Insulin stimulates sodium-dependent phosphate transport by osteoblast-like cells

1991 ◽  
Vol 260 (5) ◽  
pp. E751-E755 ◽  
Author(s):  
K. J. Kunkler ◽  
L. M. Everett ◽  
D. K. Breedlove ◽  
S. A. Kempson
Keyword(s):  
De Novo ◽  
Insulin Receptors ◽  
Phosphate Transport ◽  
Osteosarcoma Cell ◽  
Maximal Velocity ◽  
Stimulatory Action ◽  
Cotransport System ◽  
Umr 106 ◽  
Dose Dependent ◽  
De Novo Protein Synthesis

The rat osteosarcoma cell line UMR-106-01 has an osteoblast-like phenotype. When grown in monolayer culture, these cells transport Pi via a Na(+)-dependent carrier. The Na(+)-Pi cotransport system is stimulated by parathyroid hormone (PTH). Because there are insulin receptors on osteoblast-like cells, we determined possible effects of insulin on Na(+)-Pi cotransport in UMR-106-01 cells. Incubation of cells with 10(-8) M insulin for 3 h produced a 73% increase (P less than 0.025) in Na(+)-Pi cotransport. There was no significant change in Na(+)-L-alanine cotransport or in Na(+)-independent uptake of Pi and alanine. The stimulatory action of insulin on Na(+)-Pi cotransport was present within 2 h and was dose dependent in the range 10(-10) to 10(-7) M. The increase in Na(+)-Pi cotransport was accompanied by an increase in apparent maximal velocity with no change in apparent Michaelis constant for Pi. Use of cycloheximide to block de novo protein synthesis did not interfere with this action of insulin. We conclude that insulin, like PTH, directly stimulates the Na(+)-Pi cotransport system in osteoblast-like cells. The mechanism remains to be determined.

scholarly journals Norepinephrine, tri-iodothyronine and insulin upregulate glyceraldehyde-3-phosphate dehydrogenase mRNA during Brown adipocyte differentiation

1999 ◽  
pp. 169-179 ◽  
Author(s):  
I Barroso ◽  
B Benito ◽  
C Garci-Jimenez ◽  
A Hernandez ◽  
MJ Obregon ◽  
...  
Keyword(s):  
De Novo ◽  
Precursor Cells ◽  
Brown Adipocyte ◽  
Mrna Levels ◽  
Brown Adipocytes ◽  
Gapdh Gene ◽  
Gapdh Mrna ◽  
Brown Adipose ◽  
Dose Dependent ◽  
De Novo Protein Synthesis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was studied in differentiating brown adipocytes. Northern blot analysis showed that GAPDH mRNA levels increased during differentiation of precursor cells into mature adipocytes, mainly in the initial stages of the differentiation process. Insulin, tri-iodothyronine (T(3)) and norepinephrine, the main regulators of brown adipose tissue function, upregulated GAPDH mRNA levels, whereas retinoic acid inhibited them. The effect of insulin was present on all culture days examined, was time- and dose-dependent, and was exerted through its own receptors, as demonstrated by comparing insulin and insulin-like growth factor (IGF)-I and -II potencies in this system. Using the transcriptional inhibitor, actinomycin D, we demonstrated that T(3), and to a lesser extent insulin, stabilized GAPDH mRNA. Experiments with cycloheximide indicated that both hormones require de novo protein synthesis to achieve their effects. Using cAMP analogs, we showed that the effect of norepinephrine is probably exerted through this second messenger. Co-operation was elucidated between norepinephrine- and insulin-mediated induction of GAPDH mRNA levels. In summary, we have demonstrated that GAPDH mRNA is subjected to multifactorial regulation in differentiating brown adipocytes that includes differentiation of precursor cells and the lipogenic/lipolytic regulators of the tissue.

