Disassembly of Subplasmalemmal Actin Filaments Induces Cytosolic Ca2+ Increases in Astropecten aranciacus Eggs

Background/Aims: Eggs of all animal species display intense cytoplasmic Ca2+ increases at fertilization. Previously, we reported that unfertilized eggs of Astropecten aranciacus exposed to an actin drug latrunculin A (LAT-A) exhibit similar Ca2+ waves and cortical flashes after 5-10 min time lag. Here, we have explored the molecular mechanisms underlying this unique phenomenon. Methods: Starfish eggs were pretreated with various agents such as other actin drugs or inhibitors of phospholipase C (PLC), and the changes of the intracellular Ca2+ levels were monitored by use of Calcium Green in the presence or absence of LAT-A. The concomitant changes of the actin cytoskeleton were visualized with fluorescent F-actin probes in confocal microscopy. Results: We have shown that the LAT-A-induced Ca2+ increases are related to the disassembly of actin flaments: i) not only LAT-A but also other agents depolymerizing F-actin (i.e. cytochalasin B and mycalolide B) induced similar Ca2+ increases, albeit with slightly lower efficiency; ii) drugs stabilizing F-actin (i.e. phalloidin and jasplakinolide) either blocked or significantly delayed the LAT-A-induced Ca2+ increases. Further studies utilizing pharmacological inhibitors of PLC (U-73122 and neomycin), dominant negative mutant of PLC-ɣ, specific sequestration of PIP2 (RFP-PH), InsP3 uncaging, and quantitation of endogenous InsP3 all indicated that LAT-A induces Ca2+ increases by stimulating PLC rather than sensitizing InsP3 receptors. In support of the idea, it bears emphasis that LAT-A timely increased intracellular contents of InsP3 with concomitant decrease of PIP2 levels in the plasma membrane. Conclusion: Taken together, our results suggest that suboolemmal actin filaments may serve as a scaffold for cell signaling and modulate the activity of the key enzyme involved in intracellular Ca2+ signaling.

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Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide

Biochemical Journal ◽

10.1042/bj20061704 ◽

2007 ◽

Vol 404 (2) ◽

pp. 179-190 ◽

Cited By ~ 130

Author(s):

Mark Windheim ◽

Christine Lang ◽

Mark Peggie ◽

Lorna A. Plater ◽

Philip Cohen

Keyword(s):

Protein Kinase ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

Muramyl Dipeptide ◽

Dominant Negative ◽

Signalling Pathways ◽

Dominant Negative Mutant ◽

Protein Kinase Activity ◽

P38α Mapk

MDP (muramyl dipeptide), a component of peptidoglycan, interacts with NOD2 (nucleotide-binding oligomerization domain 2) stimulating the NOD2–RIP2 (receptor-interacting protein 2) complex to activate signalling pathways important for antibacterial defence. Here we demonstrate that the protein kinase activity of RIP2 has two functions, namely to limit the strength of downstream signalling and to stabilize the active enzyme. Thus pharmacological inhibition of RIP2 kinase with either SB 203580 [a p38 MAPK (mitogen-activated protein kinase) inhibitor] or the Src family kinase inhibitor PP2 induces a rapid and drastic decrease in the level of the RIP2 protein, which may explain why these RIP2 inhibitors block MDP-stimulated downstream signalling and the production of IL-1β (interleukin-1β) and TNFα (tumour necrosis factor-α). We also show that RIP2 induces the activation of the protein kinase TAK1 (transforming-growth-factor-β-activated kinase-1), that a dominant-negative mutant of TAK1 inhibits RIP2-induced activation of JNK (c-Jun N-terminal kinase) and p38α MAPK, and that signalling downstream of NOD2 or RIP2 is reduced by the TAK1 inhibitor (5Z)-7-oxozeaenol or in TAK1-deficient cells. We also show that MDP activates ERK1 (extracellular-signal-regulated kinase 1)/ERK2 and p38α MAPK in human peripheral-blood mononuclear cells and that the activity of both MAPKs and TAK1 are required for MDP-induced signalling and production of IL-1β and TNFα in these cells. Taken together, our results indicate that the MDP–NOD2/RIP2 and LPS (lipopolysaccharide)–TLR4 (Toll-like receptor 4) signalling pathways converge at the level of TAK1 and that many subsequent events that lead to the production of pro-inflammatory cytokines are common to both pathways.

