Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, Biophysics, Preadipocyte, Adipogenesis, Obesity, Matrix metalloproteinase, Gelatinase, ADIPOSE-TISSUE DEVELOPMENT, MATRIX-METALLOPROTEINASE INHIBITION, NUTRITIONALLY INDUCED OBESITY, ADIPOCYTE DIFFERENTIATION, EXTRACELLULAR-MATRIX, IN-VIVO, MICE, INVOLVEMENT, PREADIPOCYTES, ORGANIZATION, 3T3 Cells, Adipocytes, Adiponectin, Animals, Cell Differentiation, Embryo, Mammalian, Enzyme-Linked Immunosorbent Assay, Fatty Acid-Binding Proteins, Fibroblasts, Gene Expression, Male, Matrix Metalloproteinase 2, Mice, Mice, Inbred BALB C, Mice, Knockout, Mice, Nude, Microscopy, Polarization, PPAR gamma, RNA Interference, Reverse Transcriptase Polymerase Chain Reaction, 0601 Biochemistry and Cell Biology, 1115 Pharmacology and Pharmaceutical Sciences, 3101 Biochemistry and cell biology

Abstract:

Expansion of adipose tissue is associated with hypertrophy and/or hyperplasia (through adipogenesis), together with angiogenesis and intensive remodeling of the extracellular matrix. Research over the past decade suggests a prominent role in these processes for the gelatinase subtypes of the matrix metalloproteinase (MMP) family. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are secreted by adipocytes and their activity is modulated during adipose tissue expansion. In the present study, we have evaluated the role of the gelatinases in adipogenesis. Murine embryonic fibroblasts (MEFs) derived from wild type or MMP-2 knockout mice were subjected to a hormonal cocktail to stimulate adipogenic differentiation. At the end of a 12-day period, significantly less differentiation was observed in MMP-2 knockout MEFs compared to wild type MEFs, as shown by 90% reduced Oil Red O staining (intracytoplasmatic lipid content). In accordance, gene expression of the pro-adipogenic markers aP2 and PPAR-γ2 was decreased with 72% and 54%, respectively. Alternatively, selective shRNA-mediated gene silencing was performed in murine 3T3-F442A preadipocytes (a well established committed cell line). Stable knockdown of MMP-2 was confirmed by i) up to 90% decreased mRNA expression ii) reduced antigen levels in cultured medium at experimental day 0 (10.7 ± 0.7 versus 45.9 ± 10.6 ng/ml for scrambled shRNA; p=0.002) and iii) 60% less gelatinolytic activity. Loss of MMP-2 significantly decreased differentiation, as demonstrated by Oil Red O staining (reduction by 65%) and mRNA expression of the adipogenic markers aP2 and PPAR-γ. Upon selective MMP-9 knockdown in 3T3-F442A cells, no significant differences in adipogenesis were observed as compared to control cells. Thus, in vitro differentiation of preadipocytes into mature adipocytes is promoted by MMP-2. Next, we extended our in vitro findings to an in vivo model of de novo adipogenesis. Nude athymic BALB/c mice were subcutaneously injected with 3T3-F442A cells with/without MMP-2 knockdown. De novo fat pad formation was analyzed after 4 weeks of high fat diet feeding. Fat pad mass and adipocyte size and density were not significantly affected by MMP-2 knockdown. Expression of adipogenic markers (aP2, PPAR-γ and adiponectin) was, however, significantly decreased. Similar experiments are ongoing in MMP-2 knockout mice. Thus, our data indicate that MMP-2 is a key player in early preadipocyte differentiation.