antagonistic action, investigated in the presence of increasing concentrations of serum, appeared to resemble non-competitive inhibition which would suggest different sites of action for SM and inhibitors. Incubation of the liver explants with cortisol (0.01—1 μg/ml) increased the sulphation-inhibiting activity of the culture medium and the effect was dose-dependent. Simultaneous addition of cycloheximide suppressed the inhibition. Gel filtration of the culture medium on Sephadex G 75 showed that: a) at pH 7.9, inhibitors eluted in the same fractions as [125I]SM-A bound to its carrier (apparent molecular weight ∼45 000); b) at pH 2.4, inhibitors still eluted as large molecules, but SM activity appeared in the same fractions as the dissociated [125I]SM-A. The question arises whether the cartilage sulphation inhibitor might not be the same molecule as the SM-carrier protein released by the liver.

Inhibitory Effects of Purple Sweet Potato Leaf Extract on the Proliferation and Lipogenesis of the 3T3-L1 Preadipocytes

2015 ◽  
Vol 43 (05) ◽  
pp. 915-925 ◽  
Author(s):  
Shou-Lun Lee ◽  
Hsien-Kuang Lee ◽  
Ting-Yu Chin ◽  
Ssu-Chieh Tu ◽  
Ming-Hsun Kuo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Sweet Potato ◽  
Early Stage ◽  
Water Extract ◽  
Potato Leaf ◽  
Purple Sweet Potato ◽  
Pre Treatment ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Purple sweet potato leaves (PSPLs) are healthy vegetable that is rich in anti-oxidants. A solution of boiling water extract of PSPL (PSPLE) is believed to be able to prevent obesity and metabolic syndrome in the countryside of Taiwan, but its efficacy has not yet been verified. The purpose of this study was to investigate the possible anti-adipogenesis effect of PSPLE in vitro. PSPLE was used to treat the 3T3-L1 cells, and the effects on cell proliferation and adipogenesis were investigated. The results showed that PSPLE caused a dose-dependent decrease in the cell proliferation of 3T3-L1 preadipocytes, but did not alter the cell viability. In addition, PSPLE induced ERK inactivation in the 3T3-L1 preadipocytes. Furthermore, pre-treatment of confluent 3T3-L1 cells with PSPLE led to reduced lipid accumulation in differentiated 3T3-L1 cells. The inhibition of lipogenesis could result from the PSPLE-induced down-regulation of the expression of the C/EBPα and SREBP-1 transcription factors during 3T3-L1 adipocyte differentiation. These results suggest that PSPLE not only inhibits cell proliferation at an early stage but also inhibits adipogenesis at a later stage of the differentiation program.

scholarly journals Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells

1989 ◽  
Vol 262 (2) ◽  
pp. 449-456 ◽  
Author(s):  
C Hanekom ◽  
A Nel ◽  
C Gittinger ◽  
A Rheeder ◽  
G Landreth
Keyword(s):  
T Cells ◽  
Time Course ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Jurkat Cells ◽  
Serine Kinase ◽  
Transient Activation ◽  
Normal Human ◽  
Dose Dependent

Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells.

scholarly journals Effect of micromolar concentrations of manganese ions on calcium-ion cycling in rat liver mitochondria

1983 ◽  
Vol 212 (3) ◽  
pp. 773-782 ◽  
Author(s):  
B P Hughes ◽  
J H Exton
Keyword(s):  
Rat Liver ◽  
Calcium Ion ◽  
Incubation Medium ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Manganese Ions ◽  
Dose Dependent Decrease ◽  
Incubation Temperatures ◽  
Dose Dependent

The effects of micromolar concentrations of Mn2+ on the rat liver mitochondrial Ca2+ cycle were investigated. It was found that the addition of Mn2+ to mitochondria which were cycling 45Ca2+ led to a rapid dose dependent decrease in the concentration of extramitochondrial 45Ca2+ of about 1 nmol/mg of protein. The effect was complete within 30 s, was half maximal with 10 microM Mn2+ and was observed in the presence of 3 mM Mg2+ and 1 mM ATP. It occurred over a broad range of incubation temperatures, pH and mitochondrial Ca2+ loads. It was not observed when either Mg2+ or phosphate was absent from the incubation medium, or in the presence of Ruthenium Red. These findings indicate that micromolar concentrations of Mn2+ stimulate the uptake of Ca2+ by rat liver mitochondria, and provide evidence for an interaction between Mg2+ and Mn2+ in the control of mitochondrial Ca2+ cycling.

Comparison of somatostatin-28 and somatostatin-14 clearance by the perfused rat liver

1985 ◽  
Vol 63 (1) ◽  
pp. 62-67 ◽  
Author(s):  
M. Seno ◽  
Y. Seino ◽  
Y. Takemura ◽  
S. Nishi ◽  
H. Ishida ◽  
...  
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Perfused Rat Liver ◽  
Half Time ◽  
Hepatic Extraction Ratio ◽  
Plasma Extract ◽  
Second Peak ◽  
Somatostatin 14

