The protective effect of simvastatin against low dose streptozotocin induced type 1 diabetes in mice is independent of inhibition of HMG-CoA reductase Tobias Rydgren * , Stellan Sandler Department of Medical Cell Biology, Uppsala University, Husargatan 3, P.O. Box 571, Biomedicum, SE-75123 Uppsala, Sweden article info Article history: Received 19 December 2008 Available online 14 January 2009 Keywords: Simvastatin Type 1 diabetes Mevalonic acid CD-1 mice HMG-CoA reductase Lymphocyte function associated antigen-1 Streptozotocin abstract Besides a cholesterol-lowering effect, simvastatin possesses anti-inflammatory properties attributed to inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and/or direct binding to, and inhibition of, the integrin lymphocyte function associated antigen-1 (LFA-1). We have shown that simvastatin protects against multiple low dose streptozotocin (MLDS) induced type 1 diabetes in mice. Presently, we examined if this effect could be abolished by co-administration of mevalonic acid, thus determining if the protective effect is dependent or independent of inhibition of HMG-CoA reductase. Mevalonic acid did not affect the protective effect of simvastatin against MLDS diabetes. Moreover, spleens from these mice did not show any signs of toxic side-effects, thus excluding the possibility that the protective effect is secondary to a general inflammatory response. We suggest that simvastatin’s pro- tective effect mainly is independent of HMG-CoA reductase inhibition. This implies that inhibition of LFA- 1 activation is important for the protective effect exerted by simvastatin. Ó 2009 Elsevier Inc. All rights reserved. Simvastatin belongs to a class of drugs called statins, or 3-hydro- xy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, and are widely used for the treatment of hypercholesterolemia since it blocks the conversion of HMG-CoA to mevalonate, the rate limiting step in the cholesterol synthesis pathway. Lately, detailed analysis of different clinical trials suggests that statins have direct anti-inflam- matory effects independent of their effects on plasma cholesterol levels [1–5]. Studies in animal models have also showed that statins exert such effects by e.g., interfering with endothelial adhesion and transendothelial migration of leukocytes to sites of inflammation [6–10]. These effects may be dependent and/or independent of the inhibition of HMG-CoA reductase. The HMG-CoA reductase depen- dent anti-inflammatory effects by statins might prevent the mem- brane localization and function of a variety of small GTP binding proteins (e.g., Ras, Rho and Rab), since mevalonate is a precursor not only for cholesterol but also for isoprenoids, necessary for the post-translational lipid modification of these proteins [11]. This in turn has been shown to e.g., prevent expression of intercellular adhesion molecule-1 (ICAM-1) [12] and inhibit integrin activation [13]. The HMG-CoA reductase independent anti-inflammatory ef- fects of some statins, including simvastatin, leads to inhibition of the integrin lymphocyte function associated antigen-1 (LFA-1) by di- rect binding to an allosteric site of the protein, which results in de- creased LFA-1 mediated leukocyte adhesion to ICAM-1 and decreased T-cell co-stimulation [14]. Type 1 diabetes is considered to be a complex autoimmune disease in which macrophages and T-cells invade the pancreatic islets and selectively destroy the insulin producing b-cells [15]. We have re- cently shown that the administration of simvastatin can delay disease onset in two different mouse models for type 1 diabetes [16]. One of these was the multiple low-dose streptozotocin (MLDS) model, which has been shown to be T-lymphocyte dependent [17–20]. In this mod- el, the mice receive five daily i.p. injections of streptozotocin (STZ), and a progression of hyperglycemia and insulitis evolve during a 2- week-period. In this study, we used the MLDS model to investigate if the protective effect of simvastatin treatment could be abolished by administration of mevalonic acid, thus aiming to determine whether the protective effect of simvastatin in this model is mainly dependent or independent of HMG-CoA reductase inhibition. Materials and methods Reagents. Simvastatin was kindly provided by Merck Research Laboratories (Rahway, NJ). Simvastatin was activated by alkaline hydrolysis, prior to experiments, as described previously [16]. Mevalonic acid lactone was purchased from Sigma–Aldrich Swe- den AB (Stockholm, Sweden), and converted to sodium mevalonic acid by solubilization in 0.1 M sodium hydroxide, incubation at 50 °C for 2 h, and adjustment of the pH to 7.4 with hydrochloric acid (adapted from a study by Wagner et al. [21]). Streptozotocin (STZ) was purchased from Sigma Chemicals (St. Louis, MO). Animals and treatment. All experiments were approved by the local animal ethics committee for Uppsala University. Outbred 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.017 * Corresponding author. Fax: +46 18 471 4059. E-mail address: Tobias.Rydgren@mcb.uu.se (T. Rydgren). Biochemical and Biophysical Research Communications 379 (2009) 1076–1079 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc