Study of the 3T3-L1 adipocyte responses to a mild mitochondrial uncoupling : analyses of the modifications affecting the mitochondrial population and the release of adipokines.

Résumé

As obesity results, at least partly, from a chronically positive energy balance, increasing energy expenditure through mitochondrial uncoupling in adipocytes has been considered as an attractive approach to fight obesity. Currently, mitochondrial uncoupling in adipocytes is known to trigger a reduction in triglyceride (TG) content, a phenotype also found in adipocytes incubated with TNFα (tumor-necrosis factor α), another condition known to induce the “dedifferentiation” of adipocytes. However, the impact of a mitochondrial uncoupling on the abundance/composition of mitochondria and its connection with triglyceride content in adipocytes is largely unknown. In addition, while mitochondrial activity has been linked to adipokine expression, the effects of mitochondrial uncoupling on adipokine expression and secretion has been poorly investigated. However, this is a very important question to address as adipokines (referring to hormones, cytokines and chemokines released by adipocytes acting on peripheral tissues) are thought to be the molecular links between obesity and associated diseases such as insulin resistance, type 2 diabetes, cardiovascular diseases and inflammation, brought altogether in the metabolic syndrome. In the first part of this study, the effects of a mild mitochondrial uncoupling triggered by carbonyl cyanide (p-trifluoromethoxy) phenylhydrazone (FCCP) on the mitochondrial population of 3T3-L1 adipocytes were investigated using both quantitative and qualitative (biochemical, bioenergetics, genomics, proteomics) approaches. We found that chronic but mild mitochondrial uncoupling does not stimulate mitochondrial biogenesis in adipocytes, but induces a subtle adaptive cell response characterized by modifications of mitochondrial protein content. Subproteomic analysis revealed that the abundance of pyruvate carboxylase was significantly reduced in mitochondria of TNFα- and FCCP-treated adipocytes. A functional study showed that overexpression of this key enzyme in lipid metabolism is able to prevent the TG content reduction in adipocytes exposed to mitochondrial uncoupling or TNFα. These results provide a new mechanism by which the effects of mitochondrial uncoupling might limit triglyceride accumulation in adipocytes. We also found that a mild and chronic mitochondrial uncoupling down-regulates the expression and the secretion of leptin, Acrp30, and resistin and modifies the abundance of active PAI-1 in the supernatant of 3T3-L1 adipocytes. In addition, adipocytes treated with TNFα (a pro-inflammatory cytokine abundantly found in the adipose tissue of obese individuals) display similar modifications. However and unexpectedly, we observed that conditioned media from FCCP-treated adipocytes impair insulin-stimulated glucose uptake in differentiated C2C12 cells while conditioned media from TNFα-treated adipocytes do not impair insulin-stimulated glucose uptake in these cells. Additionally, we demonstrated, using a DNA array (“DualChip inflammation” allowing gene expression analysis for 253 inflammatory genes), that mitochondrial uncoupling does not trigger a molecular signature supporting the initiation of an inflammatory response by adipocytes. Indeed, the expression of many important pro-inflammatory effectors was not affected in adipocytes exposed to FCCP. All together, these sets of data strongly suggest that insulin resistance is acquired by myocytes exposed to conditioned media from adipocytes themselves exposed to a mitochondrial uncoupling and probably triggered by modifications in the set of adipokines (still to be identified) released by adipocytes in these conditions. In conclusion, we demonstrated, for the first time, that a mild and chronic mitochondrial uncoupling affects mitochondrial population without triggering mitochondrial biogenesis and that pyruvate carboxylase might play a key role in mitochondrial uncouplinginduced triglyceride content reduction in adipocytes. We also found that mitochondrial uncoupling affects the expression and the release of several adipokines by adipocytes, which in turn could trigger insulin resistance in C2C12 myocytes. These results open the way to further investigation on the role of mitochondrial uncoupling on the secretory function of adipocytes and bring some highlights on the comprehension of adipocyte cell responses to a mitochondrial uncoupling. This information is needed before thinking of using controlled and specifically targeted mitochondrial uncoupling in white adipose tissue as a therapeutic strategy to tackle obesity and its associated disorders.

la date de réponse	25 nov. 2010
langue originale	Anglais
L'institution diplômante	Universite de Namur
Superviseur	Thierry Arnould (Promoteur), Martine Raes (Copromoteur), Yves Poumay (Président), Xavier De Bolle (Jury), Paul Holvoet (Jury) & Rudolf J. WIESNER (Jury)