1266 Research Article CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes Sébastien Vankoningsloo1, Aurélia De Pauw1, Andrée Houbion1, Silvia Tejerina1, Catherine Demazy1, Françoise de Longueville2, Vincent Bertholet2, Patricia Renard1, José Remacle1,2, Paul Holvoet3, Martine Raes1 and Thierry Arnould1,* 1Laboratory of Biochemistry and Cellular Biology, University of Namur (F.U.N.D.P.), Rue de Bruxelles, 61, 5000 Namur, Belgium 2Eppendorf Array Technologies, Rue du Séminaire, 12, 5000 Namur, Belgium 3Cardiovascular Research Unit of the Center for Experimental Surgery and Anesthesiology, Katholieke Universiteit Leuven (KUL), Belgium *Author for correspondence (e-mail: [email protected]) Accepted 12 December 2005 Journal of Cell Science 119, 1266-1282 Published by The Company of Biologists 2006 doi:10.1242/jcs.02848 Summary Several mitochondrial pathologies are characterized by protein ␣ (C/EBP␣), C/EBP homologous protein-10 lipid redistribution and microvesicular cell phenotypes (CHOP-10), mitochondrial glycerol-3-phosphate resulting from triglyceride accumulation in lipid- dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 metabolizing tissues. However, the molecular mechanisms (SCD1). We also demonstrate that overexpression of two underlying abnormal fat distribution induced by dominant negative mutants of the cAMP-response element- mitochondrial dysfunction remain poorly understood. In binding protein CREB (K-CREB and M1-CREB) and this study, we show that inhibition of respiratory complex siRNA transfection, which disrupt the factor activity and III by antimycin A as well as inhibition of mitochondrial expression, respectively, inhibit antimycin-A-induced protein synthesis trigger the accumulation of triglyceride triglyceride accumulation. Furthermore, CREB knock- vesicles in 3T3-L1 fibroblasts. We also show that treatment down with siRNA also downregulates the expression of with antimycin A triggers CREB activation in these cells. several genes that contain cAMP-response element (CRE) To better delineate how mitochondrial dysfunction induces sites in their promoter, among them one that is potentially triglyceride accumulation in preadipocytes, we developed a involved in synthesis of triglycerides such as SCD1. These low-density DNA microarray containing 89 probes, which results highlight a new role for CREB in the control of Journal of Cell Science allows gene expression analysis for major effectors and/or triglyceride metabolism during the adaptative response of markers of adipogenesis. We thus determined gene preadipocytes to mitochondrial dysfunction. expression profiles in 3T3-L1 cells incubated with antimycin A and compared the patterns obtained with differentially expressed genes during the course of in vitro Supplementary material available online at adipogenesis induced by a standard pro-adipogenic http://jcs.biologists.org/cgi/content/full/119/7/1266/DC1 cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with Key words: CREB, Lipid metabolism, Mitochondrial dysfunction, antimycin A, among them CCAAT/enhancer-binding Gene expression, SiRNA, Adipocytes Introduction accumulation in muscles and liver of healthy and lean elderly The large variety of metabolic disorders related to bio- people (Petersen et al., 2003). Reduced mitochondrial activity energetical stress underlines the essential role of mitochondrial and intramyocytic accumulation of TG were also found in the activity in cellular physiology. More particularly, several lines young and insulin-resistant offspring of parents with type 2 of evidence now show that defects in ␤-cells insulin secretion diabetes. Insulin-resistant patients usually have a lower ratio of as well as systemic insulin resistance in type 2 diabetes could type 1 muscle fibers to the more glycolytic type 2 muscle be attributable to mitochondrial dysfunction (Lowell and fibers, an observation that could be explained by the reduced Shulman, 2005). In human muscle cells, the major effectors expression of members of the peroxisome proliferator- involved in insulin resistance include adipokines (Lazar, 2005) activated receptor ␥ (PPAR␥)-coactivator-1 (PGC-1) family and fatty acids, which cause a direct inhibition of insulin- and the downregulation of their target genes involved in stimulated glucose transport activity through a decrease in mitochondrial biogenesis (Wu et al., 1999; Mootha et al., phosphoinositide 3-kinase (PI 3-kinase) signaling (Dresner et 2003). Hypoxic conditions that inhibit mitochondrial al., 1999). Furthermore, impairment of mitochondrial activity respiration also lead to TG accumulation in cardiomyocytes associated with ageing, which could result from alterations in (Huss et al., 2001). In