ABSTRACT
SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.
Subject(s)
Adipocytes/physiology , Cell Membrane/physiology , Glucose Transporter Type 4/metabolism , Membrane Fusion , Qa-SNARE Proteins/metabolism , Qb-SNARE Proteins/metabolism , Qc-SNARE Proteins/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , 3T3-L1 Cells , Adipocytes/drug effects , Adipocytes/metabolism , Animals , Cell Membrane/metabolism , Cytoplasmic Vesicles/metabolism , Glucose/metabolism , Insulin/pharmacology , Mice , Protein Transport , Qa-SNARE Proteins/genetics , Qb-SNARE Proteins/genetics , Qc-SNARE Proteins/genetics , RNA Interference , Vesicle-Associated Membrane Protein 2/geneticsABSTRACT
White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPARgamma in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity.
Subject(s)
Adipocytes/metabolism , Adipose Tissue, White/metabolism , Energy Metabolism/physiology , Lipolysis/physiology , Proteins/metabolism , 3T3-L1 Cells , Adipocytes/cytology , Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Adipose Tissue, Brown/ultrastructure , Adipose Tissue, White/cytology , Adipose Tissue, White/ultrastructure , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Crosses, Genetic , Gene Expression Regulation , Heterozygote , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Molecular Weight , Proteins/chemistry , Proteins/genetics , RNA, Small Interfering/metabolismABSTRACT
White adipose tissue (WAT) is important as an energy reservoir in mammals, but the precise mechanism by which energy storage in WAT is controlled remains unclear. It is well known that representative anabolic hormone insulin efficiently stores triglyceride in adipocytes. We showed that insulin inhibited beta-agonist-induced lipolysis at least in part by inhibiting phosphorylation of perilipin and hormone-sensitive lipase (HSL) in 3T3-L1 adipocytes. Furthermore, insulin inhibited beta-agonist-induced increase of PGC-1alpha expression, which is important for mitochondrial biogenesis and energy expenditure. These results suggest the possibility that insulin efficiently stores triglyceride in adipocytes by decreasing lipolysis and repressing energy expenditure.
Subject(s)
Adipocytes/metabolism , Insulin/pharmacology , Lipolysis/drug effects , Trans-Activators/antagonists & inhibitors , Triglycerides/metabolism , 3T3 Cells , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Adipocytes/drug effects , Adrenergic beta-Agonists/pharmacology , Animals , Blotting, Western , Carrier Proteins , Energy Metabolism/drug effects , Gene Expression/drug effects , Mice , Perilipin-1 , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phosphoproteins/metabolism , Phosphorylation/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Sterol Esterase/metabolism , Trans-Activators/genetics , Transcription FactorsABSTRACT
Peroxisome proliferator-activated receptor (PPAR)-gamma plays an important role in adipogenesis. However, the functions of PPAR-gamma in differentiated adipocytes have remained unclear. The role of PPAR-gamma in mature 3T3-L1 adipocytes was therefore investigated by overexpression of a dominant negative mutant of this protein (PPAR-gamma-DeltaC) that lacks the 16 COOH-terminal amino acids and that has been shown to prevent the thiazolidinedione-induced differentiation of 3T3-L1 cells into adipocytes. Overexpression of PPAR-gamma-DeltaC in mature 3T3-L1 adipocytes by adenovirus gene transfer resulted in a decrease in both cell size and intracellular triglyceride content, an increase in the extent of lipolysis, and a reduction in the rate of free fatty acid uptake. Furthermore, overexpression of this mutant reduced the abundance of mRNAs for several key enzymes that contribute to triglyceride and free fatty acid metabolism as well as the amounts of GLUT4, insulin receptor, insulin receptor substrate (IRS), and C/EBPalpha mRNAs. It also reduced both the concentration of IRS2 and the insulin-stimulated glucose uptake. These results suggest that PPAR-gamma plays an important role in mature 3T3-L1 adipocytes at least in part by maintaining the expression of genes that confer the characteristics of mature adipocytes.
