Biomedical subjects

Soazig Le Lay

Publications and source records attributed to Soazig Le Lay.

9 recordsLinked to original sources

PubMed2006

Integrin-mediated adhesion regulates membrane order.

The properties of cholesterol-dependent domains (lipid rafts) in cell membranes have been controversial. Because integrin-mediated cell adhesion and caveolin both regulate trafficking of raft components, we investigated the effects of adhesion and caveolin on membrane order. The fluorescent probe Laurdan and two-photon microscopy revealed that focal adhesions are highly ordered; in fact, they are more ordered than caveolae or domains that stain with cholera toxin subunit B (CtxB). Membrane order at focal adhesion depends partly on phosphorylation of caveolin1 at Tyr14, which localizes to focal adhesions. Detachment of cells from the substratum triggers a rapid, caveolin-independent decrease in membrane order, followed by a slower, caveolin-dependent decrease that correlates with internalization of CtxB-stained domains. Endocytosed CtxB domains also become more fluid. Thus, membrane order is highly dependent on caveolae and focal adhesions. These results show that lipid raft properties are conferred by assembly of specific protein complexes. The ordered state within focal adhesions may have important consequences for signaling at these sites.

Animals↗

PubMed2006

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts.

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.

Animals↗

PubMed2006

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: a role for caveolar endocytosis.

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.

3T3-L1 Cells↗

PubMed2005

Getting rid of caveolins: phenotypes of caveolin-deficient animals.

The elucidation of the role of caveolae has been the topic of many investigations which were greatly enhanced after the discovery of caveolin, the protein marker of these flask-shaped plasma membrane invaginations. The generation of mice deficient in the various caveolin genes (cav-1, cav-2 and cav-3) has provided physiological models to unravel the role of caveolins or caveolae at the whole organism level. Remarkably, despite the essential role of caveolins in caveolae biogenesis, all knockout mice are viable and fertile. However, lack of caveolae or caveolins leads to a wide range of phenotypes including muscle, pulmonary or lipid disorders, suggesting their implication in many cellular processes. The aim of this review is to give a broad overview of the phenotypes described for the caveolin-deficient mice and to link them to the numerous functions so far assigned to caveolins/caveolae.

Animals↗

PubMed2005

Caveolin-1 is not essential for biosynthetic apical membrane transport.

Caveolin-1 has been implicated in apical transport of glycosylphosphatidylinositol (GPI)-anchored proteins and influenza virus hemagglutinin (HA). Here we have studied the role of caveolin-1 in apical membrane transport by generating caveolin-1-deficient Madin-Darby canine kidney (MDCK) cells using retrovirus-mediated RNA interference. The caveolin-1 knockdown (cav1-KD) MDCK cells were devoid of caveolae. In addition, caveolin-2 was retained in the Golgi apparatus in cav1-KD MDCK cells. However, we found no significant alterations in the apical transport kinetics of GPI-anchored proteins or HA upon depletion of caveolin-1. Similar results were obtained using embryonic fibroblasts from caveolin-1-knockout mice. Thus, we conclude that caveolin-1 does not play a major role in lipid raft-mediated biosynthetic membrane trafficking.

Adenoviridae↗

PubMed2003

Regulation of ABCA1 expression and cholesterol efflux during adipose differentiation of 3T3-L1 cells.

Adipose cells specialized in energy storage, contain large intracellular triglyceride-rich lipid droplets, are enriched with free cholesterol, and express sterol-regulated transcription factors such as liver X receptor (LXR). The recent identification of the LXR-dependent ATP binding cassette transporter A1 (ABCA1) pathway for cholesterol release from peripheral cells has led us to address the question of the expression and function of ABCA1 in adipocytes. In 3T3-L1 adipose cells, we observed a strong induction of ABCA1 mRNA during adipose differentiation, but only limited variations in ABCA1 protein. Lipid efflux onto apolipoprotein A-I (apoA-I), which depends on ABCA1, was comparable in adipocytes and preadipocytes, demonstrating a differential regulation of ABCA1 mRNA and cholesterol efflux. We also found that total cell cholesterol remained stable during differentiation of 3T3-L1 cells, but membrane cholesterol was lower in adipocytes than in preadipocytes, suggesting redistribution of cholesterol to the lipid droplet. Finally, we show that under standard lipolytic stimulation, 3T3-L1 adipocytes do not release cholesterol onto apoA-I, a process that required long exposures to lipolytic agents (24 h). In conclusion, despite large induction of ABCA1 mRNA during differentiation, cholesterol efflux through the ABCA1 pathway remains limited in adipocytes and requires prolonged lipolysis. This is consistent with the view of the adipocyte behaving as a cholesterol sink, with plasma cholesterol-buffering properties.

