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M Benito

Publications and source records attributed to M Benito.

At least 37 records · Page 2Linked to original sources
PubMed2000

Essential role of insulin receptor substrate-2 in insulin stimulation of Glut4 translocation and glucose uptake in brown adipocytes.

Insulin and insulin-like growth factor I signals are mediated via phosphorylation of a family of insulin receptor substrate (IRS) proteins, which may serve both complementary and overlapping functions in the cell. To study the metabolic effects of these proteins in more detail, we established brown adipocyte cell lines from wild type and various IRS knockout (KO) animals and characterized insulin action in these cells in vitro. Preadipocytes derived from both wild type and IRS-2 KO mice could be fully differentiated into mature brown adipocytes. In differentiated IRS-2 KO adipocytes, insulin-induced glucose uptake was decreased by 50% compared with their wild type counterparts. This was the result of a decrease in insulin-stimulated Glut4 translocation to the plasma membrane. This decrease in insulin-induced glucose uptake could be partially reconstituted in these cells by retrovirus-mediated re-expression of IRS-2, but not overexpression of IRS-1. Insulin signaling studies revealed a total loss of IRS-2-associated phosphatidylinositol (PI) 3-kinase activity and a reduction in phosphotyrosine-associated PI 3-kinase by 30% (p < 0.05) in the KO cells. The phosphorylation and activity of Akt, a major downstream effector of PI 3-kinase, as well as Akt-dependent phosphorylation of glycogen synthase kinase-3 and p70S6 kinase were not affected by the lack of IRS-2; however, there was a decrease in insulin stimulation of Akt associated with the plasma membrane. These results provide evidence for a critical role of IRS-2 as a mediator of insulin-stimulated Glut4 translocation and glucose uptake in adipocytes. This occurs without effects in differentiation, total activation of Akt and its downstream effectors, but may be caused by alterations in compartmentalization of these downstream signals.

Adipocytes↗
PubMed2000

Okadaic acid inhibits insulin-induced glucose transport in fetal brown adipocytes in an Akt-independent and protein kinase C zeta-dependent manner.

In the present study we have investigated the effect of increased serine/threonine phosphorylation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) by okadaic acid pretreatment on brown adipocyte insulin signalling leading to glucose transport, an important metabolic effect of insulin in brown adipose tissue. Okadaic acid pretreatment before insulin stimulation decreased IRS-1 and IRS-2 tyrosine phosphorylation in parallel to a decrease in their sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. IRS-1/IRS-2-associated p85alpha and phosphatidylinositol (PI) 3-kinase enzymatic activity were partly reduced in brown adipocytes pretreated with okadaic acid upon stimulation with insulin. Furthermore, insulin-induced glucose uptake was totally abolished by the inhibitor in parallel with a total inhibition of insulin-induced protein kinase C (PKC) zeta activity. However, activation of Akt/PKB or p70 S6 kinase (p70(s6k)) by insulin remained unaltered. Our results suggest that downstream of PI 3-kinase, insulin signalling diverges into at least two independent pathways through Akt/PKB and PKC zeta, the PKC zeta pathway contributing to glucose transport induced by insulin in fetal brown adipocytes.

Adipose Tissue, Brown↗
PubMed2000

Noradrenaline induces brown adipocytes cell growth via beta-receptors by a mechanism dependent on ERKs but independent of cAMP and PKA.

It has been well established that the key role of noradrenaline is the induction of uncoupling-protein-1 (UCP-1) expression, the unique marker of brown adipocytes. However, its implication on proliferation and the pathways involved are not as well characterized. By using rat fetal brown adipocytes as a model, we show that, although noradrenaline activates extracellular regulated kinases (ERKs) through beta-, alpha1-, and alpha2-receptors, only beta-receptors mediate cell growth by a mechanism that requires ERKs activation but is independent of cyclic-adenosine-monophosphate/protein kinase A (cAMP/PKA). Conversely, the cAMP/PKA cascade mediates noradrenaline-induced UCP-1 expression, whereas ERKs pathway attenuates thermogenic differentiation. On the other hand, alpha1- and alpha2-receptors have an antiproliferative effect that is enhanced by ERK inhibition.

Adipocytes↗
PubMed2000

Fibronectin regulates morphology, cell organization and gene expression of rat fetal hepatocytes in primary culture.

