Adipocyte Produces Matrix Metalloproteinases 2 and 9 Involvement in Adipose Differentiation Anne Bouloumie ´ , Coralie Sengene ` s, Ghyslaine Portolan, Jean Galitzky, and Max Lafontan Adipocyte hypertrophy and hyperplasia together with angiogenesis contribute to the growth of the fat mass. Because changes in the extracellular matrix (ECM) components are often associated with such cellular remodeling, we studied the adipocyte expression of the matrix metalloproteinases (MMPs) 2 and 9, two key enzymes involved in the modulation of ECM. The pres- ent study provides the first evidence that human adi- pose tissue produces and secretes MMP-2 and -9 as shown by gelatin zymography analysis performed on media conditioned by human subcutaneous adipose tis- sue and human preadipocytes in primary cultures and by reverse transcriptase–polymerase chain reaction (RT- PCR) analysis on transcripts from mature human adipo- cytes. The further characterization performed on the murine 3T3F442A preadipocyte cell line demonstrates that MMP expression, assessed by RT-PCR and Western blot analysis, as well as activity, assessed by gelatin zymography analysis, increased during the adipocyte differentiation, whereas the expression of tissue inhib- itor metalloproteinases 1 and 2 were abolished or not affected, respectively. Finally, preadipocyte treatment with MMP inhibitors such as batimastat and captopril, as well as neutralizing antibodies, markedly decreased adipocyte differentiation as demonstrated by the inhi- bition in the appearance of lipogenic (triglycerides) and lipolytic (glycerol release and hormone-sensitive lipase expression) markers. These data suggest that MMP-2 and -9 could be important key regulators of adipocyte differentiation. Thus, the adipocyte-derived MMPs might represent a new target for the inhibition of adipose tissue growth. Diabetes 50:2080 –2086, 2001 O besity is associated with an excessive growth of adipose tissue, the development of which is dependent on cellular events concerning both adipose tissue cell populations, i.e., adipocytes and their precursor cells, preadipocytes, as well as micro- vascular endothelial cells. Indeed, during the settlement of obesity, enhanced lipogenesis together with decreased lipolysis will result in a net increase in lipid storage within the adipocyte, leading to adipocyte hypertrophy. Adipo- cyte hyperplasia due to preadipocyte proliferation and differentiation will contribute to adipose mass expansion (1). Concomitant with both hypertrophic and hyperplasic events, stimulation of angiogenic process will provide new blood vessels in the growing tissue, thus permitting the supply of oxygen and nutrients necessary for adipose tissue metabolism (2). It is quite obvious that dramatic tissue remodeling occurs within the adipose tissue during the fat mass development. However, whereas mechanisms leading to adipocyte hypertrophy and hyperplasia have been exten- sively studied, few data are available concerning the regulation of the angiogenic processes as well as the modification of the extracellular matrix (ECM) during fat mass increment and the settlement of obesity. Adipocytes, besides their metabolic activities, are able to produce several factors, such as growth factors and cytokines, which may play a role in the paracrine regulation of the adipose tissue remodeling (3). Indeed, adipocytes also secrete proangiogenic factors, such as vascular endothe- lial growth factor (VEGF) (4), tumor necrosis factor– (5), monobutyrin (6), and leptin (7). These factors, originating from adipocytes, may contribute to the formation of new blood vessels inside the fat pad. Moreover, because these secretions are increased during adipocyte differentiation, it further strengthens the hypothesis of the existence of a paracrine loop between adipocyte differentiation and the stimulation of angiogenic process. Besides these secreted factors, proteases and ECM components might also play an important role in regu- lating adipose tissue remodeling. Indeed, it is now well established that degradation of ECM represents the first step in the angiogenic process. Matrix metalloproteinases (MMPs), especially MMP-2 and -9, have been shown to be necessary for this event (8). No data report the presence of MMPs in adipose tissue and adipocytes. However, ECM components are synthesized and degraded during the process of adipocyte differentiation (9–11), and some studies have shown that different ECM context modulates adipocyte differentiation (12). We performed the present study to determine whether adipocytes and preadipocytes produce MMPs. We provide the first evidence that human adipose tissue releases MMP-2 and -9 and that this secretion is modulated during adipocyte differentiation. Moreover, MMP activities are directly involved in the regulation of adipocyte differenti- ation, since their inhibition resulted in a blockade of adipocyte differentiation. It is then tempting to speculate that the adipocyte-derived MMPs might represent a new From the Institut National de la Sante ´ et de la Recherche Me ´ dicale (INSERM) U317, Laboratoire de Pharmacologie Me ´ dicale et Clinique, Toulouse, France. Address correspondence and reprint requests to Dr. Anne Bouloumie ´, INSERM U317, Laboratoire de Pharmacologie Me ´ dicale et Clinique, 37 alle ´es Jules Guesde F-31073 Toulouse cedex, France. E-mail: bouloumi@cict.fr. Received for publication 6 February 2001 and accepted 18 May 2001. BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; F12, nutrient mix F12; GAPDH, glyceraldehyde- 3-phosphate dehydrogenase; HSL, hormone-sensitive lipase; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; RT-PCR, reverse tran- scriptase–polymerase chain reaction; TIMP, tissue inhibitor metalloprotein- ase; VEGF, vascular endothelial growth factor. 2080 DIABETES, VOL. 50, SEPTEMBER 2001