Matrix metalloproteinase inhibition affects adipose tissue mass in obese mice Matthias Van Hul,* Florea Lupu, † Tom Dresselaers, ‡ Johan Buyse § and H Roger Lijnen* *Center for Molecular and Vascular Biology, ‡ Biomedical Nuclear Magnetic Resonance Unit/MoSAIC, § Laboratory of Livestock Physiology, Immunology and Genetics, Katholieke Universiteit Leuven, Leuven, Belgium and † Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA SUMMARY 1. Because the development of adipose tissue involves remodelling of the extracellular matrix (ECM), which requires matrix metalloproteinase (MMP) activity, we exam- ined whether MMP inhibitors may have the potential to affect adipose tissue mass in obese mice. 2. Administration of the relatively gelatinase-specific MMP inhibitor tolylsam ((R)-3-methyl-2-[4-(3-p-tolyl-[1,2,4]oxa- diazol-5-yl)-benzenesulphonylamino]-butyric acid; 100 mg/kg per day) for 7 weeks to obese wild-type mice on a high-fat diet resulted in significantly lower bodyweight (P < 0.05), lower subcutaneous (SC) and gonadal (GON) adipose tissue mass (both P < 0.05) and smaller adipocytes in both SC (P < 0.005) and GON (P < 0.0005) adipose tissues. 3. Magnetic resonance imaging confirmed a lower total body fat content in tolylsam-treated mice (P < 0.0005). In addition, tolylsam treatment of wild-type mice was associated with a marked enhancement in metabolic rate. 4. Electron microscopy analysis of tissue sections at the end of the 7 week feeding period revealed significantly higher collagen accumulation in the ECM of SC adipose tissues of tolylsam-treated mice (P < 0.001). 5. Thus, the relatively gelatinase-specific MMP inhibitor tolylsam has the potential to affect fat tissue growth in obese mice. Key words: adipocyte, adipose tissue, gelatinase, matrix metalloproteinases, obesity. INTRODUCTION The development of adipose tissue is a complex process in which several growth factors, cytokines, hormones and proteinases con- tribute to adipogenesis, angiogenesis and proteolytic remodelling of the extracellular matrix (ECM). 1 The matrix metalloproteinase (MMP) system plays an important role in these processes, suggesting that modulation of the activity of specific MMPs may affect adipose tissue development. 2 Several lines of evidence indicate a functional role for the gelatinases (gelatinase A or MMP-2 and gelatinase B or MMP-9). Thus, in vitro studies with proteinase inhibitors and neutralizing antibodies have revealed that MMP-2 and MMP-9 play a key role in adipocyte differentiation. 3 Furthermore, MMP-2-deficient, but not MMP-9-deficient, mice kept on a high-fat diet (HFD) develop significantly less adipose tissue than their wild-type littermates. 4,5 Previous studies have shown the potential to impair adipose tissue development in young mice by using broad-spectrum MMP inhibitors, such as galardin, 6 Bay 12- 9566 7 or Ro 28-2653. 8 We have shown that the more gelatinase-specific inhibitors tolylsam 4 and ABT-518 9 impair bodyweight gain and adipose tissue development in young wild- type mice kept on an HFD. However, it is not known whether such inhibitors have the potential to affect established obesity, which would be more relevant clinically. In the present study, we investigated the potential of tolylsam (IC 50 5 nmol/L for MMP-2, 49 nmol/L for MMP-9 and 34 nmol/L for MMP-12) 10 to affect adipose tissue mass and composition in mice with established obesity. METHODS Diet-induced obesity model Five-week-old male wild-type mice (C57Bl/6 genetic back- ground) were kept in microisolation cages on a 12 h light–dark cycle and fed ad libitum with an HFD (TD 88137; Harlan Teklad, Zeist, The Netherlands; 42% kcal as fat, 20.1 kJ/g) for 20 weeks. Then, five obese mice were given the HFD supple- mented with 100 mg/kg per day tolylsam for an additional period of 7 weeks, with another five mice continuing on the HFD alone (control group). Tolylsam ((R)-3-methyl-2-[4-(3-p-tolyl-[1,2,4] oxadiazol-5-yl)-benzenesulphonylamino]-butyric acid), an MMP-2, MMP-9 and MMP-12 inhibitor, 10 was the kind gift of Shionogi (Osaka, Japan). The weight and food intake of the mice were measured at weekly intervals. Fat pad volumes were measured 1 week after the start (i.e. 26 weeks of age) and at the end (i.e. 32 weeks of age) of the experiments using non-invasive magnetic resonance imaging (MRI), performed as described previously. 11 After an overnight fast, mice were killed by intraperitoneal injec- tion of 60 mg/kg Nembutal (Abbott Laboratories, North Chicago, IL, USA). Blood was collected via the retro-orbital sinus on triso- dium citrate (final concentration 0.01 mol/L) and plasma was stored at À80°C. Intra-abdominal (gonadal; GON) and inguinal subcutaneous (SC) fat pads were removed and weighed, and Correspondence: HR Lijnen, Center for Molecular and Vascular Biology, KU Leuven, Campus Gasthuisberg, O&N 1, Herestraat 49, Box 911, B-3000 Leuven, Belgium. Email: roger.lijnen@med.kuleuven.be Received 27 January 2012; revision 10 April 2012; accepted 8 April 2012. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Blackwell Publishing Asia Pty Ltd Clinical and Experimental Pharmacology and Physiology (2012) 39, 544–550 doi: 10.1111/j.1440-1681.2012.05714.x