The hepatic clearances of somatostatin (SS)-28 and SS-14 by the perfused rat liver were compared, using a recirculating, plasma-free, erythrocyte-containing perfusion system. The disappearance rate constant, half time, clearance, and hepatic extraction ratio when 1.2 nM SS-28 was added to the perfusate were 0.0221 ± 0.0051 min−1, 36.6 ± 7.6 min, 0.34 ± 0.08 mL/min, and 17.2 ± 3.9%, respectively. The corresponding values obtained when SS-14 was added to the perfusate were 0.0405 ± 0.0022 min−1, 17.3 ± 1.0 min, 0.71 ± 0.05 mL/min, and 35.4 ± 2.6%, respectively. The differences between the SS-28 and SS-14 indices were all statistically significant. In addition, the perfusates with SS-28 added were eluted on Sephadex G-25 fine columns and somatostatinlike immunoreactivity (SLI) was determined. No SS-14 was found in perfusate containing SS-28 at both 5 and 30 min after the beginning of the perfusion. To investigate whether or not the liver plays an important role in the clearance of SS-28 or the conversion of SS-28 to SS-14 in vivo, the plasma disappearance of 2 μg SS-28 was compared in the whole rat and the functionally hepatectomized model. The half time of plasma SS-28 was 1.43 ± 0.12 min in the whole rat, significantly shorter than the 2.20 ± 0.14 min in the hepatectomized model. Gel filtration of plasma extract samples at 0.5 min after the SS-28 injection showed two major peaks of SLI: a first peak corresponding to SS-28 and a second peak coeluted in the position of SS-14 in both the whole rat and the hepatectomized model. At 4 min after the SS-28 injection, the first peak disappeared and only a small second peak was observed. These results suggest that SS-28 is cleared by the rat liver in vivo and in vitro and that it is cleared more slowly than SS-14. Furthermore, we find that little, if any, conversion of SS-28 to SS-14 occurs in the liver.

scholarly journals Identification of a new metalloproteinase inhibitor that forms tight-binding complexes with collagenase

1990 ◽  
Vol 269 (1) ◽  
pp. 183-187 ◽  
Author(s):  
T E Cawston ◽  
V A Curry ◽  
I M Clark ◽  
B L Hazleman
Keyword(s):  
Connective Tissue ◽  
Culture Media ◽  
Tight Binding ◽  
Gel Filtration ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Metalloproteinase Inhibitor ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Extracellular Activity

Connective-tissue cells produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that the levels of this inhibitor controlled the extracellular activity of these enzymes. We recently detected a new metalloproteinase inhibitor present in culture media of WI-38 fibroblasts. The inhibitor, named ‘large inhibitor of metalloproteinases’ (LIMP), can be separated from TIMP by gel filtration on Ultrogel AcA 44, where it is eluted with an apparent Mr of 76,000. A portion of this inhibitor-containing peak binds to concanavalin A-Sepharose, indicating that at least some of the inhibitor contains carbohydrate. LIMP inhibits collagenase (MMP-1), stromelysin (MMP-3) and gelatinase (MMP-2) in a dose-dependent fashion. Collagenase forms tight-binding complexes with LIMP, which can be separated from free collagenase on gel-filtration columns. The complex is eluted with Mr 81,600 (AcA 44) or Mr 60,000 (Superose 12). This complex is larger than that formed between collagenase and TIMP, which has Mr 52,800 (Aca 44) or 41,000 (Superose 12). Polyclonal antibody to TIMP does not recognize LIMP by immunoblotting, and will not block the inhibition of collagenase by LIMP, showing that LIMP is not a multimeric form of TIMP. The role of this new inhibitor in connective-tissue breakdown studies and its relationship to previously described inhibitors of metalloproteinases is discussed.

scholarly journals Simvastatin Results in a Dose-Dependent Toxic Effect on Spiral Ganglion Neurons in anIn VitroOrganotypic Culture Assay

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Katharina Leitmeyer ◽  
Andrea Glutz ◽  
Cristian Setz ◽  
Leonie Wieland ◽  
Sulamith Egloff ◽  
...  
Keyword(s):  
Mevalonate Pathway ◽  
Neuronal Survival ◽  
Spiral Ganglion ◽  
Spiral Ganglion Neurons ◽  
Supporting Cells ◽  
Simvastatin Treatment ◽  
Dose Dependent Decrease ◽  
Ganglion Neurons ◽  
Dose Dependent

Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an enzyme necessary for the production of mevalonate. They are widely used as cholesterol-lowering drugs. However, conflicting data about the effect of statins on neuronal cells has been published. To explore the effect of simvastatin on spiral ganglion neurons (SGNs), SG explants of 5-day-old rats were treated with increasing concentrations of simvastatin. In addition, SG explants were treated with mevalonate and with the combination of simvastatin and mevalonate. SGN number, length of the neurites, area of nonneuronal supporting cells, and neuronal survival were analyzed. Simvastatin treatment results in a significant dose-dependent decrease of SG neurite number, length of neurites, area of supporting cells, and SG neuronal survival compared to control. Interestingly, treatment with mevalonate in addition to simvastatin increased SG neuronal survival compared to simvastatin treatment only. However, treatment with mevalonate in addition to simvastatin did not influence SG neurite number, length of neurites, and area of supporting cells compared to simvastatin treatment only. Our results suggest a neurotoxic effect of simvastatin on SGNsin vitro. Neurotoxicity seems to be at least partially mediated by the mevalonate pathway. Therefore, caution is warranted to use simvastatin as a potential otoprotective drug.

Sign in / Sign up

Export Citation Format

Share Document