BACKGROUND/AIMS: The extracellular matrix regulates hepatic development and regeneration, modulating the maintenance of liver architecture in the differentiated state. The aim of this work was to analyze how different extracellular matrix molecules modulate fetal hepatocyte morphology, growth and differentiation. METHODS: We cultured fetal hepatocytes either on plastic or on different extracellular matrix proteins, i.e., collagen I, fibronectin or E-C-L (entactin-collagen IV-laminin) and we analyzed cell attachment, morphological organization, proliferative response and gene expression. RESULTS: Cell attachment was increased by all the extracellular matrix proteins to a similar extent. However, only fibronectin facilitated the formation of elongated cord-like structures, reminiscent of liver plate organization. Immunocytochemical analysis of the cells in these structures revealed high levels of albumin and cytokeratin 18, phenotypical markers of parenchymal hepatocytes. Fibronectin did not block the mitogenic stimuli induced by epidermal growth factor in these cells and the elongated structures appeared either in the absence or in the presence of the mitogen. Cells cultured on fibronectin, regardless of whether epidermal growth factor was present or not, also presented the maximal levels of expression for liver specific genes, such as albumin or alpha-fetoprotein. This expression was coincident with an increased expression of hepatocyte nuclear factor (HNF)-4 and a higher HNF-1alpha/HNF-1beta ratio, when compared with those cells that were cultured on collagen or E-C-L extracellular matrix. CONCLUSIONS: These results suggest that fibronectin might play a differential role, as compared to other extracellular matrix proteins, in fetal hepatocyte organization and gene expression.

Animals↗
PubMed2000

Epidermal growth factor impairs the cytochrome C/caspase-3 apoptotic pathway induced by transforming growth factor beta in rat fetal hepatocytes via a phosphoinositide 3-kinase-dependent pathway.

Transforming growth factor beta (TGF-beta)-mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-beta-induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-beta disrupts the mitochondrial transmembrane potential (DeltaPsi(m))( )and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase-dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-beta produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-x(L), an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-x(L) levels even in the presence of TGF-beta. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-beta-induced bcl-x(L )down-regulation. We conclude that PI 3-kinase mediates the survival effect of EGF on TGF-beta-induced death by acting upstream from the mitochondrial changes, i.e., preventing bcl-x(L) down-regulation, cytochrome c release, and activation of caspase-3.

Animals↗
PubMed2000

Akt mediates insulin rescue from apoptosis in brown adipocytes: effect of ceramide.

We have recently shown that insulin can rescue serum deprived adipocytes from apoptosis in a PI 3 kinase and MAP kinase dependent manner. This study investigated the contribution of Akt and p70S6-kinase in insulin rescue from two different apoptotic triggers, serum deprivation and ceramide treatment. Insulin rescued serum-deprived immortalized brown adipocytes from apoptosis through phosphatidylinositol (PI) 3-kinase and Akt pathways, but independently of p70S6-kinase, as demonstrated by the use of inhibitors such as LY294002 or Rapamycin, and transfection experiments with dominant-negative constructs of Akt or p85 subunit of PI 3-kinase. A constitutively active Akt construct mimicked the insulin survival effect, decreasing the percentage of hypodiploid cells, the percentage of apoptopic cells and precluding the formation of apoptotic nuclei. We propose that the insulin survival effect on immortalized brown adipocytes is mediated through activation of Akt. However, insulin and EGF failed to rescue brown adipocytes from ceramide-induced apoptosis, as determined by DNA laddering, hypodiploid cells and apoptotic nuclei. Ceramide treatment blunted Akt activity but not PI 3-kinase activity, and insulin and EGF were unable to activate Akt. Ceramide also caused apoptosis in cells transfected with a constitutively active Akt construct, since phosphorylation of Akt was impaired under these experimental conditions. This study suggests that activation of Akt may be an absolute requirement for the survival of brown adipocytes.

Adipocytes↗
PubMed2000

p38 mitogen-activated protein kinase mediates tumor necrosis factor-alpha-induced apoptosis in rat fetal brown adipocytes.

