Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Insulin and tri-iodothyronine induce glucokinase mRNA in primary cultures of neonatal rat hepatocytes.

ABSTRACT: Glucokinase (EC 2.7.1.2) first appears in the liver of the rat 2 weeks after birth and increases after weaning on to a high-carbohydrate diet. We investigated the hormonal regulation of glucokinase (GK) mRNA in primary cultures of hepatocytes from 10-12-day-old suckling rats. GK mRNA was undetectable in such cells after 48 h of culture in serum-free medium devoid of hormones. Addition of insulin or tri-iodothyronine (T3) to the medium resulted in induction of GK mRNA. The effects of insulin and T3 were dose-dependent and additive. Dexamethasone alone did not induce GK mRNA, but enhanced the response to insulin and decreased the response to T3. Induction of GK mRNA by insulin was not affected when the medium glucose concentration was varied between 5 and 15 mM, nor when culture was conducted in glucose-free medium supplemented with lactate and pyruvate or galactose. The time course of initial accumulation of GK mRNA in response to insulin was characterized by a lag of 12 h and an induction plateau reached after 36 h. If hepatocytes were then withdrawn from insulin for 24 h and subsequently subjected to a secondary stimulation by insulin, GK mRNA re-accumulated with much faster kinetics and reached the fully induced level within 8 h. Both primary and secondary responses to insulin were abolished by actinomycin D. These results provide insight into the role of hormonal stimuli in the ontogenic development of hepatic glucokinase.

SUBMITTER: Narkewicz MR

PROVIDER: S-EPMC1149602 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Glucose-6-phosphate dehydrogenase mRNA sequence abundance in primary cultures of rat hepatocytes. Effect of insulin and dexamethasone.

Project description:Hepatic glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is subject to nutritional regulation. To assess the possible role of hormones in this regulation, the amounts of G6PDH mRNA were studied in primary cultures of rat hepatocytes treated with insulin and dexamethasone, alone or in combination. Relative concentrations of G6PDH mRNA were directly assessed by a dot-blot hybridization procedure with nick-translated cDNA probes. G6PDH sequence abundance increased when the cultures were treated with insulin or dexamethasone, but the G6PDH mRNA induced by dexamethasone was not expressed at the protein level as active enzyme. In cultures treated with insulin and dexamethasone in combination, enzyme activity and G6PDH sequence abundance were greater than those induced by insulin alone. Our results directly demonstrate that G6PDH mRNA amounts are modulated in liver by these two classes of hormones and can partially account for the dietary induction of the enzyme observed in vivo.

| S-EPMC1147031 | biostudies-other

The separate roles of glucose and insulin in the induction of glucokinase in hepatocytes isolated from neonatal rats.

Project description:1. The specificity of the effect of glucose on the induction of glucokinase activity that occurs when hepatocytes freshly isolated from 13-day-old rats are incubated in Medium 199 together with insulin [Wakelam & Walker (1980) FEBS Lett. 111, 115-119] was examined. A pattern that is different from other known effects of glucose is found, and metabolism of this compound is not necessarily to account for this particular effect. 2. The effects of a raised glucose concentration and of insulin on the induction can be separated. The hexose initiates the process in the absence of insulin in a manner that is sensitive to actinomycin D but not to cycloheximide. The subsequent effect of insulin is dependent on the prior effect of glucose or other positive analogue, does not require the presence of glucose and is inhibited by cycloheximide but not by actinomycin D. 3. Induction of glucokinase in vitro in hepatocytes from neonatal animals is inhibited by adrenaline, glucagon and dibutyryl cyclic AMP, but not by vasopressin or angiotensin II. The inhibition by cyclic AMP is on the stage requiring insulin and is comparatively specific, because total protein synthesis is not apparently diminished. 4. The implications of these results are discussed with reference to possible mechanisms of induction and to the situation in vivo.

| S-EPMC1163009 | biostudies-other

Selective stimulation by tri-iodothyronine of myocardial deoxyribonucleic acid polymerase-alpha in neonatal rats.

