CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes

Abstract

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid-metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation of triglyceride vesicles in 3T3-L1 fibroblasts. We also show that treatment with antimycin A triggers CREB activation in these cells. To better delineate how mitochondrial dysfunction induces triglyceride accumulation in preadipocytes, we developed a low-density DNA microarray containing 89 probes, which allows gene expression analysis for major effectors and/or markers of adipogenesis. We thus determined gene 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 adipogenesis induced by a standard pro-adipogenic cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with antimycin A, among them CCAAT/enhancer-binding protein α (C/EBPα), C/EBP homologous protein-10 (CHOP-10), mitochondrial glycerol-3-phosphate dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 (SCD1). We also demonstrate that overexpression of two dominant negative mutants of the cAMP-response element-binding protein CREB (K-CREB and M1-CREB) and siRNA transfection, which disrupt the factor activity and expression, respectively, inhibit antimycin-A-induced triglyceride accumulation. Furthermore, CREB knockdown with siRNA also downregulates the expression of several genes that contain cAMP-response element (CRE) sites in their promoter, among them one that is potentially involved in synthesis of triglycerides such as SCD1. These results highlight a new role for CREB in the control of triglyceride metabolism during the adaptative response of preadipocytes to mitochondrial dysfunction.

The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry

“…Reduced content and functional capacity of mitochondria are involved in a wide range of human pathologies, including obesity and diabetes (Hojlund et al 2003, Ritov et al 2005. The decrease in ATPB expression in obese placentas may lead to mitochondrial dysfunction, leading to an accumulation of lipid metabolites (Vankoningsloo et al 2006, Mailloux et al 2007). An accumulation of lipid metabolites could, in turn, partially explain the insulin signalling defects reported in placentas from obese pregnant women (Colomiere et al 2009).…”

Mild mitochondrial uncoupling induces 3T3-L1 adipocyte de-differentiation by a PPARγ-independent mechanism, whereas TNFα-induced de-differentiation is PPARγ dependent

“…We took advantage of a low-density cDNA microarray to analyze gene expression of key molecular markers and effectors of adipogenesis (Vankoningsloo et al, 2006). Differentiated cells were thus treated for 24, 72 or 144 hours with 0.5 μM FCCP, 10 ng/ml TNFα or 10 μM isoproterenol and gene expression was analyzed in triplicate for each experimental condition.…”

“…Mouse 3T3-L1 preadipocytes from ATCC, were differentiated as previously described (Vankoningsloo et al, 2006). Cell differentiation lasted for 10 or 12 days to obtain more than 90% of differentiated cells, as estimated by phenotype analysis and Oil red O (ORO) staining.…”

“…Glycerol release was measured in the 24-hour-old conditioned medium as an index of lipolysis using the enzymatic INT kit (Sigma) according to the manufacturer's instructions. TG content was determined after lipid extraction in methanol-chloroform as previously described (Vankoningsloo et al, 2006) and quantified with the same INT kit. Results were then normalized for protein content, determined by the BCA method (Pierce).…”

The Role of Creb in Adipogenesis through Mammalian Target of Rapamycin of Complex 1 (mTORC1) Pathyway

“…Other study by Vankoningsloo proved that the activation of CREB induced by mithochondrial dysfunction has triggered the accumulation of triglyceride in preadipocyte 3T3-L1. It established the role of CREB in adipogenesis process [14]. However, those studies did not associate the activation of CREB and p70S6K1.…”