Tumor necrosis factor-alpha (TNFalpha) induces apoptosis and cell growth inhibition in primary rat fetal brown adipocytes. Here, we examine the role played by some members of the mitogen-activated protein kinase (MAPK) superfamily. TNFalpha activates extracellular regulated kinase-1/2 (ERK1/2) and p38MAPK. Inhibition of p38MAPK by either SB203580 or SB202190 highly reduces apoptosis induced by TNFalpha, whereas ERK inhibition potentiates it. Moreover, cotransfection of an active MKK3 mutant and p38MAPK induces apoptosis. p38MAPK inhibition also prevents TNFalpha-induced cell cycle arrest, whereas MEK1 inhibition enhances this effect, which correlates with changes in proliferating cell nuclear antigen expression, but not in cyclin D1. c-Jun and activating transcription factor-1 are potential downstream effectors of p38MAPK and ERKs upon TNFalpha treatment. Thus, TNFalpha-induced c-Jun messenger RNA expression requires ERKs activation, whereas p38MAPK inhibition enhances its expression. In addition, TNFalpha-induced activating transcription factor-1 phosphorylation is extensively decreased by SB203580. However, TNFalpha-induced NF-kappaB DNA-binding activity is independent of p38MAPK and ERK activation. On the other hand, C/EBP homology protein does not appear to mediate the actions of TNFalpha, because its expression is almost undetectable and even reduced by TNFalpha. Finally, although TNFalpha induces c-Jun N-terminal kinase (JNK) activation, transfection of a dominant negative of either JNK1 or JNK2 had no effect on TNFalpha-induced apoptosis. These results suggest that p38MAPK mediates TNFalpha-induced apoptosis and cell cycle arrest, whereas ERKs do the opposite, and JNKs play no role in this process of apoptosis.

Adipocytes↗
PubMed2000

Insulin and the beta3-adrenoceptor differentially regulate uncoupling protein-1 expression.

Cross-talk between insulin and the adrenergic system is important in the regulation of energy homeostasis. In cultured, differentiated mouse brown adipocytes, beta3-adrenergic stimulation induced a 4.5-fold increase in uncoupling protein-1 (UCP-1) expression, which was diminished by 25% in the presence of insulin. Beta3-adrenergic stimulation also activated mitogen-activated protein (MAP) kinase by 3.5-fold and caused a decrease in basal phosphoinositide (PI) 3-kinase activity detected in p110gamma- and Gbeta-subunit-immunoprecipitates in a time-dependent manner, whereas insulin stimulated p110alpha- and phosphotyrosine-associated PI 3-kinase activity. Inhibition of MAP kinase or PI 3-kinase potentiated the beta3-adrenergic effect on UCP-1 expression, both alone and in the presence of insulin. Thus, insulin inhibits beta3-adrenergic stimulation of UCP-1, and both MAP kinase and PI 3-kinase are negative regulatory elements in the beta3-adrenergic control of UCP-1 expression. Cross-talk between the adrenergic and insulin signaling systems and impaired regulation of UCP-1 might contribute to the development of a reduced energy balance, resulting in obesity and insulin resistance.

Adipocytes↗
PubMed2000

Detection of telomerase activity in human carcinomas using a trap-ELISA method: correlation with hTR and hTERT expression.

We have evaluated telomerase activity in a tumour population of 65 human cancers by using a TRAP-based method, in which detection is performed by an enzyme immunoassay (ELISA). We have corroborated that sensitivity and specificity of this new procedure can be considered similar to that of classical TRAP method, having the advantage of a rapid and reproducible analysis of large pools of samples. Thus, telomerase activity was detected in 83% of the tumours included in our population. Moreover, we found a significant association between enzyme activity and both hTR and hTERT expression (P=0.004 and P=0.04, respectively).

DNA-Binding Proteins↗
PubMed1999

beta(3)-adrenergic stimulation differentially inhibits insulin signaling and decreases insulin-induced glucose uptake in brown adipocytes.

Activity of the sympathetic nervous system is an important factor involved in the pathogenesis of insulin resistance and associated metabolic and vascular abnormalities. In this study, we investigate the molecular basis of cross-talk between beta(3)-adrenergic and insulin signaling systems in mouse brown adipocytes immortalized by SV40 T infection. Insulin-induced tyrosine phosphorylation of the insulin receptor, insulin receptor substrate 1 (IRS-1), and IRS-2 was reduced by prestimulation of beta(3)-adrenergic receptors (CL316243). Similarly, insulin-induced IRS-1-associated and phosphotyrosine-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity, but not IRS-2-associated PI 3-kinase activity, was reduced by beta(3)-adrenergic prestimulation. Furthermore, insulin-stimulated activation of Akt, but not mitogen-activated protein kinase, was diminished. Insulin-induced glucose uptake was completely inhibited by beta(3)-adrenergic prestimulation. These effects appear to be protein kinase A-dependent. Furthermore inhibition of protein kinase C restored the beta(3)-receptor-mediated reductions in insulin-induced IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. Together, these findings indicate cross-talk between adrenergic and insulin signaling pathways. This interaction is protein kinase A-dependent and, at least in part, protein kinase C-dependent, and could play an important role in the pathogenesis of insulin resistance associated with sympathetic overactivity and regulation of brown fat metabolism.