Regulation of quail oviduct phospholipase A2 activity by estradiol

1994 ◽  
Vol 131 (2) ◽  
pp. 205-212 ◽  
Author(s):  
JM Fayard ◽  
S Chanal ◽  
B Felouati ◽  
O Macovschi ◽  
M Lagarde ◽  
...  
Keyword(s):  
De Novo ◽  
Gene Activation ◽  
Hydrolytic Activity ◽  
Estradiol Benzoate ◽  
Phospholipase A ◽  
Calcium Dependent ◽  
Dose Dependent ◽  
De Novo Protein Synthesis ◽  
Phospholipase A2 Activity ◽  
Stimulation Of

Fayard JM, Chanal S, Felouati B, Macovschi O, Lagarde M, Pageaux JF, Laugier C. Regulation of quail oviduct phospholipase A2 activity by estradiol. Eur J Endocrinol 1994;131:205–12. ISSN 0804–4643 The phospholipase A2 (PLA2) activity was measured in the oviduct of immature and estradiol benzoate (EB)-treated quails. The pH profiles demonstrate the presence of two PLA2 isoforms in the avian oviduct: a neutral isoform, optimally active at pH 7–7.5 and calcium independent, responsible for most of the hydrolytic activity in the immature oviduct and poorly stimulated by estradiol; and an alkaline isoform, optimally active at pH 8–9.5 and calcium dependent, with little activity in the immature tissue but markedly stimulated by EB. After EB injection, PLA2 activation occurs at first during the prereplicative period of oviduct cells (+172% at 6 h), it is dose dependent from 0.01 to 1 mg/kg EB and can be prevented by cycloheximide together with ornithine decarboxylase activation. Moreover, estradiol was inactive on cell-free extracts of immature oviducts. These results suggest that EB increases PLA2 activity through gene activation and de novo protein synthesis. The correlation between the early stimulation of PLA2 activity and the proliferation of oviduct cells is discussed. JM Fayard, INSERM U.352, Laboratoire de Physiologie–Pharmacodynamie, INSA 406, 69621 Villeurbanne Cedex, France

Parathyroid hormone action on phosphate transport is inhibited by high osmolality

1990 ◽  
Vol 258 (5) ◽  
pp. F1336-F1344 ◽  
Author(s):  
S. A. Kempson ◽  
C. Helmle ◽  
M. I. Abraham ◽  
H. Murer
Keyword(s):  
Parathyroid Hormone ◽  
Monolayer Culture ◽  
De Novo ◽  
Fluid Phase ◽  
Phosphate Transport ◽  
Normal Medium ◽  
Opossum Kidney ◽  
Cell Monolayers ◽  
Phosphate Cotransport ◽  
De Novo Protein Synthesis

Parathyroid hormone (PTH) produces rapid inhibition of Na(+)-phosphate cotransport, characterized by a decreased maximal rate of transport, and the inhibition is independent of de novo protein synthesis. The present study determined whether the action of PTH on Na(+)-phosphate cotransport is mediated, at least in part, by rapid endocytic internalization of Na(+)-phosphate cotransporters present in the plasma membrane. Horseradish peroxidase, a fluid-phase marker, was used to demonstrate the presence of endocytosis in opossum kidney (OK) epithelial cells in monolayer culture. An increase in medium osmolality to 500 mosmol/kgH2O, by addition of sucrose, produced 80% inhibition of endocytosis within 1 h. The inhibition was reversed on returning the cells to normal medium. Incubation of OK cell monolayers with PTH (10(-8) M) for 3 h at normal osmolality (281 mosmol/kgH2O) inhibited Na(+)-phosphate cotransport (4 min uptakes) by 56-67%. In hyperosmolar medium (513 mosmol/kgH2O), when endocytosis was inhibited, PTH inhibited Na(+)-phosphate cotransport by only 25-39%, a change that was significantly different from the inhibition in normal medium. Hyperosomolality had no effect on PTH inhibition of Na(+)-H+ exchange or on PTH stimulation of intracellular adenosine 3',5'-cyclic monophosphate. We conclude that the full inhibitory action of PTH on Na(+)-phosphate cotransport may require an intact endocytic mechanism.