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Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression

Blood ◽

10.1182/blood-2004-12-4832 ◽

2005 ◽

Vol 106 (5) ◽

pp. 1808-1816 ◽

Cited By ~ 64

Author(s):

Erwan Le Scolan ◽

Dimitri Pchejetski ◽

Yoshiko Banno ◽

Nicole Denis ◽

Patrick Mayeux ◽

...

Keyword(s):

Transgenic Mice ◽

Molecular Mechanisms ◽

Sphingosine Kinase ◽

Dominant Negative ◽

Recurrent Event ◽

Dominant Negative Mutant ◽

Apoptosis Resistance ◽

Kinase Gene ◽

Extracellular Signal

Abstract The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia. (Blood. 2005;106:1808-1816)

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Oleamide, a Sleep-Inducing Supplement, Upregulates Doublecortin in Hippocampal Progenitor Cells via PPARα

Journal of Alzheimer s Disease ◽

10.3233/jad-215124 ◽

2021 ◽

pp. 1-16

Author(s):

Avik Roy ◽

Madhuchhanda Kundu ◽

Sudipta Chakrabarti ◽

Dhruv R. Patel ◽

Kalipada Pahan

Keyword(s):

Progenitor Cells ◽

Neuronal Differentiation ◽

Chromatin Immunoprecipitation ◽

Molecular Mechanisms ◽

Hippocampal Neurogenesis ◽

Dominant Negative ◽

Site Directed Mutagenesis ◽

Lipid Lowering ◽

Dominant Negative Mutant ◽

Wild Type

Background: Doublecortin (DCX), a microtubule associated protein, has emerged as a central biomarker of hippocampal neurogenesis. However, molecular mechanisms by which DCX is regulated are poorly understood. Objective: Since sleep is involved with the acquisition of memory and oleamide or 9-Octadecenamide (OCT) is a sleep-inducing supplement in human, we examined whether OCT could upregulate DCX in hippocampal progenitor cells (HPCs). Methods: We employed real-time PCR, western blot, immunostaining, chromatin immunoprecipitation, lentiviral transduction in HPCs, and the calcium influx assay. Results: OCT directly upregulated the transcription of Dcx in HPCs via activation of peroxisome proliferator-activated receptor α (PPARα), a lipid-lowering transcription factor. We observed that, HPCs of Ppara-null mice displayed significant impairment in DCX expression and neuronal differentiation as compared to that of wild-type mice. Interestingly, treatment with OCT stimulated the differentiation process of HPCs in wild-type, but not Ppara-null mice. Reconstruction of PPARα in mouse Ppara-null HPCs restored the expression of DCX, which was further stimulated with OCT treatment. In contrast, a dominant-negative mutant of PPARα significantly attenuated the stimulatory effect of OCT on DCX expression and suppressed neuronal differentiation of human neural progenitor cells. Furthermore, RNA microarray, STRING, chromatin immunoprecipitation, site-directed mutagenesis, and promoter reporter assay have identified DCX as a new target of PPARα. Conclusion: These results indicate that OCT, a sleep supplement, directly controls the expression of DCX and suggest that OCT may be repurposed for stimulating the hippocampal neurogenesis.

