scholarly journals Insulin-mediated inhibition of apolipoprotein B secretion requires an intracellular trafficking event and phosphatidylinositol 3-kinase activation: studies with brefeldin A and wortmannin in primary cultures of rat hepatocytes

1996 ◽  
Vol 313 (2) ◽  
pp. 567-574 ◽  
Author(s):  
Janet D. SPARKS ◽  
Thuy L. PHUNG ◽  
Mary BOLOGNINO ◽  
Charles E. SPARKS
Keyword(s):  
Apolipoprotein B ◽  
Enzyme Inhibitor ◽  
Primary Cultures ◽  
Brefeldin A ◽  
Rat Hepatocytes ◽  
Phosphatidylinositol 3 Kinase ◽  
Apo B ◽  
Insulin Effect ◽  
Insulin Inhibition ◽  
Phosphatidylinositol 3

Insulin inhibition of the secretion of apolipoprotein B (apo B) was studied in primary cultures of rat hepatocytes by using brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and by using the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Incubation of hepatocytes with BFA (10 μg/ml) for 1 h inhibited the subsequent secretion of apo B, albumin and transferrin for up to 3 h. BFA treatment resulted in the time-dependent accumulation in cells of [14C]leucine-labelled proteins and apo B. Under conditions where insulin decreased total apo B (cell plus secreted), BFA blocked the insulin-dependent effect. These results suggest that export of apo B from the ER is a prerequisite for the observed insulin effect. Treatment of hepatocytes with wortmannin for 20 min abolished insulin inhibition of apo B secretion, suggesting that the insulin effect on the apo B pathway involves activation of PI 3-K. Enzyme inhibitor studies indicate that chymostatin and (+)-(2S,3S)-3-[(S)-methyl-1-(3-m e t h y l b u t y l c a r b a m o y l) - b u t y l c a r b a m o y l] - 2-oxiranecarboxylate (E-64-c) partially block insulin effects on apo B compared with leupeptin, which had no discernible effect. The cell-permeable derivative of E-64-c, EST, and N-Ac-Leu-Leu-norleucinal (ALLN) were most effective in blocking insulin effects on apo B. These results suggest that insulin action on apo B in primary rat hepatocytes involves (1) vesicular movement of apo B from the ER; (2) activation of PI 3-K and (3) a cellular protease that is either a cysteine- or calcium-activated neutral protease.

scholarly journals Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence.

1986 ◽  
Vol 34 (9) ◽  
pp. 1223-1230 ◽  
Author(s):  
G A Keller ◽  
C Glass ◽  
D Louvard ◽  
D Steinberg ◽  
S J Singer
Keyword(s):  
Serum Albumin ◽  
Apolipoprotein B ◽  
Intracellular Transport ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Cell System ◽  
Secretory Proteins ◽  
Transport Pathway ◽  
Apo B ◽  
Normal Rat

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.

scholarly journals Direct activation of telomerase by GH via phosphatidylinositol 3′-kinase

2005 ◽  
Vol 185 (3) ◽  
pp. 421-428 ◽  
Author(s):  
L Gómez-García ◽  
F M Sánchez ◽  
M T Vallejo-Cremades ◽  
I A Gómez de Segura ◽  
E De Miguel del Campo
Keyword(s):  
Kinase Inhibitor ◽  
Primary Cultures ◽  
Chinese Hamster ◽  
Signal Transduction Pathway ◽  
Rat Hepatocytes ◽  
Telomerase Activity ◽  
Cell System ◽  
Phosphatidylinositol 3 Kinase ◽  
Direct Activation ◽  
Phosphatidylinositol 3

Telomerase is a ribonucleoprotein DNA polymerase that has been associated with cell proliferation, cell survival and apoptosis inhibition. Telomerase is regulated by specific growth factors, cytokines and hormones. The present study examines the effect of GH on telomerase activity and identifies the signal transduction pathway involved in this process in Chinese hamster ovary (CHO)4 cells, which express rat GH receptor cDNA. Telomeric repeat amplification protocol assays demonstrated that treating CHO4 cells with increasingly high doses of GH up-regulated telomerase activity with the maximum activation at 24 h. Similarly, GH activated telomerase in another cell system, primary cultures of rat hepatocytes. The telomerase activation in CHO4 cells was produced with an increase in hamster telomerase catalytic subunit (hamTERT) mRNA expression. The telomerase activity induced by GH was specifically blocked by the phosphatidylinositol 3′-kinase (PI3-K) inhibitor, LY294002, but not by the MAP kinase kinase inhibitor, PD98059. These findings suggest that GH could activate telomerase through the direct activation of TERT transcription, as well as through the PI3-K signalling pathway.

scholarly journals Regulation of Hepatic Insulin-Like Growth Factor-Binding Protein-1 Gene Expression by Insulin: Central Role for Mammalian Target of Rapamycin Independent of Forkhead Box O Proteins

Endocrinology ◽  
2006 ◽  
Vol 147 (5) ◽  
pp. 2383-2391 ◽  
Author(s):  
Catherine Mounier ◽  
Victor Dumas ◽  
Barry I. Posner
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
Binding Protein ◽  
Mammalian Target Of Rapamycin ◽  
Pi3 Kinase ◽  
Target Of Rapamycin ◽  
Mrna Levels ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Inhibition ◽  
Phosphatidylinositol 3