Nicotinamide as a rapid-acting inhibitor of renal brush-border phosphate transport

1988 ◽  
Vol 255 (1) ◽  
pp. F15-F21 ◽  
Author(s):  
K. I. Wu ◽  
R. A. Bacon ◽  
H. A. Al-Mahrouq ◽  
S. A. Kempson
Keyword(s):  
Protein Synthesis ◽  
Transport System ◽  
Brush Border ◽  
Time Course ◽  
De Novo ◽  
Phosphate Transport ◽  
Renal Brush Border Membrane ◽  
Dependent Inhibition ◽  
De Novo Protein Synthesis ◽  
Renal Brush Border

Administration of nicotinamide to rats produces specific dose-dependent inhibition of Na+-dependent phosphate transport across the renal brush-border membrane (BBM) and an increase in urinary excretion of phosphate. The intracellular mechanism of action of nicotinamide is not well established. As a step in this direction, the present studies determined whether nicotinamide was a rapid- or slow-acting regulator of the BBM phosphate transport system. Nicotinamide (0.5 g/kg) inhibited Na+-dependent BBM phosphate transport under conditions when de novo protein synthesis was inhibited by cycloheximide (1.0 mg/kg). Furthermore, the degree of inhibition was not different from that achieved by nicotinamide alone, suggesting that the action of nicotinamide does not require de novo protein synthesis. Studies on the time course of the onset of nicotinamide action revealed inhibition of BBM phosphate transport within 1 h after injection of nicotinamide, even in rats pretreated with cycloheximide. The rapid response to nicotinamide and its independence of de novo protein synthesis characterize nicotinamide as a rapid-acting regulator of the Na+-dependent phosphate transport system in renal BBM.

Acute and early effects of aldosterone on Na-K-ATPase activity in Madin-Darby canine kidney epithelial cells

1993 ◽  
Vol 264 (6) ◽  
pp. F1021-F1026 ◽  
Author(s):  
M. Shahedi ◽  
K. Laborde ◽  
L. Bussieres ◽  
C. Sachs
Keyword(s):  
Atpase Activity ◽  
Time Course ◽  
De Novo ◽  
Mdck Cells ◽  
Early Effect ◽  
Catalytic Sites ◽  
Early Effects ◽  
Dose Dependent ◽  
De Novo Protein Synthesis ◽  
Darby Canine Kidney

The time course and mechanism of early effects of aldosterone on renal Na-K-adenosinetriphosphatase (Na-K-ATPase) activity and number of units were studied in MDCK cells. Aldosterone induced a time- and dose-dependent stimulation of Na-K-ATPase activity. The stimulatory effect of aldosterone on activity and number of pump units increased progressively and was inhibited by spironolactone. In presence of cycloheximide, the stimulatory effect of aldosterone on activity and number of catalytic sites persisted to the same extent until 30 min and decreased by 20% after 60 min. In these cells, dimethylamiloride addition during preincubation abolished the aldosterone-induced stimulation in Na-K-ATPase activity up to 60 min. In contrast, furosemide addition did not alter the effect of aldosterone on Na-K-ATPase activity. The present study demonstrates an early effect of aldosterone on Na-K-ATPase activity that can be separated into the following two successive periods: 1) increase in pump number due to insertion of presynthetized units secondary to Na entry through an amiloride-sensitive apical pathway; and 2) an increase in pump number by de novo protein synthesis.

Regulation of Na-K-2Cl cotransport in osteoblasts

1991 ◽  
Vol 261 (3) ◽  
pp. C433-C440 ◽  
Author(s):  
N. Whisenant ◽  
B. X. Zhang ◽  
M. Khademazad ◽  
P. Loessberg ◽  
S. Muallem
Keyword(s):  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Hypotonic Medium ◽  
Cell Shrinkage ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Maximal Stimulation ◽  
Cotransport System ◽  
Umr 106 ◽  
Stimulation Of