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Dual suppression of adipogenesis by cigarette smoke through activation of the aryl hydrocarbon receptor and induction of endoplasmic reticulum stress

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90829.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. E721-E730 ◽

Cited By ~ 13

Author(s):

Tsuyoshi Shimada ◽

Nobuhiko Hiramatsu ◽

Kunihiro Hayakawa ◽

Shuhei Takahashi ◽

Ayumi Kasai ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Aryl Hydrocarbon Receptor ◽

Cigarette Smoke ◽

Molecular Mechanisms ◽

Adipocyte Differentiation ◽

Suppressive Effect ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Aryl Hydrocarbon

Cigarette smoking decreases body weight, whereas molecular mechanisms underlying this phenomenon have not been elucidated. In this report, we investigated regulation of adipogenesis by cigarette smoke and involvement of aryl hydrocarbon receptor (AhR) and endoplasmic reticulum (ER) stress. We found that cigarette smoke extract (CSE) inhibited differentiation of preadipocytes into adipocytes dose dependently. It was associated with a decrease in lipid accumulation, blunted expression of adipocyte markers (adiponectin, PPAR-γ, and C/EBPα), and sustained expression of a preadipocyte marker MCP-1. CSE markedly induced activation of AhR, and AhR agonists (2,3,7,8-tetrachlorodibenzo- p-dioxin, benzo[ a]pyrene and 3-methylcholanthrene) reproduced the inhibitory effect of CSE on adipocyte differentiation. Furthermore, knockout of the AhR gene or blockade of AhR by a dominant-negative mutant attenuated the suppressive effects of CSE on adipocyte differentiation. We also found that CSE induced ER stress in preadipocytes, and ER stress inducers (thapsigargin, tunicamycin, and A23187) reproduced the suppressive effect of CSE on the differentiation of preadipocytes. Interestingly, AhR agonists did not cause ER stress, and ER stress inducers did not activate AhR. These results suggested that cigarette smoke has the potential to inhibit adipocyte differentiation via dual, independent mechanisms, i.e., through activation of the AhR pathway and induction of the unfolded protein response.

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Molecular mechanisms of invadopodium formation

The Journal of Cell Biology ◽

10.1083/jcb.200407076 ◽

2005 ◽

Vol 168 (3) ◽

pp. 441-452 ◽

Cited By ~ 446

Author(s):

Hideki Yamaguchi ◽

Mike Lorenz ◽

Stephan Kempiak ◽

Corina Sarmiento ◽

Salvatore Coniglio ◽

...

Keyword(s):

Rna Interference ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Egf Receptor ◽

De Novo ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Cell Periphery ◽

Matrix Degradation ◽

Degradation Activity

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.

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Abstract MP170: Increased Ga o Expression Regulated by NRSF Plays a Key Role in the Development of Heart Failure Through the Impairment of Ca 2+ Homeostasis

Circulation Research ◽

10.1161/res.127.suppl_1.mp170 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Hideaki Inazumi ◽

Yasuaki Nakagawa ◽

Kenji Moriuchi ◽

Koichiro Kuwahara

Keyword(s):

Heart Failure ◽

Cardiac Function ◽

Cardiac Dysfunction ◽

Intracellular Signaling ◽

Molecular Mechanisms ◽

Troponin T ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Cardiac Gene ◽

Normal Cardiac Function

Background: In the development of heart failure, pathological intracellular signaling reactivates fetal cardiac gene program, which leads to pathological cardiac remodeling. We previously reported that a transcriptional repressor, neuron restrictive silencer factor (NRSF) represses fetal cardiac gene program and maintains normal cardiac function, while pathological stimuli de-repress this NRSF mediated repression via activation of CaMKII. Molecular mechanisms by which NRSF maintains cardiac function remains to be determined, however. Purpose: To elucidate molecular mechanisms by which NRSF maintains normal cardiac function. Methods and Results: Newly generated cardiac-specific NRSF knockout mice (NRSF-cKO) showed cardiac dysfunction and premature deaths accompanied with lethal arrhythmias, as was observed in our previously reported cardiac-specific dominant-negative mutant of NRSF transgenic mice (dnNRSF-Tg). Expression of Gnao1 gene encoding Gα o , a member of inhibitory G proteins, was commonly increased in ventricles of dnNRSF-Tg and NRSF-cKO. ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay identified that NRSF transcriptionally regulates Gnao1 gene expression. Genetic Knockdown of Gα o in dnNRSF-Tg and NRSF-cKO ameliorated the reduced systolic function, increased arrhythmogenicity and reduced survival rates. Conversely cardiac-specific GNAO1 overexpression was sufficient to show impaired cardiac function. Mechanistically, Gα o increases current density in surface sarcolemmal L-type Ca 2 + channel and then activates CaMKII without affecting protein kinase A activity, which finally leads to impaired Ca 2+ handling and systolic dysfunction. Furthermore, expression of Gα o is also increased in ventricles of transverse aortic constriction model mice and cardiac troponin T mutant DCM model mice, in both of which, genetic reduction of Gα o prevented the progression of cardiac dysfunction. Conclusions: Increased expression of Gα o , induced by attenuation of NRSF-mediated repression forms a pathological circuit via activation of CaMKII and progresses heart failure by impairing Ca 2+ homeostasis. Gα o is a potential therapeutic target for heart failure.