Adipocytes↗
PubMed1999

Apoptotic response to TGF-beta in fetal hepatocytes depends upon their state of differentiation.

Transforming growth factor-beta (TGF-beta1) induces death of fetal hepatocytes by an apoptotic mechanism. However, even when very high concentrations and/or long-term exposure to the cytokine is used, 40-50% of cells always survive. The process of cell survival is coincident with changes in morphology and phenotype, with cells showing a fibroblastic appearance and eliciting an epithelial-fibroblastic transition. Surviving cells continue responding to TGF-beta in terms of growth control. Expression of liver-specific genes is very low in these cells; this effect is due to the decrease in their rate of transcription as soon as 2 h after the addition of the factor. Surviving cells present a decreased DNA binding activity for liver-enriched transcription factors, an increased DNA binding activity for AP-1, and a high expression of protooncogenes. These cells are immature hepatocytes since in the presence of the appropriate signal (i.e., epidermal growth factor), they can differentiate, organizing in cell clusters and increasing both liver-specific mRNA expression and liver-enriched transcription factor activity. In accord with these results, TGF-beta, secreted at high concentrations during liver carcinogenesis, might induce death of normal cells while providing a selective advantage for the survival of cells that are "partially transformed" or "less differentiated" and unresponsive to the factor.

Animals↗
PubMed1999

DNA amplification on chromosome 6p12 in non small cell lung cancer detected by arbitrarily primed polymerase chain reaction.

Gene amplification is clearly an important aspect of tumour growth and development and has prognostic significance in certain tumours. The identification and genetic characterisation of new areas of amplification in human malignancy remains an important goal in understanding the underlying genetic lesions within these tissues. In the present work, arbitrarily primed-PCR (AP-PCR) has been applied to detect and characterise amplified DNA fragments in human non small cell lung cancer (NSCLC). Our results show that gains of genomic sequences occur at high frequency (64% of all genomic changes analysed). Moreover, we succeeded in detecting a genomic sequence that is highly amplified in one of the tumours analysed. The amplification intensity of this DNA fragment was also increased in 29 (45%) of the 65 NSCLC patients from our study. The amplified DNA fragment was isolated and identified as a 600 bp sequence mapped to chromosome 6p12. This sequence did not show significant homology with known human DNA sequences. Interestingly, a gene related to cancer processes, the pim-1 oncogene, is placed neighbouring to this region on chromosome 6. Survival studies revealed that disease-free interval of NSCLC patients was shorter in patients bearing the amplified sequence (p = 0.05 by the Breslow test). Our findings suggest that the amplified sequence located on chromosome 6 might be relevant in the pathogenesis of human NSCLC. Int. J. Cancer (Pred. Oncol.), 84:344-349, 1999.

Animals↗
PubMed1999

Activated Ha-ras induces apoptosis by association with phosphorylated Bcl-2 in a mitogen-activated protein kinase-independent manner.

Serum deprivation of Ha-ras-transformed brown adipocyte cell line resulted in a dramatic apoptotic cell death, as detected either by DNA laddering or by an increase in the percentage of hypodiploid cells or by nuclei condensation and fragmentation, as compared with immortalized cell line or primary fetal brown adipocytes. Moreover, transient transfection of immortalized brown adipocytes with a constitutively active ras gene (Ha-raslys12) mimics the high rate of apoptosis detected in the transformed cell line. On the other hand, transient transfection of the dominant-negative construct of raf-1 rescued serum-deprived Ha-ras-transformed brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the external display of phosphatidylserine, and the DNA laddering. However, inhibition of mitogen-activated protein kinase with PD098059 did not preclude apoptosis and in fact increased the rate of apoptosis observed in serum-deprived Ha-ras-transformed cells, indicating that the Ras/Raf-1 pathway induced apoptosis throughout a mitogen-activated protein kinase kinase 1 (MEK-1)-independent pathway. Furthermore, apoptosis in Ha-ras-transformed brown adipocytes is concurrent with an up-regulation in the expression of the pro-apoptotic protein Bcl-xS, the expression of the anti-apoptotic protein Bcl-2 being down-regulated. Finally, an association of Ras and Raf with phosphorylated Bcl-2 protein was demonstrated in immunoprecipitates from apoptotic cells. Thus, we propose a mechanism of apoptosis in Ha-ras-transformed adipocytes under serum deprivation involving Raf-1 association with phosphorylated Bcl-2, down-regulation of Bcl-2 expression, and up-regulation of Bcl-xS expression.

Adipocytes↗
PubMed1999

Inhibition of caspases rescues brown adipocytes from apoptosis downregulating BCL-XS and upregulating BCL-2 gene expression.