Uptake of 86Rb was used to follow the activity of Na-K-2Cl cotransport in the osteosarcoma cell line UMR-106-01. The ouabain-resistant fraction of 86Rb uptake was sensitive to bumetanide and furosemide. Furosemide-sensitive 86Rb uptake required the presence of Na+, K+, and Cl- in the incubation medium. These observations indicate the presence of a Na-K-2Cl cotransport system in osteoblasts. Cotransporter activity was stimulated by agonists which increase adenosine 3',5'-cyclic monophosphate (cAMP), cytosolic free Ca2+ ([Ca2+]i), and protein kinase C (PKC) activity such as parathyroid hormone (PTH) and prostaglandin E2 (PGE2). However, endothelin, which increases [Ca2+]i and PKC activity without affecting cellular levels of cAMP, was ineffective in stimulating the cotransporter. Accordingly, increasing cellular cAMP with forskolin was as effective as PTH and PGE2 in stimulating the cotransporter. Stimulation of PKC with TPA inhibited the cotransporter in a time- and concentration-dependent manner. No stimulation of cotransport could be demonstrated at any 12-O-tetradecanoyl-phorbol-13-acetate (TPA) concentration or incubation time. The Na-K-2Cl cotransporter was stimulated by cell shrinkage. Maximal stimulation was observed after swelling the cells in hypotonic medium and subsequent shrinkage in isotonic medium. Stimulation by cell shrinkage can be demonstrated in control, agonist-, cAMP-, and TPA-treated cells. These observations suggest that 1) the osteoblastic Na-K-2Cl cotransporter is activated by calciotropic hormones predominantly through an increase in cellular cAMP, and 2) in osteoblasts, the cotransporter is independently regulated by different biochemical pathways.

Mechanisms of regulation of G11α protein by dexamethasone in osteoblastic UMR 106–01 cells

2002 ◽  
Vol 282 (1) ◽  
pp. E24-E30 ◽  
Author(s):  
Ricky Cheung ◽  
Jane Mitchell
Keyword(s):  
De Novo ◽  
Mrna Level ◽  
Time Lag ◽  
Regulatory Protein ◽  
Protein Synthesis Inhibitor ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Synthesis Inhibitor ◽  
Glucocorticoid Treatment ◽  
Umr 106

We have previously demonstrated that glucocorticoids increased Gq/11α protein expression and phospholipase C activity in the rat osteosarcoma cell line UMR 106–01. In this study, we demonstrated that G11α is the primary Gq-subtype family member expressed in UMR cells. Dexamethasone treatment increased the expression of G11α protein in both a time- and a dose-dependent manner. Glucocorticoid treatment significantly increased the half-life of G11α protein from 20.3 to 63 h. Steady-state G11α mRNA level was also increased by glucocorticoid treatment by ∼70%. This change was not the result of changes in RNA stability but rather the result of increased transcription, because the glucocorticoid-mediated upregulation of G11α mRNA was blocked by the transcription inhibitor actinomycin D. The dexamethasone induction of G11α mRNA occurred after a time lag of 12–24 h and was blocked by the protein synthesis inhibitor cycloheximide. These results suggest that the dexamethasone-induced rise in G11α protein results primarily from changes in the degradation rate of the protein, whereas changes in G11α mRNA play a smaller role and require de novo synthesis of regulatory protein(s).

Involvement of protein kinase C in the stimulation of sodium-dependent phosphate transport by parathyroid hormone in osteoblast-like cells

1994 ◽  
Vol 131 (6) ◽  
pp. 646-651 ◽  
Author(s):  
Makoto Arao ◽  
Toru Yamaguchi ◽  
Toshitsugu Sugimoto ◽  
Masaaki Fukase ◽  
Kazuo Chihara
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Phosphate Transport ◽  
Osteosarcoma Cell ◽  
Kinase C ◽  
Umr 106