addition, we recently showed that, the mitochondrial DNA (mtDNA), also leads to triglyceride (TG) impairment of mitochondrial activity by inhibitors of Mitochondrial dysfunction triggers triglyceride accumulation 1267 respiratory complexes also triggers TG accumulation in murine adipogenic markers that are potentially responsible for TG 3T3-L1 preadipocytes, resulting from a decrease in fatty acid accumulation in AA-treated preadipocytes. These results were ␤-oxidation and an increase in glucose uptake enhancing compared with differentially expressed genes obtained for glycerol 3-phosphate synthesis (Vankoningsloo et al., 2005). differentiating 3T3-L1 cells in the presence of a standard Changes in cellular metabolism due to a loss of hormone cocktail. The DNA microarray used in this study mitochondrial oxidative capacity lead to the activation of cell allows gene expression profiling for 89 genes related to signaling pathways and modifications in the expression of adipogenesis and lipid metabolism. These markers have been many nuclear genes. This process, known as mitochondria- carefully selected, based on the literature reporting on nucleus retrograde communication, has mainly been studied in differentiation-specific gene expression during 3T3-L1 yeast (Liao et al., 1991; Butow and Avadhani, 2004) and more adipogenesis (Burton et al., 2004; Guo and Liao, 2000; recently in mammalian cells depleted in mitochondrial DNA Kratchmarova et al., 2002). We also evidenced that, inhibition (mtDNA) (Amuthan et al., 2002; Biswas et al., 1999; Biswas of CREB expression with small interfering RNA (siRNA) and et al., 2005; Arnould et al., 2002). For example, we have reduction of its activity by overexpression of two dominant recently shown that depletion of mtDNA or inhibition of negative mutants (K-CREB and M1-CREB) diminishes the TG mitochondrial respiration activates the cAMP-response- accumulation induced by AA in 3T3-L1 preadipocytes. Finally, element binding protein (CREB) by phosphorylation of Ser133 combining RNA interference (RNAi) and microarray mediated through a Ca2+/calmodulin-dependent kinase IV technology, we identified several CREB-target genes that are (CaMK IV) pathway (Arnould et al., 2002). differentially regulated when 3T3-L1 cells are incubated with CREB is a transcription factor with pleiotropic effects that AA. Taken together, these results not only extend the role of has already been reported to play a role in the control of CREB in adipocyte biology and lipid metabolism but also memory (Scott et al., 2002), cell proliferation (Della Fazia et highlight the 3T3-L1 preadipocyte response to mitochondrial al., 1997), and in glucose and lipid metabolism (Zhou et al., dysfunction leading to TG accumulation, which might not only 2004; Reusch et al., 2000; Herzig et al., 2003). Previous impair adipocyte metabolism but also the physiology of studies showed that this transcription factor is also an early insulin-dependent tissues. regulator of adipocyte differentiation because it is activated by phosphorylation of Ser133 in the presence of adipogenic Results inducers, such as glucocorticoids, cyclic AMP analogues and Mitochondrial dysfunction induces TG accumulation in insulin-like growth factor-1 (IGF-1), or high concentrations of 3T3-L1 preadipocytes insulin (MacDougald and Lane, 1995). Furthermore, ectopic Differentiation of 3T3-L1 preadipocytes into adipocytes is expression of the chimeric and constitutively active VP16- easily triggered by a standard adipogenic cocktail comprising CREB is sufficient to trigger adipogenesis, whereas insulin, a cAMP-elevating agent and dexamethasone overexpression of a dominant negative form of CREB (K- (MacDougald and Lane, 1995). The main morphological CREB) inhibits the adipogenic program (Reusch et al., 2000) characteristic of adipogenesis is the progressive storage of and leads to apoptosis of mature adipocytes (Reusch and large amounts of cytosolic TG, whereas TG that accumulate in Journal of Cell Science Klemm, 2002). These data suggest that CREB acts as pro- 3T3-L1 cells incubated with 10 nM AA form small but adipogenic and survival factor. It has also been reported that numerous vesicles, as revealed after the staining of neutral the positive effect of activated CREB on adipogenesis is lipids with Oil Red O (Fig. 1A) (Vankoningsloo et al., 2005). mediated by the overexpression of the CCAAT/enhancer- Quantitative analysis showed that TG accumulation in 3T3-L1 binding protein ␤ (C/EBP␤) gene, a key transcription factor cells treated for 8 days with AA depends on the concentration in the differentiation program that contains dual cAMP- of this metabolic inhibitor (Fig. 1B). A similar phenotype was response element