Persistent correction of hyperglycemia in streptozotocin-nicotinamide-induced diabetic mice by a non-conventional radical scavenger

M. Novelli B. Bonamassa M. Masini N. Funel D. Canistro V. De Tata M. Martano A. Soleti D. Campani M. Paolini P. Masiello 0 ) Dipartimento di Patologia Sperimentale , Biotecnologie Mediche, Infettivologia ed Epidemiologia, Universit degli Studi di Pisa , Via Roma, 55-Scuola Medica, 56126 Pisa, Italy We previously reported that in a diabetes mouse model, characterised by moderate hyperglycaemia and reduced -cell mass, the radical scavenger bis(1-hydroxy-2,2,6,6tetramethyl-4-piperidinyl)decandioate di-hydrochloride (IAC), a non-conventional cyclic hydroxylamine derivative, improves metabolic alterations by counteracting -cell dysfunction associated with oxidative stress. The aims of this study were to ascertain whether the beneficial effects of IAC treatment could be maintained after its discontinuation and further elucidate the underlying mechanisms. Diabetes was induced in C57Bl/6J mice by streptozotocin (STZ) and nicotinamide (NA) administration. Diabetic mice were treated for 7 weeks with various doses of IAC (7.5, 15, or 30 mg/kg b. w./die i.p.) and monitored for additional 8 weeks after suspension of IAC. Then, pancreatic tissue was used for determination of -cell mass by immunohistochemistry and cell ultrastructural analysis. STZ-NA mice showed moderate - hyperglycaemia, glucose intolerance and reduced -cell mass (25% of controls). IAC-treated STZ-NA mice (at both doses of 15 and 30 mg/kg b.w.) showed long-term reduction of hyperglycaemia even after discontinuation of treatment, attenuation of glucose intolerance and partial preservation of -cell mass. The lowest IAC dose was much less effective. Plasma nitrotyrosine levels (an oxidative stress index) significantly increased in untreated diabetic mice and were lowered upon IAC treatment. At ultrastructural level, cells of IACtreated diabetic mice were protected against degranulation and mitochondrial alterations. In the STZ-NA diabetic mouse model, the radical scavenger IAC induces a prolonged reduction of hyperglycaemia associated with partial restoration of -cell mass and function, likely dependent on blockade of oxidative stress-induced damaging mechanisms. Type 2 diabetes (T2D) is characterised by a combination of peripheral insulin resistance and defective insulin secretion (Kahn 2003). Usually, the disease arises due to the progressive failure of insulin secretion by cells to overcome reduced sensitivity to the hormone (Kahn 2001; Prentki and Nolan 2006). This leads to hyperglycaemia, which can in turn exert deleterious effects on cells (Robertson 2004). Among the various mechanisms likely responsible for -cell dysfunction and damage induced by hyperglycaemia, oxidative stress is considered to play a major role (Kaneto et al. 2005; Prentki and Nolan 2006; Robertson 2004). Actually, in T2D, increased plasma glucose levels, often associated with high circulating free fatty acid (FFA) concentrations, result in enhanced mitochondrial superoxide production and increased exposure of cells to reactive oxygen species (ROS) (Brownlee 2003; Lowell and Shulman 2005), which can also be generated through the glycation reaction (Kaneto et al. 1996) and the hexosamine pathway (Kaneto et al. 2001). Moreover, the enhanced superoxide production is usually accompanied by increased NO generation due to induction of NO synthase (Beckman and Koppenol 1996). This favours the production of radical nitrogen species (RNS), such as the cytotoxic peroxynitrite anion, which in turn oxidises sulfhydryl groups in proteins, nitrates amino acids like tyrosine, aggravates lipid peroxidation and provokes DNA strand breaks, eventually leading to severe cell damage (Beckman and Koppenol 1996). It should also be considered that cells are especially sensitive to ROS excess because of a low expression of ROS-detoxifying enzymes in comparison with other cell types (Tiedge et al. 1997). We previously tested the protective effects of a low molecular weight radical scavenger, namely, bis(1-hydroxy2,2,6,6-tetramethyl-4-piperidinyl)decandioate di-hydrochloride (IAC), in a non-obese diabetes mouse model characterised by reduced pancreatic insulin content and moderate hyperglycaemic levels, like those usually occurring in human T2D. This model, obtained by the combined administration of streptozotocin (STZ) and nicotinamide (NA), has been developed in our laboratory as well as in others (Masiello et al. 2006; Matsuyama-Yokono et al. 2008; Nakamura et al. 2006) and is increasingly used for pharmacological research in diabetes (Novelli et al. 2007; Tahara et al. 2008, 2009). In STZ-NA mice, IAC was able to reduce hyperglycaemia and partially preserve pancreatic insulin content, thus counteracting -cell dysfunction associated with oxidative stress (Novelli et al. 2007). IAC is a non-conventional cyclic hydroxylamine derivative antioxidant synthesised in our laboratories (for its chemical structure, see Fig. 1). It is hydrosoluble in its protonated chloridrate form, membrane permeant and acts as an effective and self-regenerating intracellular scavenger of most oxygen, nitrogen and carbon-centred radicals at an early stage, i.e. prior to the generation of ROS- and RNSderived toxic products (Valgimigli et al. 2001). The aims of the present study were (1) to ascertain whether the beneficial effects of IAC in diabetic mice could be maintained after Fig. 1 Chemical structure of bis(1-hydroxy-2,2,6,6-tetramethyl-4piperidinyl)decandioate (IAC), showing the two opposite cyclic hydroxylamine moieties linked by a long aliphatic chain discontinuation of the treatment and (2) to provide further insights into the mechanisms of its protective effect. Materials and methods Experiments were performed in male C57Bl/6J mice, of the age of 89 weeks, weighing 2224 g, purchased from Harlan Laboratories s.r.l. (S. Pietro al Natisone, Udine, Italy). Mice were kept at a constant temperature of 2425C and were subjected to a controlled 12 h lightdark cycle; they had free access to water and pelleted diet. The experimental protocol followed the Principles of Laboratory Animal Care (US NH publication no. 83-85, revised 1985) and was approved by the Ethical Committee of the University of Pisa. Induction of diabetes Nicotinamide (210 mg/kg b.w.) (Sigma, Saint Louis, MO, USA), dissolved in saline, was injected intraperitoneally 15 min before administration of STZ (Sigma, 180 mg/kg b. w., i.p.), which was dissolved in buffer citrate (pH 4.5) immediately before use. Controls received both vehicles. During the experimental period, the animals food intake and body weight were monitored once a week. Glycaemia was measured 7, 14, and 21 days after STZ-NA treatment to assess stability of glycaemic values, and on the 22nd day, diabetic mice were homogeneously distributed into four subgroups of 1012 mice each. Pharmacological treatment was randomly assigned to the four diabetic groups, three of them receiving intraperitoneal administration of IAC at the dosage of 7.5, 15 or 30 mg/kg (...truncated)