The expression of IGF-binding protein-1 (IGFBP-1) is induced in rat liver by dexamethasone and glucagon and is completely inhibited by 100 nm insulin. Various studies have implicated phosphatidylinositol 3-kinase, protein kinase B (Akt), phosphorylation of the transcription factors forkhead in rhabdomyosarcoma 1 (Foxo1)/Foxo3, and the mammalian target of rapamycin (mTOR) in insulin’s effect. In this study we examined insulin regulation of IGFBP-1 in both subconfluent and confluent hepatocytes. In subconfluent hepatocytes, insulin inhibition of IGFBP-1 mRNA levels was blocked by inhibiting PI3 kinase activation, and there was a corresponding inhibition of Foxo1/Foxo3 phosphorylation. In these same cells, inhibition of the insulin effect by rapamycin occurred in the presence of insulin-induced Foxo1/Foxo3 phosphorylation. In confluent hepatocytes, insulin could not activate the phosphatidylinositol 3-kinase (PI3 kinase)-Akt-Foxo1/Foxo3 pathway, but still inhibited IGFBP-1 gene expression in an mTOR-dependent manner. In subconfluent hepatocytes, the serine/threonine phosphatase inhibitor okadaic acid (100 nm) partially inhibited IGFBP-1 gene expression by 40%, but did not produce phosphorylation of either Akt or Foxo proteins. In contrast, 1 nm insulin inhibited the IGFBP-1 mRNA level by 40% and correspondingly activated Akt and Foxo1/Foxo3 phosphorylation to a level comparable to that observed with 100 nm insulin. These results suggest a potential role for a serine/threonine phosphatase(s) in the regulation of IGFBP-1 gene transcription, which is not downstream of mTOR and is independent of Akt. In conclusion, we have found that in rat liver, insulin inhibition of IGFBP-1 mRNA levels can occur in the absence of the phosphorylation of Foxo1/Foxo3, whereas activation of the mTOR pathway is both necessary and sufficient.

Metabolic regulation of Na+/Pi-cotransporter-1 gene expression in H4IIE cells

2000 ◽  
Vol 278 (4) ◽  
pp. E648-E655 ◽  
Author(s):  
Zijian Xie ◽  
Hui Li ◽  
Liqin Liu ◽  
Barbara B. Kahn ◽  
Sonia M. Najjar ◽  
...  
Keyword(s):  
Metabolic Regulation ◽  
Mapk Pathway ◽  
Rat Hepatocytes ◽  
Mek Inhibitor ◽  
Dominant Negative ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Effect ◽  
Pyruvate Oxidation ◽  
H4iie Cells ◽  
Ribosomal S6 Kinase

We showed that the rat Na+/Pi cotransporter-1 (RNaPi-1) gene was regulated by insulin and glucose in rat hepatocytes. The aim of this work was to elucidate signaling pathways of insulin-mediated metabolic regulation of the RNaPi-1 gene in H4IIE cells. Insulin increased RNaPi-1 mRNA abundance in the presence of glucose and decreased RNaPi-1 mRNA in the absence of glucose, clearly establishing an involvement of metabolic signals for insulin-induced upregulation of the RNaPi-1 gene. Pyruvate and insulin increased RNaPi-1 expression but downregulated L-pyruvate kinase, indicating the existence of gene-specific metabolic signals. Although fructose, glycerol, and lactate could support insulin-induced upregulation of the RNaPi-1 gene, compounds entering metabolism beyond pyruvate oxidation, such as acetate and citrate, could not, suggesting that RNaPi-1-specific metabolic signals are generated at or above pyruvate oxidation. Wortmannin, LY-294002, and rapamycin abolished the insulin effect on the RNaPi-1 gene, whereas expression of dominant negative Asn17 Ras and mitogen-activating protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 exhibited no effect. Thus we herein propose that metabolic regulation of RNaPi-1 expression by insulin is mediated through the phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase pathways, but not the Ras/MAPK pathway.

scholarly journals Effects of nonketotic streptozotocin diabetes on apolipoprotein B synthesis and secretion by primary cultures of rat hepatocytes.

1988 ◽  
Vol 82 (1) ◽  
pp. 37-43 ◽  
Author(s):  
J D Sparks ◽  
C E Sparks ◽  
M Bolognino ◽  
A M Roncone ◽  
T K Jackson ◽  
...  
Keyword(s):  
Apolipoprotein B ◽  
Primary Cultures ◽  
Streptozotocin Diabetes ◽  
Rat Hepatocytes

scholarly journals Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem

1989 ◽  
Vol 263 (2) ◽  
pp. 411-415 ◽  
Author(s):  
T C Kwong ◽  
J D Sparks ◽  
D J Pryce ◽  
J F Cianci ◽  
C E Sparks
Keyword(s):  
Molecular Mass ◽  
Apolipoprotein B ◽  
Channel Blocker ◽  
Rat Hepatocytes ◽  
Acetate Incorporation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Apo B ◽  
Cellular Lipids ◽  
Mass Form

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.

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