Arao M, Yamaguchi T, Sugimoto T, Fukase M, Chihara K. Involvement of protein kinase C in sodiumdependent phosphate transport by parathyroid hormone in osteoblast-like cells. Eur J Endocrinol 1994;131:646–51. ISSN 0804–4643 The rat osteosarcoma cell line UMR-106 has an osteoblast-like phenotype and possesses parathyroid hormone (PTH)-responsive dual signal transduction systems [adenosine 3′,5′-cyclic monophosphatedependent protein kinase (PKA) and calcium-protein kinase C (Ca-PKC)]. These cells transport inorganic phosphate (Pi) by a Na+-dependent carrier under stimulation by PTH. The present study aimed to clarify PTH-responsive signal transduction mechanisms in the regulation of Na+-dependent Pi transport by PTH in UMR-106 cells. Exposure of these cells to 10−7 mol/l PTH induced a significant increase in Pi uptake within 30 min of incubation and it became maximal after 2 h. Parathyroid hormone (10−9 –10−7 mol/l) stimulated Pi uptake dose dependently. Activation of PKC by 12-O-tetradecanoyl phorbol- 13-acetate (TPA) also increased Pi uptake in time- and dose-dependent manners similar to PTH In contrast, neither PKA activation by 10 mol/l forskolin or by 10−4 mol/l dibutyryladenosine 3′,5′-cyclic monophosphate nor calcium ionophore treatment with 10−7 mol/l A23187 or with 10−7 mol/l ionomycin during 3-h incubations affect Pi uptake, except its increase by 10−4 mol/l forskolin at a 3-h incubation. These agents had no influence on Pi uptake even in combined treatments with TPA. The PTH-induced increase in Pi uptake was abolished almost completely by pretreating cells with PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) (50 μmol/l) or staurosporin (10 and 50 nmol/l), and by down-regulating PKC with a prolonged TPA treatment. These results indicate that the messenger system mediated by PKC, rather than by PKA or by cytosolic calcium, plays a crucial role in the regulation of Na+-dependent Pi transport by PTH within a few hours of exposure of the hormone in the osteoblast-like cells. Toru Yamaguchi, Third Division, Department of Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan

1,25-Dihydroxyvitamin D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells

1993 ◽  
Vol 264 (2) ◽  
pp. C287-C295 ◽  
Author(s):  
J. Green ◽  
K. V. Luong ◽  
C. R. Kleeman ◽  
L. H. Ye ◽  
C. Chaimovitz
Keyword(s):  
Vitamin D3 ◽  
Phosphate Uptake ◽  
Phosphate Transport ◽  
Osteoblastic Cells ◽  
Maximal Velocity ◽  
Uptake System ◽  
Dihydroxyvitamin D3 ◽  
1,25 Dihydroxyvitamin D3 ◽  
Pi Transport ◽  
Umr 106

In the present work we investigated the influence of vitamin D3 metabolites on Na(+)-dependent phosphate (Pi) transport in the clonal osteoblastic cell line UMR-106. The vitamin D3 metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] dose-dependently inhibited Pi transport with a half-maximal concentration of approximately 5 x 10(-11) M. The effect of 1,25(OH)2D3 was first observed after 8 h of preincubation period. Inhibition of phosphate uptake was relatively specific for the 1,25(OH)2D3 analogue of vitamin D3. The potency order was 1,25(OH)2D3 >> 24,25-dihydroxyvitamin D3 > 25-[3H]hydroxyvitamin D3. Kinetically, 1,25(OH)2D3 decreased the maximal velocity of the phosphate uptake system, whereas the affinity for phosphate was unaffected. Activation of protein kinase C (PKC) in UMR-106 cells stimulated Na(+)-dependent Pi transport. Nonetheless, the inhibitory effect of 1,25(OH)2D3 on Pi transport was not related to downregulation of PKC. Chemical determination of intracellular Pi showed a 50% reduction after 24-h preincubation with 10(-8) M 1,25(OH)2D3. We conclude that 1,25(OH)2D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells. This in turn leads to intracellular Pi depletion. The physiological implication of this phenomenon on the effects of vitamin D on osteoblasts in situ is discussed.

scholarly journals Glucocorticoids increase insulin binding and the amount of insulin-receptor mRNA in human cultured lymphocytes

1988 ◽  
Vol 249 (3) ◽  
pp. 715-719 ◽  
Author(s):  
Y Shibasaki ◽  
H Sakura ◽  
M Odawara ◽  
M Shibuya ◽  
Y Kanazawa ◽  
...  
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
De Novo ◽  
Insulin Receptors ◽  
Fold Increase ◽  
Insulin Binding ◽  
Dependent Manner ◽  
Receptor Mrna ◽  
Dose Dependent ◽  
Receptor Mrnas

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.

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