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Cross-talk between RhoH and Rac1 in regulation of actin cytoskeleton and chemotaxis of hematopoietic progenitor cells

Blood ◽

10.1182/blood-2007-06-093237 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2597-2605 ◽

Cited By ~ 49

Author(s):

Hee-Don Chae ◽

Katherine E. Lee ◽

David A. Williams ◽

Yi Gu

Keyword(s):

Progenitor Cells ◽

Molecular Mechanisms ◽

Green Fluorescence Protein ◽

Dominant Negative ◽

Hematopoietic Progenitor Cells ◽

Dominant Negative Mutant ◽

Active Member ◽

Actin Assembly ◽

Filamentous Actin ◽

Hematopoietic Progenitor

RhoH, a hematopoietic-specific and constitutively active member of the Rho guanosine triphosphatase (GTPase) family, has been implicated in the negative regulation of Rac GTPase-mediated signaling in hematopoietic cells. However, the molecular mechanisms underlying the functional interaction between RhoH and Rac in primary cells are poorly understood. Here we show that deletion of Rhoh in hematopoietic progenitor cells (HPCs) leads to increased stromal-derived factor-1α (SDF-1α)–induced chemotaxis and chemokinesis (random migration). The abnormally enhanced migration of Rhoh−/− HPCs is associated with increased Rac1 activity and translocation of Rac1 protein to the cell membrane, where it colocalizes with cortical filamentous-actin (F-actin) and lipid rafts. Expression of the dominant-negative mutant Rac1N17 inhibits the cortical F-actin assembly and chemotaxis of wild-type and Rhoh−/− HPCs to the same extent. Conversely, overexpression of RhoH in HPCs blocks the membrane translocation of Rac1–enhanced green fluorescence protein (EGFP) and active Rac1V12–EGFP proteins and impairs cortical F-actin assembly and chemotaxis in response to SDF-1α stimulation. Furthermore, we demonstrate that the subcellular localization and inhibitory function of RhoH in HPCs are regulated by C-terminal motifs, including a CKIF prenylation site. Together, we have identified an antagonistic role of RhoH in regulation of cortical F-actin assembly and chemotaxis via suppressing Rac1 membrane targeting and activation in primary HPCs.

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Filamin A (FLNA) Plays an Essential Role in Somatostatin Receptor 2 (SST2) Signaling and Stabilization After Agonist Stimulation in Human and Rat Somatotroph Tumor Cells

Endocrinology ◽

10.1210/en.2014-1063 ◽

2014 ◽

Vol 155 (8) ◽

pp. 2932-2941 ◽

Cited By ~ 25

Author(s):

E. Peverelli ◽

E. Giardino ◽

D. Treppiedi ◽

E. Vitali ◽

V. Cambiaghi ◽

...