3T3-L1 Cells↗

PubMed2002

Neuropeptide AF and FF modulation of adipocyte metabolism. Primary insights from functional genomics and effects on beta-adrenergic responsiveness.

The presence of a neuropeptide AF and FF receptor (NPFF-R2) mRNA in human adipose tissue (Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres, P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D. J., and Sarau, H. M. (2000) J. Biol. Chem. 275, 25965-25971) suggested these peptides, principally recognized for their pain modulating effects, may also impact on adipocyte metabolism, an aspect that has not been explored previously. Our aim was thus to obtain more insights into the actions of these peptides on adipocytes, an approach initially undertaken with a functional genomic assay. First we showed that 3T3-L1 adipocytes express both NPFF-R1 and NPFF-R2 transcripts, and that NPAF binds adipocyte membranes with a nanomolar affinity as assessed by surface plasmon resonance technology. Then, and following a 24-h treatment with NPFF or NPAF (1 microm), we have measured using real-time quantitative reverse transcriptase-PCR the mRNA steady state levels of already well characterized genes involved in key pathways of adipose metabolism. Among the 45 genes tested, few were modulated by NPFF ( approximately 10%) and a larger number by NPAF ( approximately 27%). Interestingly, NPAF increased the mRNA levels of beta2- and beta3-adrenergic receptors (AR), and to a lesser extent those of beta1-ARs. These variations in catecholamine receptor mRNAs correlated with a clear induction in the density of beta2- and beta3-AR proteins, and in the potency of beta-AR subtype-selective agonists to stimulate adenylyl cyclase activity. Altogether, these data show that NPFF-R1 and NPFF-R2 are functionally present in adipocytes and suggest that besides their well described pain modulation effects, NPAF and to a lesser extent NPFF, may have a global impact on body energy storage and utilization.

3T3 Cells↗

PubMed2002

Insulin and sterol-regulatory element-binding protein-1c (SREBP-1C) regulation of gene expression in 3T3-L1 adipocytes. Identification of CCAAT/enhancer-binding protein beta as an SREBP-1C target.

We evaluated the hypothesis of sterol-regulatory element-binding protein (SREBP)-1c being a general mediator of the transcriptional effects of insulin, with a focus on adipocytes, in which insulin profoundly influences specific gene expression. Using real time quantitative reverse transcriptase-PCR to monitor changes in the expression of about 50 genes that cover a wide range of adipocyte functions, we have compared the impact of insulin treatment with that of adenoviral overexpression of either dominant positive or dominant negative SREBP-1c mutants in 3T3-L1 adipocytes. As expected, insulin up-regulated, dominant positive stimulated, and dominant negative decreased previously characterized direct SREBP targets (FAS, SCD-1, and low density lipoprotein receptor). We also identified three novel SREBP-1c transcriptional targets in adipocytes, which were confirmed by run-on assays: plasminogen activator inhibitor 1, CCAAT/enhancer-binding protein delta (C/EBPdelta), and C/EBPbeta. Because most insulin-regulated genes were also modulated by SREBP-1c mutants, our data establish that 1) SREBP-1c is an important mediator of insulin transcriptional effects in adipocytes, and 2) C/EBPbeta is under the direct control of SREBP-1c, as demonstrated by the ability of SREBP-1c to activate the transcription from C/EBPbeta promoter through canonical SREBP binding sites. Thus, some of the effects of insulin and/or SREBP-1c in mature fat cells might require C/EBPbeta or C/EBPdelta as transcriptional relays.

3T3 Cells↗

PubMed2002

Sterol regulatory element binding protein-1c expression and action in rat muscles: insulin-like effects on the control of glycolytic and lipogenic enzymes and UCP3 gene expression.

Sterol regulatory element binding protein-1c (SREBP-1c) is a transcription factor that mediates insulin effects on hepatic gene expression. It is itself transcriptionally stimulated by insulin in hepatocytes. Here we show that SREBP-1c mRNA is expressed in adult rat skeletal muscles and that this expression is decreased by diabetes. The regulation of SREBP-1c expression was then assessed in cultures of adult muscle satellite cells. These cells form spontaneously contracting multinucleated myotubes within 7 days of culture. SREBP-1c mRNA is expressed in contracting myotubes. A 4-h treatment with 100 nmol/l insulin increases SREBP-1c expression and nuclear abundance by two- to threefold in myotubes. In cultured myotubes, insulin increases the expression of glycolytic and lipogenic enzyme genes and inhibits the 9-cis retinoic acid-induced UCP3 expression. These effects of insulin are mimicked by adenovirus-mediated expression of a transcriptionally active form of SREBP-1c. We conclude that in skeletal muscles, SREBP-1c expression is sensitive to insulin and can transduce the positive and negative actions of the hormone on specific genes and thus has a pivotal role in long-term muscle insulin sensitivity.

Alitretinoin↗