Serum deprivation of the immortalized brown adipocyte cell line resulted in growth arrest in G0/G1 phases of the cell cycle and apoptosis, as detected by DNA laddering, nuclei condensation and fragmentation, and an increase in the percentage of hypodiploid cells. In addition, apoptosis in these cells is accompanied by an induction of the expression of the apoptotic form of the Bcl-x gene, the isoform Bcl-xS, and by a decrease of Bcl-2 expression, Bcl-xL remaining almost undetectable. The loss of mitochondrial membrane potential was associated with apoptosis. Z-VAD, a cell-permeable inhibitor of caspases, but not cycloheximide, precludes DNA laddering under serum deprivation. Moreover, Z-VAD rescues serum-deprived brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the percentage of apoptotic cells under Tunnel assay, and the external display of phosphatidylserine. More importantly, Z-VAD survival effects on immortalized brown adipocytes concur with a downregulation of Bcl-xS mRNA/protein and an upregulation of Bcl-2 protein content. Ultimately, Z-VAD prevents the loss of mitochondrial membrane potential.

Adipocytes↗
PubMed1999

Insulin and insulin-like growth factor I up-regulate GLUT4 gene expression in fetal brown adipocytes, in a phosphoinositide 3-kinase-dependent manner.

Fetal brown adipocytes cultured in a serum-free medium, containing 5 mM glucose, expressed both GLUT4 and GLUT1 glucose transporters at the mRNA and protein level. Treatment with either insulin or insulin-like growth factor (IGF)-I at physiological concentrations up-regulates the expression of the GLUT4 gene, producing a time-dependent mRNA accumulation (7-fold increase at 24 h) and a 2.5-fold increase in the amount of protein in the total membrane fraction. However, insulin treatment down-regulates GLUT1 mRNA and protein expression. Moreover, either insulin or IGF-I transactivates a full-promoter GLUT4-chloramphenicol acetyltransferase gene (CAT) construct transiently transfected to the cells, without affecting GLUT1-CAT activity. In consequence, insulin treatment for 24 h increased by 3-fold the basal glucose uptake. Inhibition of phosphoinositide (PI) 3-kinase activity with chemical agents such as wortmannin or LY294002 partially blocked insulin-induced GLUT4 mRNA accumulation, insulin-induced GLUT4 protein content, GLUT4-CAT transactivation and glucose uptake. Furthermore, co-transfection of brown adipocytes with a dominant-negative form of PI 3-kinase precluded the transactivation of the GLUT4 promoter by insulin. However, inhibition of p70S6 kinase (p70(s6k)) with rapamycin or of mitogen-activated protein kinase (MAPK) with PD098059 does not preclude insulin effects on GLUT4 gene expression or glucose uptake. Our results show for the first time a positive effect of insulin on GLUT4 gene expression in fetal brown adipocytes, suggesting the existence of insulin response element(s) in its promoter. Moreover, PI 3-kinase, but not p70(s6k) or MAPK, is an essential requirement for insulin regulation of GLUT4 gene expression.

Adipose Tissue↗
PubMed1999

Glucocorticoid receptor down-regulates c-Jun amino terminal kinases induced by tumor necrosis factor alpha in fetal rat hepatocyte primary cultures.

The effect of dexamethasone on Jun N-terminal kinase (JNK) activity was assayed by using fetal hepatocytes in primary culture. The addition of tumor necrosis factor alpha (TNF-alpha) caused an increase in JNK in a dose- and time-dependent manner. We show that activation of JNK by this extracellular signal is inhibited by dexamethasone in a dose-dependent fashion. This inhibitory effect was observed in cells treated for 10 minutes with dexamethasone in the presence of protein phosphatase inhibitors such as orthovanadate or okadaic acid, or in cells previously treated with actinomycin D. Glucocorticoid receptor (GR) can be precipitated with the fusion protein, GST-c-Jun (1-79), bound to agarose beads. However, the inhibitory effect of glucocorticoids on JNK activity was also observed using ATF-2 as substrate. In addition, dexamethasone inhibits JNK phosphorylation induced by TNF-alpha. Finally, we show that GR can also be phosphorylated in tyrosine residues in response to TNF-alpha and epidermal growth factor (EGF) upon ligand-binding. Our results suggest that the anti-inflammatory effect of glucocorticoids on the inflammatory pathways induced by TNF-alpha can be explained, at least in part, by modulating JNK activity through a direct protein-protein interaction; the JNK phosphorylation and tyrosine-phosphorylation state of GR may be regulatory steps also involved in that effect.

Animals↗