Project description:Three forms of DNA polymerase (alpha, beta and gamma) were separated from isolated rat myocardial cells on the basis of template, pH and ionic requirements, sensitivity to N-ethylmaleimide and position on sucrose gradients. Tri-iodothyronine administration (20mug/100g intraperitoneally) to 3-week-old rats resulted in selective stimulation of DNA polymerase-alpha (198+/-7.1 versus 102+/-5.8pmol of [(3)H]dTMP/30min per mg of protein in untreated controls, P<0.01), with no change in polymerases-beta and -gamma. [(3)H]Thymidine incorporation into myocardial DNA was also enhanced in tri-iodothyronine-treated neonatal rats (132+/-11.2 versus 53+/-4.1c.p.m./mug of DNA in controls, P<0.001). Increased incorporation was associated with an expansion of deoxyribonucleoside 5'-triphosphate pools, especially that of dTTP (24+/-1.6 versus 10+/-1.1pmol/mg of DNA, P<0.01). Neither DNA polymerase activities nor [(3)H]thymidine incorporation were changed in 6-month-old rats in response to tri-iodothyronine. Unstimulated adult myocardial cells had DNA polymerase activities comparable with those in 3-week-old animals, but significantly lower [(3)H]-thymidine incorporation and deoxyribonucleoside triphosphate concentrations. Enhancement of both DNA polymerase-alpha activity and [(3)H]thymidine incorporation in tri-iodothyronine-treated young rats was prevented by concomitant administration of either vinblastine (1mug/g) or daunomycin (2mug/g); actinomycin D (0.1mug/g) or cycloheximide (8mug/g), on the other hand, prevented the increase in [(3)H]thymidine incorporation, but not DNA polymerase-alpha activation. These results demonstrate an age-dependent stimulation of myocardial DNA replication by tri-iodothyronine and suggest an inter-relationship between DNA synthesis and subsequent entry into mitosis.

| S-EPMC1161019 | biostudies-other

Regulation of fatty acid and carbohydrate metabolism by insulin, growth hormone and tri-iodothyronine in hepatocyte cultures from normal and hypophysectomized rats.

Project description:The interactions of insulin, growth hormone (somatotropin) and tri-iodothyronine (T3) in the long-term (24 h) regulation of fatty acid and carbohydrate metabolism were studied in hepatocyte primary cultures isolated from normal or hypophysectomized Sprague-Dawley rats. Hepatocytes from hypophysectomized rats had similar rates of palmitate metabolism, but lower rates of ketogenesis, than hepatocytes from normal rats. They also had a lower endogenous triacylglycerol content and lower activities of NADP-linked dehydrogenases than did cells from normal rats. The inhibitions of ketogenesis and gluconeogenesis by insulin were more marked in hepatocytes from hypophysectomized than from normal rats. Insulin caused a 7-10-fold increase in cellular glycogen in hepatocytes from hypophysectomized rats, compared with a 2-3-fold increase in cells from normal rats, and it increased cellular triacylglycerol by 65% in cells from hypophysectomized rats, compared with 11% in cells from normal rats. In hepatocytes from hypophysectomized rats, growth hormone and T3 increased ketogenesis both separately and in combination (12% and 23% respectively; P less than 0.05), whereas in hepatocytes from normal rats only the combination of growth hormone and T3 caused a significant increase in ketogenesis. In cells from hypophysectomized rats, T3 and growth hormone had different effects on carbohydrate metabolism: T3, but not growth hormone, potentiated the anti-gluconeogenic and glycogenic effects of insulin. It is concluded that hypophysectomy increases the responsiveness of hepatocytes to insulin, growth hormone and T3, and that growth hormone and T3 regulate fatty acid and carbohydrate metabolism by different mechanisms.

| S-EPMC1138396 | biostudies-other

Insulin-like growth factor I and insulin induce adipogenic-related gene expression in fetal brown adipocyte primary cultures.

Project description:Fetal rat brown adipocytes show high-affinity binding sites for both insulin-like growth factor I (IGF-I) and insulin. Cell culture for 24 h in the presence of IGF-I or insulin, independently, up-regulated the mRNA expression of adipogenic-related genes, such as fatty acid synthase (FAS), glycerol-3-phosphate de-hydrogenase and insulin-regulated glucose transporter Glut4, and down-regulated the expression of phosphoenolpyruvate carboxykinase mRNA in a dose-dependent manner. Moreover, both IGF-I and insulin increased the FAS gene transcription rate at 2 h, producing a time-dependent accumulation of FAS mRNA. Furthermore IGF-I or insulin increased glucose uptake and lipid content throughout the 24 h culture period. Our results suggest that both IGF-I and insulin are major signals involved in initiating and/or maintaining the expression of adipogenic-related genes in fetal rat brown adipocytes.

| S-EPMC1217813 | biostudies-other

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

Project description:Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the viable cell number is a very valuable reference parameter. Indirect methods of cell number/viability measurements may have up to 10 % standard deviation. This can lead to undesirable large deviations in the analysis of "-omics" data as well as time course studies. Such data should be preferably normalized to the exact viable cell number at a given time to allow meaningful interpretation and understanding of the biological processes. Manual counting of cell number is very laborious and not possible in certain experimental setups. We therefore, developed a simple and reliable fluorescence based method with an accuracy of 95-98 % for the determination of the viable cell number in situ. We optimized the seeding cell densities for primary rat hepatocytes for optimal cell adhesion. This will help in efficient use of primary cells which are usually limited in availability. The method will be very useful in the application of "-omics" techniques, especially metabolome analysis where the specific rates of uptake/production of metabolites can be reliably calculated.

| S-EPMC4960120 | biostudies-literature

The effects of glucocorticoids on insulin-stimulated lipogenesis in primary cultures of rat hepatocytes.

Project description:We used primary cultures of rat hepatocytes to evaluate the effects of glucocorticoids on insulin-responsive hepatic lipogenesis. The data indicate that hepatocytes incubated for 20 h with dexamethasone (0.1 microM) alone are profoundly resistant to the ability of insulin to stimulate lipogenesis acutely. In contrast, primary cultures of hepatocytes incubated with dexamethasone plus insulin are hyper-responsive to the ability of insulin to stimulate lipogenesis chronically. This potentiation of insulin action by a glucocorticoid occurs at physiological concentrations of the two hormones. Exposure to dexamethasone plus insulin for more than 4 h is required for the two hormones to enhance insulin action either by overcoming the insulin resistance induced by dexamethasone alone or by stimulating insulin action induced by insulin alone. Despite the marked potentiation of insulin action, hepatocytes exposed to dexamethasone plus insulin are less sensitive to insulin, as demonstrated by a shift to the right in the dose-response curve for insulin-stimulated lipogenesis. The resistance of hepatocytes to the acute effects of insulin after exposure to dexamethasone alone and the potentiation of insulin action and decreased sensitivity to insulin after exposure to insulin plus dexamethasone are all mediated by post-insulin-binding events. These studies demonstrate potentiation of insulin action in the liver by physiological concentrations of glucocorticoids and may have physiological significance for the regulation of normal hepatic lipogenesis, for the hyperlipidaemia observed with the pharmacological use of glucocorticoids, and for disease states in man associated with hyperinsulinaemia and hypercortisolism.

| S-EPMC1152020 | biostudies-other

Pregnane X receptor modulates the inflammatory response in primary cultures of hepatocytes.

Project description:Bacterial sepsis is characterized by a rapid increase in the expression of inflammatory mediators to initiate the acute phase response in liver. Inflammatory mediator release is counterbalanced by the coordinated expression of anti-inflammatory molecules such as interleukin 1 receptor antagonist (IL1-Ra) through time. This study determined whether activation of pregnane X receptor (PXR, NR1I2) alters the lipopolysaccharide (LPS)-inducible gene expression program in primary cultures of hepatocytes (PCHs). Preactivation of PXR for 24 hours in PCHs isolated from wild-type mice suppressed the subsequent LPS-inducible expression of the key inflammatory mediators interleukin 1? (IL-1?), interleukin 6 (IL-6), and tumor necrosis factor ? (TNF?) but not in PCHs isolated from Pxr-null (PXR-knockout [KO]) mice. Basal expression of key inflammatory cytokines was elevated in PCHs from PXR-KO mice. Stimulation of PCHs from PXR-KO mice with LPS alone produced enhanced levels of IL-1? when compared with wild-type mice. Experiments performed using PCHs from both humanized-PXR transgenic mice as well as human donors indicate that prolonged activation of PXR produces an increased secretion of IL1-Ra from cells through time. Our data reveal a working model that describes a pivotal role for PXR in both inhibiting as well as in resolving the inflammatory response in hepatocytes. Understanding the molecular details of how PXR is converted from a positive regulator of drug-metabolizing enzymes into a transcriptional suppressor of inflammation in liver will provide new pharmacologic strategies for modulating inflammatory-related diseases in the liver and intestine.

| S-EPMC4352581 | biostudies-literature

Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures.

Project description:BackgroundHepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown.ResultsIn this study we isolated primary hepatocytes from transgenic Per2(Luc) mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2(-/-) Per2(Luc) cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes.ConclusionsOur results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals.

| S-EPMC3911982 | biostudies-literature

Glucose- and insulin-independent induction of ATP citrate lyase in primary cultures of rat hepatocytes.

Project description:ATP citrate lyase, which is involved in the translocation of the lipogenic precursor acetyl-CoA from mitochondria to cytosol, was studied in primary cultures of hepatocytes from adult rats. After an initial decrease at the first day of culture the enzyme activity was nearly constant during the following days. It could be enhanced between 24h and 48 h in culture about 1.5-fold by elevation of the insulin concentration to 10-7mol/1.22-fold by elevation of the glucose concentration from 5 to 25 mmol/l and 3.5-fold by simultaneous elevation of insulin and glucose. The increase of activity was about linear with time for 24 h and could be blocked by cycloheximide, which inhibits protein synthesis at the translational level. Both observations suggest that the enhancement of activity was due to induction rather than to activation by interconversion. The glucose-dependent induction was furthermore evidenced by immunotitration which indicated a parallel increase of activity and enzyme protein.

| S-EPMC1152010 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data