Contribution of adipose triglyceride lipase and hormone-sensitive lipase to lipolysis in hMADS adipocytes.

Lipolysis is the catabolic pathway by which triglycerides are hydrolyzed into fatty acids. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) have the capacity to hydrolyze in vitro the first ester bond of triglycerides, but their respective contributions to whole cell lipolysis in human adipocytes is unclear. Here, we have investigated the roles of HSL, ATGL, and its coactivator CGI-58 in basal and forskolin-stimulated lipolysis in a human white adipocyte model, the hMADS cells. The hMADS adipocytes express the various components of fatty acid metabolism and show lipolytic capacity similar to primary cultured adipocytes. We show that lipolysis and fatty acid esterification are tightly coupled except in conditions of stimulated lipolysis. Immunocytochemistry experiments revealed that acute forskolin treatment promotes HSL translocation from the cytosol to small lipid droplets and redistribution of ATGL from the cytosol and large lipid droplets to small lipid droplets, resulting in enriched colocalization of the two lipases. HSL or ATGL overexpression resulted in increased triglyceride-specific hydrolase capacity, but only ATGL overexpression increased whole cell lipolysis. HSL silencing had no effect on basal lipolysis and only partially reduced forskolin-stimulated lipolysis. Conversely, silencing of ATGL or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Altogether, these results suggest that ATGL/CGI-58 acts independently of HSL and precedes its action in the sequential hydrolysis of triglycerides in human hMADS adipocytes.

The transcriptional co-activator PGC-1alpha up regulates apelin in human and mouse adipocytes.

By using pangenomic microarray, we identified apelin as a unique adipokine up regulated by the transcriptional co-activator peroxisome proliferator-activated receptor gamma (PPARgamma) co-activator 1alpha (PGC-1alpha) in human white adipocytes. We investigated its regulation in vitro and in vivo. Overexpression of PGC-1alpha by adenovirus in human adipocytes induces apelin expression and secretion. Pharmacological induction of cAMP, an upstream regulator of endogenous PGC-1alpha expression, up regulates apelin gene expression and also apelin secretion in human and mice adipocytes. Moreover, during cold exposure in mice, a physiological situation known to induce both cAMP and PGC-1alpha, apelin expression in adipocytes and plasma levels were increased. This is the first demonstration that PGC-1alpha is involved in the regulation of an adipokine gene expression and release.

[RNA interference: towards a functional genomics in mammalian cells?]. / L'interférence par l'ARN: vers une génomique fonctionnelle chez les mammifères ?

The discovery of the induction of RNA degradation by double stranded RNA in C. elegans, "RNA interference", makes it possible to envision systematic studies of gene function in mammalian cells. Indeed, in spite of the existence in mammals of the interferon response to double stranded RNA, the introduction of small interfering RNA can induce a sequence specific inhibition of gene expression either through RNA degradation or by blocking translation. Although the inhibition is transient and usually not complete, strategies have been developed to achieve long term gene silencing. The issue of target specificity is still not completely clear and will probably constitute a limitation of this approach. However, because of the unprecedented ease with which large scale screens can be performed, RNA interference has already established itself as the tool of choice to initiate functional genomics in mammalian cells.

Peroxisome proliferator-activated receptor-alpha control of lipid and glucose metabolism in human white adipocytes.

This work aimed at characterizing the role of peroxisome proliferator-activated receptors (PPAR)alpha in human white adipocyte metabolism and at comparing PPAR alpha and PPAR gamma actions in these cells. Primary cultures of human fat cells were treated with the PPAR alpha agonist GW7647 or the PPAR gamma agonist rosiglitazone. Changes in gene expression were determined using DNA microarrays and quantitative RT-PCR. Western blot and metabolic studies were performed to identify the biological effects elicited by PPAR agonist treatments. GW7647 induced an up-regulation of beta-oxidation gene expression and increased palmitate oxidation. Unexpectedly, glycolysis was strongly reduced at transcriptional and functional levels by GW7647 leading to a decrease in pyruvate and lactate production. Glucose oxidation was decreased. Triglyceride esterification and de novo lipogenesis were inhibited by the PPAR alpha agonist. GW7647-induced alterations were abolished by a treatment with a PPAR alpha antagonist. Small interfering RNA-mediated extinction of PPAR alpha gene expression in hMADS adipocytes attenuated GW7647 induction of palmitate oxidation. Rosiglitazone had no major impact on glycolysis and beta-oxidation. Altogether these results show that PPAR alpha can selectively up-regulate beta-oxidation and decrease glucose utilization in human white adipocytes.

[RNA interference in mammalian cells]. / L'interférence par l'ARN dans les cellules de mammifère.

RNA interference was the first regulation by small RNA to be described in detail. It was initially identified in C. elegans as a sequence-specific post-transcriptional silencing induced by double stranded RNA. There are two main steps in the process, the cleavage of long double stranded RNA molecules into small interfering RNA of about twenty nucleotides and the incorporation of these small molecules into a protein complex to which it confers a sequence specific interaction with RNA substrates. The "classical" RNA interference is associated with the cleavage and the subsequent degradation of the targeted RNA which in its simplest form can be carried out by a single protein (Argonaute 2 in mammals) and a small interfering RNA. The cleavage requires a near perfect complementarity between the substrate and the small guide present in the complex; this sequence specificity and the catalytic nature of the process create an almost ideal tool to silence any gene for which the sequence is known. However several considerations limit the efficacy and the specificity of this process. Foremost is our current inability to restrict the activity of small regulatory RNA to this RNA cleavage pathway which leads to the activation of other cellular regulations some of which have a lower level of sequence specificity than RNA interference. A better understanding of these regulatory pathways will be necessary in order to achieve the specific and efficient silencing that experimentalists dream of.