Supplementary MaterialsData_Sheet_1. cells during obesity-induced diabetogenesis, we developed transgenic mice selectively overexpressing galectin 3 in cells and tested their susceptibility to obesity-induced type-2 diabetes. Obesity was induced with a 16-week high-fat diet regime. Pancreatic cells CW-069 were tested for susceptibility to apoptosis induced by non-esterified fatty acids and cytokines as well as parameters of oxidative stress. Results: Our results demonstrated that overexpression of galectin 3 increases -cell apoptosis in HFD conditions and increases the percentage of proinflammatory F4/80+ macrophages in islets that express galectin 3 and TLR4. In isolated islets, we have shown that galectin 3 overexpression increases cytokine and palmitate-triggered -cell apoptosis and also increases NO2?-induced oxidative stress of cells. Also, in pancreatic lymph nodes, macrophages were shifted toward a proinflammatory TNF–producing phenotype. Conclusions/Interpretation: CW-069 By complementary and approaches, we have shown that galectin 3-overexpression facilitates -cell damage, enhances cytokine and palmitate-triggered -cell apoptosis, and increases NO2?-induced oxidative stress in cells. Further, the results suggest KITH_HHV1 antibody that increased expression of galectin 3 in the pancreatic cells affects the metabolism of glucose and glycoregulation in mice on a high-fat diet, affecting both fasting glycemic values and glycemia after glucose loading. is not defined. data have provided conflicting evidence. Early studies indicated that IL-1-stimulated rat islets upregulated galectin 3, which protected cells against IL-1 toxicity (19). On the other hand, a deficiency of galectin 3 due to genetic deletion or application of chemical inhibitor protects pancreatic islets from TNF-+IFN-+IL-1-triggered apoptosis (20). In this study, we aimed to investigate the role of the galectin 3 expressed in cells in HFD-induced metabolic defects. To clarify the CW-069 role of galectin 3 expression in cells during obesity-induced diabetogenesis, we used transgenic mice selectively overexpressing galectin 3 in cells. We show that overexpression of galectin 3 promotes -cell apoptosis in HFD conditions and increases the percentage of proinflammatory F4/80+ macrophages in islets that express galectin 3 and Toll-like receptor 4 (TLR4). Further, we present data that galectin 3 overexpression increases cytokine and palmitate-triggered -cell apoptosis and NO2? induced oxidative stress in cells. Thus, in complementary and approaches, we show that galectin 3 overexpression facilitates -cell damage, enhances cytokine, and palmitate-triggered -cell apoptosis, and increases NO2?-induced oxidative stress in cells. Materials and Methods Experimental Mice and Study Design We used wild-type C57BL/6J male mice (WT) CW-069 and littermate CW-069 C57BL/6J mice with transgenically enhanced galectin 3 expression in the pancreatic cells (TG), 8C10 weeks old, obtained in collaboration with Prof. Bernard Thorens (Center for Integrative Genomics, University of Lausanne). To generate transgenic mice expressing galectin 3 in pancreatic islet -cells, the galectin 3 cDNA was subcloned in front of the rat insulin promoter, as previously described (21). Transgenic mice in C57Bl/6 background were prepared by a commercial service. For testing the mouse genotype, we extracted DNA from ear tissue (KAPA Express Extract, KK7102, Kapabiosystems, USA). For PCR reaction, we used the KAPA 2G Fast Ready Mix PCR Kit (KK5102, Kapabiosystems, USA) and the primers listed in Supplementary Material. Overexpression of galectin 3 in the pancreatic cells was confirmed with 591 bp PCR product visualized on agarose gel (Supplement 1). All experimental animals were bred in our animal facilities under standard laboratory conditions in a temperature-controlled environment with a 12 h light/dark cycle and received water and a standard low-fat diet (LFD, 10% calories from fat, Mucedola, Italy) or a high-fat diet (HFD, 60% calories from fat, Mucedola, Italy) for 16 weeks. We used 15C20 animals per group in two repeated experiments. Mice were sacrificed by cervical dislocation, and blood samples, visceral adipose tissue, and pancreas were collected for further analyses. Ethics Statement The study was conducted in compliance with all the regulations and recommendations stated in the European Union Directive 2010/63/EU. All animal procedures were approved by the Ethical Committee of the Faculty of Medical Sciences, University of Kragujevac (Permit Number: 01-6675). Metabolic Parameters The body weight of mice.