Keyword(s):

Tumor Cells ◽

Molecular Mechanisms ◽

Somatostatin Receptor ◽

Pituitary Tumors ◽

Dominant Negative ◽

Gh3 Cells ◽

Dominant Negative Mutant ◽

Filamin A ◽

Variable Expression ◽

Apoptotic Effect

Somatostatin receptor type 2 (SST2) is the main pharmacological target of medical therapy for GH-secreting pituitary tumors, but molecular mechanisms regulating its expression and signaling are largely unknown. The aim of this study was to investigate the role of cytoskeleton protein filamin A (FLNA) in SST2 expression and signaling in somatotroph tumor cells. We found a highly variable expression of FLNA in human GH-secreting tumors, without a correlation with SST2 levels. FLNA silencing in human tumoral cells did not affect SST2 expression and localization but abolished the SST2-induced reduction of cyclin D1 (−37% ± 15% in control cells, P < .05 vs basal) and caspase-3/7 activation (+63% ± 31% in control cells, P < .05 vs basal). Overexpression of a FLNA dominant-negative mutant that specifically prevents SST2-FLNA binding reduced SST2 expression after prolonged agonist exposure (−55% ± 5%, P < .01 vs untreated cells) in GH3 cells. Moreover, SST2-induced apoptotic effect (77% ± 54% increase of caspase activity, P < .05 vs basal) and SST2-mediated ERK1/2 inhibition (48% ± 17% reduction of ERK1/2 phosphorylation, P < .01 vs basal) were abrogated in cells overexpressing another FLNA mutant that prevents FLNA interaction with partner proteins but not with SST2, suggesting a scaffold function of FLNA in somatotrophs. In conclusion, these data demonstrate that FLNA is involved in SST2 stabilization and signaling in tumoral somatotrophs, playing both a structural and functional role.

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β-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2017937118 ◽

2021 ◽

Vol 118 (7) ◽

pp. e2017937118

Author(s):

Silvia G. del Villar ◽

Taylor L. Voelker ◽

Maartje Westhoff ◽

Gopireddy R. Reddy ◽

Heather C. Spooner ◽

...

Keyword(s):

Ventricular Myocytes ◽

Myocardial Contraction ◽

Small Gtpase ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Rab Proteins ◽

Recycling Endosome ◽

Channel Trafficking ◽

Latrunculin A

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large “superclusters” wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered “emptying” of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac CaV1.2.

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Early biochemical events in insulin-stimulated fluid phase endocytosis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1999.276.1.e94 ◽

1999 ◽

Vol 276 (1) ◽

pp. E94-E105 ◽

Cited By ~ 3

Author(s):

Diana M. Pitterle ◽

Robert T. Sperling ◽

Martin G. Myers ◽

Morris F. White ◽

Perry J. Blackshear

Keyword(s):

Insulin Receptor ◽

Molecular Mechanisms ◽

Insulin Receptors ◽

Fluid Phase ◽

Mek Inhibitor ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Mutant ◽

Brown Adipose ◽

Fluid Phase Endocytosis

We examined the initial molecular mechanisms by which cells nonselectively internalize extracellular solutes in response to insulin. Insulin-stimulated fluid phase endocytosis (FPE) was examined in responsive cells, and the roles of the insulin receptor, insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3′-kinase (PI 3′-kinase), Ras, and mitogen-activated protein kinase kinase (MEK) were assessed. Active insulin receptors were essential, as demonstrated by the stimulation of FPE by insulin in HIRc-B cells (Rat-1 cells expressing 1.2 × 106 normal insulin receptors/cell) but not in untransfected Rat-1 cells or in Rat-1 cells expressing the inactive A/K1018 receptor. IRS-1 expression augmented insulin-stimulated FPE, as assessed in 32D cells, a hematopoietic precursor cell line lacking endogenous IRS-1. Insulin-stimulated FPE was inhibited in mouse brown adipose tissue (BAT) cells expressing the 17N dominant negative mutant Ras and was augmented in cells expressing wild-type Ras. The MEK inhibitor PD-98059 had little effect on insulin-stimulated FPE in BAT cells. In 32D cells, but not in HIRc-B and BAT cells, insulin-stimulated FPE was inhibited by 10 nM wortmannin, an inhibitor of PI 3′-kinase. The results indicate that the insulin receptor, IRS-1, Ras, and, perhaps in certain cell types, PI 3′-kinase are involved in mediating insulin-stimulated FPE.

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