This high concentration was chosen to determine the effects of CML in a relatively short incubation time of 24 hours. Since we also use FCS in this model, it is possible that CML binds to FCS and that the actual amount of free CML reacting with the cells is much lower than 0.5 mM and might even be in the in vivo range. Only a limited number of studies about

the effect of AGE on beta cell viability Cyclopamine in vivo and function have been published. A study in a mouse beta cell line found that exposure to AGEs increased superoxide production in the mitochondria, which led to an impairment of insulin secretion [29]. Increased oxidative stress via the mitochondria due to exposure to AGEs was also found in rat beta cells [30]. Exposure of different rodent beta cell lines to AGEs induced both proliferation and apoptosis in these cells [31]. In line with these studies, we also observed a decrease in beta cell viability after exposure to the AGE CML. This decreased viability was accompanied by an increase in oxidative stress which probably results from the interaction of CML with RAGE. RAGE is a multiligand transmembrane receptor which belongs to the immunoglobulin gene superfamily [32]. Activation of RAGE by AGEs transduces multiple signals resulting in activation and translocation of nuclear transcription factors like NF-κB [4]. This leads to the expression of proinflammatory cytokines, including

IL-8 and MCP-1 [19], Y-27632 solubility dmso [20] and [21]. It has been shown that CML adducts are signal-transducing ligands for RAGE, both in vitro and in vivo [33]. However, another study found that CML-modified proteins were unable to bind

to RAGE and activate Celastrol proinflammatory signaling [34]. No changes in the gene expression of RAGE after exposure to CML were found, but this may be due to the relatively short incubation time of 24 hours. However, increased concentrations of the proinflammatory cytokine MCP-1 were detected, which could be caused by RAGE signaling, as MCP-1 is known to be regulated by RAGE. MCP-1 is involved in the pathogenesis of diabetic nephropathy [35] and is also implicated in the destruction of beta cells in type 1 diabetes [36]. The rise in MCP-1 levels could explain the observed increase in intracellular oxidative stress in these cells since MCP-1 has been associated with the induction of oxidative stress in previous studies. MCP-1 enhanced ROS generation in monocytes from unstable angina patients [37]. Additionally, MCP-1-deficiency impaired ROS generation and attenuated oxidative stress in an ovariectomy rodent model (as a model for menopause) [38]. Previous research has shown that AGEs can increase GSSG levels in human neuroblastoma cells [39]. Also in vivo an association between AGEs and a decreased glutathione redox ratio in patients undergoing continuous ambulatory peritoneal dialysis was found [40].